Abstract:
The invention relates to the polynucleotide sequence of a nontypeable stain of  Haemophilus influenzae  (NTHi) and polypeptides encoded by the polynucleotides and uses thereof. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear and/or the nasopharynx.

Description:
RELATED APPLICATIONS 
       [0001]    The present application claims priority benefit from U.S. Provisional Application 60/453,134 filed Mar. 6, 2003 which is incorporated herein by reference in its entirety. 
     
    
     FIELD OF INVENTION 
       [0002]    The invention relates to the polynucleotide sequence of a nontypeable strain of  Haemophilus influenzae  (NTHi) genome, NTHi genes contained within the genome and polypeptides encoded by the polynucleotides. The invention also relates to uses of these NTHi polynucleotides and NTHi polypeptides including vaccines and methods of treating and preventing NTHi related disorders. The invention also relates to NTHi genes which are upregulated during or in response to NTHi infection of the middle ear or nasopharynx. 
       BACKGROUND 
       [0003]    Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez,  Epidemiol . Rev., 15: 444-465, 1993). Recent statistic indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980&#39;s. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child&#39;s behavior, education and development of language skills (Baldwin,  Am. J. Otol.,  14: 601-604, 1993; Hunter et al.,  Ann. Otol. Rhinol. Laryngol. Suppl.,  163: 59-61, 1994; Teele et al.,  J. Infect. Dis.,  162: 685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al.,  Pediatr. Infect. Dis. J.,  16: S9-11, 1997). 
         [0004]    Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear, the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are ˜13%; Bright et al.,  Am. J. Public Health,  83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al.,  Pediatrics,  93(3):353-63, 1994; Bright et al., supra.; Cimons,  ASM News,  60: 527-528; Paap.  Ann. Pharmacother.,  30(11): 1291-7, 1996). 
         [0005]    Progress in vaccine development is most advanced for  Streptococcus pneumoniae , the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven-valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi,  Pedriatr. Infect. Dis. J.  16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al.,  Pedriatr. Infect. Dis J.,  19: 187-195; Eskola et al.,  Pedriatr. Infect. Dis J.,  19: S72-78, 2000; Eskola et al.,  N. Engl. Med.  344: 403-409, 2001; Snow at al.,  Otol. Neurotol.,  23: 1-2, 2002). Less progress has been made for non-typeable  Haemophilus influenzae  (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein,  Pedriatr. Infect. Dis J.,  16: S5-8, 1997; Spinola et al.,  J. Infect. Dis.,  154: 100-109, 1986). Hampering development of effective vaccines against NTHi, is the currently incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay is a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host&#39;s immune response as the disease progresses from one of host immunological tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space. 
         [0006]    Currently there is a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank,  Pedriatr. Infect. Dis J.,  13(11): 1064-8, 1994: Karma et al.,  Int. J. Pedritr. Otorhinolaryngol.,  32(Suppl.): S127-34, 1995). 
       SUMMARY OF INVENTION 
       [0007]    The present invention provides for the identification and characterization of the genomic sequence of NTHi  H. influenzae  strain 86-028NP and the polypeptide sequences encoded thereby. The 3-fold analysis of the NTHi genomic sequence is set out in a series of contig sequences denoted as SEQ ID NO: 1-576, and the subsequent 8-fold analysis of the genomic sequence is set out in a series of 11 contig sequences denoted as SEQ ID NOS: 675-685. These contigs are raw data and one of skill in the art may assemble these contigs by comparing overlapping sequences to construct the complete genome of the NTHi stain 86-028NP using routine methods. 
         [0008]    The present invention also provides for antibodies specific for the NTHi polypeptides of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting NTHi polynucleotides with specific polynucleotide probes or detecting NTHi polypeptides with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria. 
         [0009]    The present invention also contemplates methods of eliciting an immune response by administering a NTHi polypeptide of the invention or a NTHi peptide thereof. These methods include administering the NTHi polypeptide or NTHi peptide as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM. The following NTHi genes are upregulated during or in response to middle ear and/or nasopharynx infections; and the polypeptides encoded by these genes and peptides thereof are contemplates as possible OM vaccine candidates and/or target of chemotherapy: hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. NTHi hisB gene is set out as nucleotide sequence SEQ ID NO: 615 and encodes the amino acid sequence set out as SEQ ID NO: 616. NTHi sapA gene is set out as nucleotide sequence SEQ ID NO: 617 and encodes the amino acid sequence set out as SEQ ID NO: 618. NTHi rbsC gene is set out as nucleotide sequence SEQ ID NO: 619 and encodes the amino acid sequence set out as SEQ ID NO: 620. NTHi purE gene is set out as nucleotide sequence SEQ ID NO: 621 and encodes the amino acid sequence set out as SEQ ID NO: 622. NTHi ribB gene is set out as nucleotide sequence SEQ ID NO: 623 and encodes the amino acid sequence set out as SEQ ID NO: 624. NTHi arcB gene is set out as nucleotide sequence SEQ ID NO: 625 and encodes the amino acid sequence set out as SEQ ID NO: 626. NTHi uxuA gene is set out as nucleotide sequence SEQ ID NO: 627 and encodes the amino acid sequence set out as SEQ ID NO: 628. NTHi dsbB gene is set out as nucleotide sequence SEQ ID NO: 629 and encodes the amino acid sequence set out as SEQ ID NO: 630. NTHi ureH gene is set out as nucleotide sequence SEQ ID NO: 631 and encodes the amino acid sequence set out as SEQ ID NO: 632. NTHi licC gene is set out as nucleotide sequence SEQ ID NO: 633 and encodes the amino acid sequence set out as SEQ ID NO: 634. NTHi HI1647 gene is set out as nucleotide sequence SEQ ID NO: 635 and encodes the amino acid sequence set out as SEQ ID NO: 636. NTHi ispZ gene is set out as nucleotide sequence SEQ ID NO: 637 and encodes the amino acid sequence set out as SEQ ID NO: 638. NTHi radC gene is set out as nucleotide sequence SEQ ID NO: 639 and encodes the amino acid sequence set out as SEQ ID NO: 640. NTHi mukF gene is set out as nucleotide sequence SEQ ID NO: 641 and encodes the amino acid sequence set out as SEQ ID NO: 642. NTHi glpR gene is set out as nucleotide sequence SEQ ID NO: 643 and encodes the amino acid sequence set out as SEQ ID NO: 644. NTHi ihfB gene is set out as nucleotide sequence SEQ ID NO: 645 and encodes the amino acid sequence set out as SEQ ID NO: 646. NTHi argR gene is set out as nucleotide sequence SEQ ID NO: 647 and encodes the amino acid sequence set out as SEQ ID NO: 648. NTHi cspD gene is set out as nucleotide sequence SEQ ID NO: 649 and encodes the amino acid sequence set out as SEQ ID NO: 650. NTHi HI1163 gene is set out as nucleotide sequence SEQ ID NO: 651 and encodes the amino acid sequence set out as SEQ ID NO: 652. NTHi HI1063 gene is set out as nucleotide sequence SEQ ID NO: 653 and encodes the amino acid sequence set out as SEQ ID NO: 654. NTHi HI0665 gene is set out as nucleotide sequence SEQ ID NO: 655 and encodes the amino acid sequence set out as SEQ ID NO: 656. NTHi HI1292 gene is set out as nucleotide sequence SEQ ID NO: 657 and encodes the amino acid sequence set out as SEQ ID NO: 658. 
         [0010]    The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx, and therefore are contemplated to encode OM vaccine candidates and/or targets of chemotherapy. In addition, the following NTHi genes are contemplated to be virulence-associated genes and therefore are contemplated to encode possible OM vaccine candidates and/or targets of chemotherapy: HI1386, HI1462, HI1369, lav, HI1598. NTHi HI1386 gene sequence is set out as SEQ ID NO: 659 and encodes the amino acid sequence set out as SEQ ID NO: 660. NTHi HI1462 gene sequence is set out as SEQ ID NO: 661 and encodes the amino acid sequence set out as SEQ ID NO: 662. NTHi HI1369 gene sequence is set out as SEQ ID NO: 665 and encodes the amino acid sequence set out as SEQ ID NO: 666. NTHi lay gene sequence is set out as SEQ ID NO: 663 and encodes the amino acid sequence set out as SEQ ID NO: 664. NTHi HI1598 gene sequence is set out as SEQ ID NO: 669 and SEQ ID NO: 671 and encodes the amino acid sequence set out as SEQ ID NO: 670 and SEQ ID NO: 672. Additional NTHi genes associated with virulence include the polynucleotide sequences presented as SEQ ID NO: 667 and SEQ ID NO: 673. 
         [0011]    As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of the NTHi polypeptides, which are upregulated or active during infection. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating NTHi protein expression by administering an antisense oligonucleotide that specifically binds to NTHi genes that are upregulated during NTHi infections, such genes include hisB, lppB, sapA, lolA, rbsC, purE, ribB, arcB, uxuA, dsbB, ureH, licC, HI1647, ispZ, radC, mukF, glpR, ihfB, argR, cspD, HI0094, HI1163, HI1063, HI0665, HI1292, HI1064. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of the proteins encoded by theses genes. The novel NTHi genes included in the polynucleotide sequences presented as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Tables 4 and 4B are also up-regulated during infection of the middle ear and/or the nasopharynx and therefore antisense oligonucleotides that specifically bind these polynucleotide sequences are also contemplated. 
       Polynucleotides and Polypeptides of the Invention 
       [0012]    The present invention provides for the sequences of the NTHi strain 86-028NP genome. This genomic sequence is presented as a series of conng sequences denoted herein as “contigs 1-576”. Each contig is assigned a sequence identification number that correlates with its “contig number”. Therefore, the contigs of the present invention as set out as SEQ ID NOS: 1-576. These contig polynucleotide sequences may be assembled into the complete genome sequence of the NTHi strain 86-028NP using routine methods. Upon completion of 8-fold sequence analysis of the NTHi strain 82-028NP genome, the genomic sequence was assembled into 11 contigs which are denoted herein as SEQ ID NOS: 675-685. 
         [0013]    The present invention provides for the NTHi polynucleotide sequences and open reading frames contained within the contigs of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5. The present invention also provides for the polypeptide sequences encoded by the NTHi polynucleotides of the present invention such as the amino acid sequences set out in Table 3B, Table 4B and Table 5. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotides sequence of SEQ ID NOS: 1-576; SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein (b) a polynucleotide which is an allelic variant of any polynucleotides recited above; (c) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (d) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention. 
         [0014]    The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to the NTHi polynucleotides recited above. 
         [0015]    Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to the NTHi nucleotide sequences of SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 413 and Table 5 herein, or compliments thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences. 
         [0016]    The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2 nd  Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). 
         [0017]    Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO 4 , (SDS), ficoll, Denhardt&#39;s solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al.,  Nucleic Acid Hybridisation: A Practical Approach , Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids. 
         [0018]    The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685, and nucleotide sequences out in Table 3B, Table 4B and Table 5 herein, preferably the open reading frames therein, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to the open reading frames within SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al.,  Nucl. Acid. Res.,  12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,  J. Mol. Biol.,  215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources ( BLAST Manual , Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well known Smith Waterman algorithm may also be used to determine identity. 
         [0019]    Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated. 
         [0020]    The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences encoded by the nucleotide sequences included within the polynucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or the corresponding full length or mature protein. The polypeptides of the invention include the amino acid sequences of SEQ ID NO: 616, SEQ ID NO: 618, SEQ ID NO: 620, SEQ ID NO: 622, SEQ ID NO: 624, SEQ ID NO: 626, SEQ ID NO: 628, SEQ ID NO: 628, SEQ ID NO: 630, SEQ ID NO: 632, SEQ ID NO: 634, SEQ ID NO: 636, SEQ ID NO: 638, SEQ ID NO: 640, SEQ ID NO: 642, SEQ ID NO: 644, SEQ ID NO: 646, SEQ ID NO: 648, SEQ ID NO: 650, SEQ ID NO: 652, SEQ ID NO: 654, SEQ ID NO: 656, SEQ ID NO: 658, SEQ ID NO: 660, SEQ ID NO: 662, SEQ ID NO: 664, SEQ ID NO: 666, SEQ ID NO: 668, SEQ ID NO: 670, SEQ ID NO: 672, SEQ ID NO: 674, SEQ ID NO: 687, SEQ ID NO: 689, SEQ ID NO: 691, SEQ ID NO: 693, SEQ ID NO: 695, SEQ ID NO: 697, SEQ ID NO: 699, SEQ ID NO: 701, SEQ ID NO: 703, SEQ ID NO: 705, SEQ ID NO: 707, SEQ ID NO: 709, SEQ ID NO: 711, SEQ ID NO: 713, SEQ ID NO:715, SEQ ID NO: 717, SEQ ID NO: 719, SEQ ID NO: 721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO: 731, SEQ ID NO: 733, SEQ ID NO: 735, SEQ ID NO: 737, SEQ ID NO: 739, SEQ ID NO: 741, SEQ ID NO: 743, SEQ ID NO: 745, SEQ ID NO: 747, SEQ ID NO: 749, SEQ ID NO: 751, SEQ ID NO: 753, SEQ ID NO: 755, SEQ ID NO: 757, SEQ ID NO: 759, SEQ ID NO: 761, 763, SEQ ID NO: 765, SEQ ID NO: 767, SEQ ID NO: 769 or SEQ ID NO: 771 which are set out in Table 3B, Table 4B and Table 5 herein. 
         [0021]    Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) an open reading frame contained within the nucleotide sequences set forth as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5, or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. 
         [0022]    The invention also provides biologically active or immunologically active variants of the amino acid sequences of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides encoded by the polynucleotides included within the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having an amino acid sequence set out in Table 3B, Table 4B and Table 5 herein 
         [0023]    NTHi peptides refer to fragments of the NTHi polypeptides encoded by the nucleotide sequences presented in SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 or the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein. The preferred NTHi peptides are biologically and/or immunologically active. 
         [0024]    The present invention further provides isolated NTHi polypeptides or NTHi peptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins. 
         [0025]    The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions which may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a normative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine General rules for amino acid substitutions are set forth in Table 1 below. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Amino Acid Substitutions 
               
             
          
           
               
                 Original Residues 
                 Exemplary Substitutions 
                 Preferred Substitutions 
               
               
                   
               
               
                 Ala 
                 Val, Leu, Ile 
                 Val 
               
               
                 Arg 
                 Lys, Gln, Asn 
                 Lys 
               
               
                 Asn 
                 Gln 
                 Gln 
               
               
                 Asp 
                 Glu 
                 Glu 
               
               
                 Cys 
                 Ser, Ala 
                 Ser 
               
               
                 Gln 
                 Asn 
                 Asn 
               
               
                 Glu 
                 Asp 
                 Asn 
               
               
                 Gly 
                 Pro, Ala 
                 Ala 
               
               
                 His 
                 Asn, Gln, Lys, Arg 
                 Arg 
               
               
                 Ile 
                 Leu, Val, Met, Ala, Phe, 
                 Leu 
               
               
                 Leu 
                 Norleucine, Ile, Val, Met, 
                 Leu 
               
               
                 Lys 
                 Arg, 1,4 Diaminobutyric 
                 Arg 
               
               
                 Met 
                 Leu, Phe, Ile 
                 Leu 
               
               
                 Phe 
                 Leu, Val, Ile, Ala, Tyr 
                 Arg 
               
               
                 Pro 
                 Ala 
                 Gly 
               
               
                 Ser 
                 Thr, Ala, Cys 
                 Thr 
               
               
                 Thr 
                 Ser 
                 Ser 
               
               
                 Trp 
                 Tyr, Phe 
                 Tyr 
               
               
                 Tyr 
                 Trp, Phe, Thr, Ser 
                 Phe 
               
               
                 Val 
                 Ile, Met, Leu, Phe, Ala, 
                 Leu 
               
               
                   
               
             
          
         
       
     
         [0026]    Antisense polynucleotides complementary to the polynucleotides encoding the NTHi polypeptides are also provided. 
         [0027]    The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells comprising polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as  E. coli, Bacillus, Streptomyces, Pseudoinonas, Salmonella  and  Serratia.    
         [0028]    The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs. 
       Antibodies and Methods for Eliciting an Immune Response 
       [0029]    The invention provides antibodies which bind to antigenic epitopes unique to (i.e., are specific for) NTHi polypeptides. Also provided are antibodies which bind to antigenic epitopes common among multiple  H. influenzae  subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and F(ab) 2  fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art. 
         [0030]    It is known in the art that antibodies to the capsular polysaccharide of  H. influenzae  exhibit the ability to kill bacteria in vitro assays. These antibodies are also known to protect against challenge with  H. influenzae  in animal model systems. These studies indicate antibody to the capsular polysaccharides are likely to elicit a protective immune response in humans. The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both  H. influenzae  bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention which reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration into the epithelium of  H. influenzae  bacteria or enhance phagocytosis of the  H. influenzae  bacteria. 
         [0031]    In vitro complement mediated bactericidal assay systems (Musher et al.,  Infect. Immun.  39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) may be used to measure the bactericidal activity of anti-NTHi antibodies. Further data on the ability of NTHi polypeptides and NTHi peptides to elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein. 
         [0032]    It is also possible to confer short-term protection to a host by passive immunotherapy via the administration of pre-formed antibody against an epitope of NTHi, such as antibodies against NTHi OMP, LOS or noncapsular proteins. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against NTHi epitopes. 
         [0033]    The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods include immune responses which kill the NTHi bacteria and immune responses which block  H. influenzae  attachment to cells. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising a NTHi protein or NTHi peptide of the invention. In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing a NTHi protein or NTHi peptide of the invention. In yet another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding a NTHi protein or NTHi peptide of the invention. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual. 
         [0034]    An “immunological dose” is a dose which is adequate to produce antibody and/or T cell immune response to protect said individual from NTHi infection, particularly NTHi infection of the middle ear and/or the nasopharynx or lower airway. Also provided are methods whereby such immunological response slows bacterial replication. A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the NTHi polypeptides encoded by the polynucleotide of the invention or antigenic peptides thereof. 
         [0035]    The invention correspondingly provides compositions suitable for eliciting an immune response to NTHi infection, wherein the antibodies elicited block binding of NTHi bacterium to the host&#39;s cells. The compositions comprise NTHi proteins or NTHi peptides of the invention, cells expressing the NTHi polypeptide, or polynucleotides encoding the polypeptides. The compositions may also comprise other ingredients such as carriers and adjuvants. 
         [0036]    Genes that are up-regulated in NTHi infection of the middle ear and/or the nasopharynx and genes that are associated with NTHi virulence are described herein. The polypeptides and peptides thereof which are encoded by these NTHi genes are contemplated to be useful for eliciting an immune response for treating or preventing disorders associated with NTHi infection, such as OM. Some of the polypeptides encoded by these genes include: histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, Phosphoribosylaminoimidazole carboxylase catalytic subunit, ornithine carbarnolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MukF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, MukF, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI064 HI186, HI0352 genes. NTHi OMPs, LOS and noncapsular proteins are also contemplated to elicit an immune response for prevention and treatment of disorders associated with NTHi infection. 
         [0037]    An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic. 
         [0038]    The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise administering antibodies or polypeptides of the invention that block binding of NTHi cellular attachment. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment. 
         [0039]    Pharmaceutical compositions comprising antibodies of the invention, polypeptides of the invention and/or small molecules of the invention that block NTHi cellular attachment are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. 
         [0040]    Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting NTHi polynucleotides of the invention in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotide may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR. 
         [0041]    The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of NTHi antigens (NTHi polypeptides and peptides thereof) in various body fluids of individuals suspected of  H. influenzae  infection. In another embodiment, the NTHi proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays. 
       Vaccines and Chemotherapeutic Targets 
       [0042]    An aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with a NTHi antigen protein or an antigenic peptide thereof. 
         [0043]    The present invention also provides for vaccine formulations which comprise an immunogenic recombinant NTHi protein or NTHi peptide of the invention together with a suitable carrier. The NTHi polypeptides and peptides thereof contemplated as vaccine candidates and/or targets of chemotherapy include, but are not limited to, histidine biosynthesis protein, lipoprotein B, peptide ABC transporter, periplasmic SapA precursor, outer membrane lipoproteins carrier protein precursor, ribose transport system permease protein, phosphoribosylaminoimidazole carboxylase catalytic subunit, PurE, 3,4-dihydroxt-2-butone 4-phosphate synthase, ornithine carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phospshocholine cytidylytransferase, putative pyridoxine biosynthesis protein, singlet oxygen resistance protein, intracellular septation protein, DNA repair protein, MUKF protein, glycerol-3-phosphate regulon repressor, integration host factor beta subunit, arginine repressor, cold shock like protein, stress response protein, LicA, RadA and those hypothetical proteins encoded by HI0094, HI1163, HI0665, HI1292, HI1064 HI1386, HI0352 genes, NTHi OMPs, NTHi LOS and NTHi noncapsular proteins and polypeptides encoded by the novel NTHi polynucleotide sequences present in the nucleotide sequences set out as SEQ ID NOS: 1-576, SEQ ID NOS: 675-685 and the nucleotide sequences set out in Table 3B, Table 4B and Table 5 herein, and the polypeptides having the amino acid sequences set out in Table 3B, Table 4B and Table 5 herein. 
         [0044]    Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation. 
         [0045]    A. Peptide Vaccines 
         [0046]    Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal or immunized human. 
         [0047]    The NTHi antigenic peptides of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al.,  The Proteins  (1976) v. 2, Academic Press, New York, p. 255). Automated solid phase synthesizers are commercially available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTIIi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al.,  J Am Chem. Soc.,  103: 3185 (1981). 
         [0048]    Some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) (See, e.g., Jansen et al.,  Immun. Rev.  62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, and coupling agent which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC). 
         [0049]    B. Vaccine Compositions and Administration 
         [0050]    A priming dose of the immunogen that is followed by one or more booster exposures to the immunogen may be necessary to be an effective vaccine (Kramp et al.,  Infect. Immun.,  25: 771-773, 1979; Davis et al.,  Immunology Letters,  14: 341-8 1986 1987). Examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases. 
         [0051]    Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well. 
         [0052]    The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g. humoral, cellular). Known adjuvants include emulsions such as Freund&#39;s Adjuvants and other oil emulsions,  Bordetella pertussis , MF59, purified saponin from  Quillaja saponaria  (QS21), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from  E. coli , holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of  E. coli  or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages. 
         [0053]    Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%. 
         [0054]    Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazenes polyphosphatazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine or amphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA, dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, dioleoyl phosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark ( Quillaja saponaria ) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery. 
         [0055]    The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine. 
         [0056]    The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject&#39;s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration. 
         [0057]    Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing the NTHi polypeptide or NTHi peptides of the invention are administered to a patient susceptible to or otherwise at risk of bacterial infection to elicit an immune response against the antigen and thus enhance the patient&#39;s own immune response capabilities. Such an amount is defined to be an “immunogenically effective dose.” In this use, the precise amounts again depend on the patient&#39;s state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70 kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi polypeptide or NTHi peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. 
         [0058]    Humoral immune response may be measured by many well known methods, such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI). In particular, SRID utilizes a layer of a gel, such as agarose, containing, the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilution of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen. 
         [0000]    Nontypeable  Haemophilus influenzae  (NTHi) 
         [0059]      H. influenzae  is a small, nonmotile gram negative bacterium. Unlike other  H. influenzae  strains, the nontypeable  H. influenzae  (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b  H. influenzae  isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, lipopolysaccharides, lipoproteins, adhesion proteins and noncapsular proteins. 
         [0060]    Humans are the only host for  H. influenzae . NTHi strains commonly reside in the upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTH 1  causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis. 
         [0061]    Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et al.,  Infect. Immun.,  36: 535-40, 1982), enzyme allotypes (Musser et al.,  Infect. Immun.,  52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contains two common OMP&#39;s with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP&#39;s within the 32,000-42,000 dalton range. The NTHi liposaccharide profile is fundamentally different than the enteric gram negative bacteria and separates into 1-4 distinct bands ranging from less than 20,000 daltons. 
         [0062]    A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al.,  Infect. Immun.,  53: 331-5, 1988; Holmes et al.,  Microb. Pathog.,  23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al.,  Vaccine,  15: 955-61, 1997; Suzuki et al.,  Infect. Immun.,  62: 1710-8, 1994: DeMaria et al.,  Infect. Imniun.,  64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear. 
       DFI Strategy 
       [0063]    A differential fluorescence induction (DFI) strategy was used herein to identify NTHi genes induced during OM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; signature-tagged mutagenesis (STM) enabling tag-specific identification of genes that alter the virulence properties of a microorganism when mutated; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses FACS analysis to select for transcriptionally active promoters (Chiang et al.  Annu. Rev. Microbiol.,  53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro. 
         [0064]    DFI has been successfully utilized in many microorganisms. For example, a GFP reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al.,  Mol. Microbiol.,  17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when Salmonellae were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow,  Mol. Microbiol.,  22: 367-378, 1996; Valdivia and Falkow,  Science,  277: 2007-2011, 1997; Valdivia and Falkow,  Curr. Opin. Microbiol.,  1: 359-363, 1998). In addition, DFI has been used to identify promoters expressed in  S. pneumoniae  and  S. aureus  when grown under varied in vitro conditions simulating infection (Marra et al.,  Infect. Immun.,  148: 1483-1491, 2002; Schneider et al.,  Proc. Natl. Acad. Sci. U.S.A.,  97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in  Bacillus cereus  in response to environmental stimuli (Dunn and Handelsman,  Gene,  226: 297-305, 1999), in  S. pneumoniae  in response to a competence stimulatory peptide (Bartilson et al.,  Mol. Microbiol.,  39: 126-135, 2001), and upon interaction with and invasion of host cells in  Bartonella henselae  Lee and Falkow,  Infect. Immun.,  66: 3964-3967, 1998),  Listeria monocytogenes  Wilson et al.,  Infect. Immun.,  69: 5016-5024, 2001),  Brucella abortus  (Eskra et al.,  Infect. Immun.,  69: 7736-7742, 2001), and  Escherichia coli  (Badger et al.,  Mol. Microbiol.,  36: 174-182, 2000). 
         [0065]    Whereas DFI has been successfully used to identify promoters active in cell culture models of infection or in vitro conditions designed to simulate an in vivo environment, few have applied DFI to identify promoters regulated in a specific biological niche within the whole animal. This is likely due to the numerous challenges associated with sorting from an in vivo environment. The host inflammatory response, dissemination and/or clearance of bacterial cells from the site of infection, as well as adherence of bacteria to epithelial cells, possibly via biofilm formation, can make bacteria inaccessible for retrieval from the living animal. These factors, among others, contribute to the complexity of the microenvironment and the heterogeneity of gene expression as the bacteria sense and respond to these changes. Recently, DFI has been used to identify promoters expressed in  S. pneumoniae  when the bacteria were screened in a mouse model of respiratory tract infection and a gerbil infection model of OM (Marra et al.,  Infect. Immun.  70: 1422-33, 2002; Marra et al.,  Microbiol.,  148: 1483-91, 2002). 
       Animal Model 
       [0066]    The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al.,  J. Infect. Dis.,  168: 865-872, 1993; Bakaletz and Holmes,  Clin. Diagn. Lab. Immunol.,  4: 223-225, 1997; Suzuki and Bakaletz,  Infect. Immun.,  62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al.,  Vaccine,  15: 955-961, 1997; Bakaletz et al.,  Infect. Immun.,  67: 2746-2762, 1999; Kennedy et al.,  Infect. Immun.,  68: 2756-2765, 2000). 
         [0067]    In particular, there is an unique in vivo model wherein adenovirus predisposes chinchillas to  H. influenzae -induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al.,  J. Infect. Dis.,  168: 865-72, 1993; Suzuki et al.,  Infect Immunity  62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al.,  Clin. Diagnostic Lab Immunol.,  4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al.,  Infect Immunity,  67(6): 2746-62, 1999; Kennedy et al.,  Infect Immun.,  68(5): 2756-65, 2000; Novotny et al.,  Infect Immunity  68(4): 2119-28, 2000; Poolman et al.,  Vaccine  19 (Suppl. 1): S108-15, 2000). 
       Genes Upregulated In Vivo in Response to NTHi Infection of the Middle Ear 
       [0068]    In order to identify differentially regulated promoters in response to NTHi infection of the middle ear, a promoter trap library was constructed and sorting parameters defined. A portion of the promoter trap library was inoculated directly into the chinchilla middle ear and OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. In addition, the middle ear fluids were recovered 24 and 48 hours after infection. Two-color FACS analysis was used to isolated bacteria that were expressing GFP from other cells and debris associated with the effusion. Following isolation, DNA sequence of the  Haemophilus  inserts 5′ of the gfpmut3 gene were determined and analyzed. In this manner, we identified genes that are up-regulated as NTHi sense and respond to the environment of the chinchilla middle ear during AOM. The following genes were identified and due to their up-regulation during NTHi infection, they may play a role in NTHi infection and virulence. 
         [0069]    As described below in Example 7, following the DFI procedure described above and subsequent FACS analysis of gfp-expressing clones, 52 candidate clones containing potential in vivo-regulated promoters were isolated. The genes these clones control were categorized based upon general description and function within the cell and include general metabolic processes, environmental informational processing and membrane transport, membrane proteins and hypothetical proteins. Eight of these 52 clones contain sequences that are unique to NTHi strain 86-028NP. Importantly, 3 clones were isolated from independent screens in more than one animal thereby verifying the method of isolation. 
         [0070]    In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Thus, wild-type gene expression without the influence of plasmid copy number on gene regulation was analyzed, allowing for the indication of false-positive clone identification by FACS. Of the 44 candidate clones containing sequence similar to that identified in  H. influenzae  strain Rd, quantitative comparison of gene expression in vitro and in vivo confirmed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear. This analysis identified in vivo-regulated promoters which drive expression of genes involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. (See Table 4 in Example 6). 
         [0071]    Quantitative RT-PCR demonstrated a two-fold increase in lolA expression, enabling lipoprotein transport from the inner membrane to the outer membrane. Bacteria grow rapidly in the middle ear environment reaching 5.0×10 8  CFU NTHi ml middle ear fluid within 48 hours. The bacteria sense and respond to the environment, acquiring or synthesizing the necessary nutrients for growth and survival. The gene encoding the membrane component in ribose sugar transport, rbsC (SEQ ID NO: 619), showed a 5-fold increase in expression in vivo compared to cells growing in vitro. In addition, many genes involved in metabolic processes show a dramatic increase in gene expression in vivo compared to cells growing in vitro. These include a riboflavin synthesis gene, ribB (SEQ ID NO: 623), a purine nucleotide biosynthetic gene purE (SEQ ID NO: 621), ornithine carbamoyltransferase, arcB (SEQ ID NO: 625), involved in arginine degradation via the urea cycle and uxuA (SEQ ID NO: 627), encoding mannonate hydrolase, required for the uptake of D-glucuronate and transformation into glyceraldehyde 3-phosphate. In addition, but to a lesser degree, genes for histidine biosynthesis (hisB; SEQ ID NO: 615), DNA repair (radC; SEQ ID NO: 639) and a putative intracellular septation transmembrane protein (ispZ; SEQ ID NO: 637) were up-regulated. 
         [0072]    Disulfide bond formation is important for folding and assembly of many secreted proteins in bacteria. In prokaryotes, DsbA and DsbB make up the oxidative pathway responsible for the formation of disulfides. DsbB reoxidizes DsbA, which donates disulfide bonds directly to unfolded polypeptides, and DsbB has been demonstrated to generate disulfides de novo from oxidized quinones (Collet and Bardwell,  Mol. Microbiol.,  44: 1-8, 2002). In  H. influenzae  strain Rd, DsbA has required for competence for transformation (Tomb,  Proc. Natl. Acad. Sci. U.S.A.,  89: 10252-10256, 1992). Herein, an approximate 3-fold increase in dsbB gene (SEQ ID NO: 629) transcription was demonstrated, illuminating an important role for disulfide interchange for NTHi growing in the middle ear environment. 
         [0073]    Bacteria colonization of the middle ear, a normally sterile environment, results in a host inflammatory response and subsequent neutrophil infiltration. Bacteria have evolved numerous strategies to combat this host response. NTHi increase gene expression (4-fold) of ureH (SEQ ID NO:631), a homologue of a gene required for expression of active urease in  Helicobacter , shown to be involved in acid tolerance (Young et al.,  J. Bacterol.,  178: 6487-6495, 1996). Recently, it has been reported that urease activity may play a role in chronic  Actinobacillus pleuropneumoniae  infection by counteracting the decrease in pH occurring upon infection (Baltes et al.,  Infect. Iminun.,  69: 472-478, 2000; Baltes et al.,  Infect. Immun.,  69: 472-478, 2001; Bosse and Maclnnes,  Can. J. Vet. Res.,  64: 145-150). A biotype analysis on NTHi isolates from middle ear effusions demonstrated that 87% are urease positive (DeMaria et al.,  J. Clin. Microbiol.,  20: 1102-1104, 1984). However, the role of urease in NTHi virulence is unknown. Similarly, an increase in expression of a gene whose product demonstrates 88% sequence identity to a pyridoxine biosynthesis protein in  S. pneumoniae  and 60% homology to a putative singlet oxygen resistance protein that may function as an antioxidant. Phosphorylcholine (ChoP) has been implicated in the pathogenesis of NTHi (Weiser et al.,  Infect. Immun.,  65: 943-950, 1997). NTHi modulates ChoP expression by phase variation, decorating the LOS on the cell surface. ChoP may contribute to NTHi persistence in the respiratory tract via decreased susceptibility to antimicrobial peptides (Lysecko et al.,  Infect. Immun.,  68: 1664-1671, 2000) and alter the sensitivity to serum killing mediated by C-reactive protein (CRP) (Weiser et al.,  J. Exp. Med.,  187: 631-640, 1998). The microenvironment of the nasopharynx and middle ear cavity may select for the ChoP +  phenotype, as ChoP +  strains show greater colonization of the chinchilla nasopharynx (Tong et al.,  Infect. Immun.,  68: 4593-4597, 2000). Expression of the licC gene (SEQ ID NO: 633) was also increased. The licC gene encodes a phosphorylcholine cytidylyltransferase that plays a role in the biosynthesis of phosphorylcholine-derivatized LOS (Rock et al.,  J. Bacterol.,  183: 4927-4931, 2001). 
         [0074]    Also included among the in vivo-induced genes is a set whose products subsequently regulate gene expression or DNA replication. These genes include transcriptional regulation of glycerol metabolism by the glp repressor, glpR (SEQ ID NO: 643), the arginine repressor gene, argR (SEQ ID NO: 647), and the integration host factor (IHF) beta subunit, ihfB (SEQ ID NO: 645). IHF is a histone-like protein that binds DNA at specific sequences, an accessory factor involved in replication, site-specific recombination and transcription, altering the activity of a large number of operons (Goosen and van de Putte,  Mol. Microbiol.  16: 1-7, 1995). In addition, CspD inhibits DNA replication during stationary phase-induced stress response in  E. coli  (Yamanaka et al.,  Mol. Microbiol.,  39: 1572-1584, 2001) and the mukF (SEQ ID NO: 641) gene protein homologue contributes to a remodeling of the nucleiod structure into a more compact form prior to cell segregation (Sawitzke and Austin,  Proc. Natl. Acad. Sci. U.S.A.,  62: 1710-1718, 2000). The DFI strategy described herein also identified promoters induced in vivo for genes of unknown function. The hypothetical protein, HI0094, demonstrated an 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of  E. coli.    
         [0075]    A high-density transposon mutagenesis strategy was used to identify  H. influenzae  genes essential for growth on rich medium (Akerley et al.,  Proc. Natl. Acad. Sci. U.S.A.,  99: 966-971, 2002). Six genes were identified in the screen described herein that are included in essential gene set described in Akerley&#39; et al., supra. (hisB, lppB, lolA, ispZ, mukF and unknown HI0665). Recently genes of non-typeable  H. influenzae  that are expressed upon interaction with two human respiratory tract-derived epithelial cell lines have been identified. These genes included those involved in metabolic processes, stress responses, gene expression, cell envelope biosynthesis, DNA-related processes, cell division and ORF&#39;s encoding proteins of unknown function. (Ulsen et al.,  Mol. Microbiol.,  45: 485-500, 2002). Similarly the stress response gene, cspD (SEQ ID NO: 649), genes involved in purine and riboflavin biosynthesis, and a protein of unknown function, vapA was identified in the screen described herein. Expression of vapA was detected in vitro, yet vapA gene expression increased two-fold in vivo. These unique approaches identified known genes that are upregulated in NTHi-induced OM and therefore are likely to play a role in NTHi infection and virulence; and may be potential candidates for vaccines and antisense therapies and other therapeutic methods of treatment of NTHi related disorders. 
         [0076]    The DFI strategy resulted in the identification of promoters induced in vivo for genes of unknown function as well. The hypothetical protein, HI0094, demonstrated a 8-fold increase in gene expression during early OM but its role remains unknown. HI1163 (SEQ ID NO: 651) showed 58% amino acid identity with the hypothetical YdiJ proteins, a putative oxidase, of  E. coli . Therefore, these hypothetical genes are likely to play a role in OM induced by NTHi infection. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES 
         [0077]      FIG. 1  depicts the LKP gene region in a panel of  Haemophilus  isolates. The strain 86-028NP sequence is identical in this region to the sequence in NTHi strain R3001. Both of these NTHi lack the hif gene cluster encoding the hemagglutinating pilus. 
           [0078]      FIG. 2  depicts the rfaD region in a panel of  Haemophilus  isolates. The gene arrangement in the rfaD region of the strain 86-028NP genome is similar to that of the strain Rd genome but different than the arrangement of these genes seen in the genome of most NTHi examined. 
           [0079]      FIGS. 3A-3M  set out the nucleotide sequences (SEQ ID NOS: 589-614) described in Table 4, which were identified to be upregulated during OM infection (see Example 6). The nucleotides (nt.) which correspond to known genes and those nt. which correspond to the contig sequences set out as SEQ ID NO: 1-576 are also presented. 
       
    
    
     DETAILED DESCRIPTION 
       [0080]    The following examples illustrate the invention wherein Example 1 describes the sequence of the NTHi genome, Example 2 describes the identified contigs and initial gene discovery, Example 3 describes construction of the NTHi promoter trap library, Example 4 describes the analyses of 86-028NP derivatives expressing GFP, Example 5 demonstrates direct labelling of bacteria from middle ear fluids, Example 6 describes identification of promoters induced in vivo in acute otitis media, Example 7 describes identification of virulence-associated genes, and Example 8 describes identification of unique NTHi gene sequences. 
       Example 1 
     Sequence of a Non-Typeable  Haemophilus influenzae  Genome 
       [0081]    NTHi strain 86-028NP is a minimally passaged clinical isolate obtained from a pediatric patient who underwent tympanostomy and tube insertion for chronic OM at Columbus Children&#39;s Hospital. (Bakaletz et al.  Infection and Immunity,  56(2): 331-335, 1988) The 86-028NP strain was deposited with the American Type Tissue Collection (Manassas, Va. 20108 USA) on Oct. 16, 2002 and assigned accession no. PTA-4764. 
         [0082]    In an effort to more broadly approach the identification of the virulence determinants in NTHi, the genome of the NTHi 86-028NP strain was sequenced to 3-fold coverage. Chromosomal DNA was prepared from strain 86-028NP using the Puregene protocol and sheared to 2-4 kb in size with a Hydroshear instrument (Gene Machines). The sheared DNA was ethanol-precipitated, end-repaired using a mixture of Klenow enzyme and T4 DNA polymerase, and size-selected by agarose gel electrophoresis to obtain 2-4 kb fragments as described in Chissoe et al. ( Methods: a Companion to Methods of Enzymology  3: 55-65, 1991) and Sambrook et al. ( Molecular Cloning: a Laboratory Manual,  2 nd  Ed. Cold Springs Harbor, N.Y., 1989). These fragments were cloned into vector pUC18 using the SmaI restriction site (phosphatase-treated) and transformed into  E. coli  XL-1 Blue, selecting for ampicillin resistance. Colonies that contain inserts were identified by blue/white screening on LB-Amp plates containing X-gal, and transferred into 96-deep well plates containing 1.5 ml of TB-Amp (TB=Terrific Broth) broth. The deep-well plate cultures were grown overnight (18-22 hours) at 37° C. Template preparation, sequencing and contig assembly were performed. 
         [0083]    Automated template preparation was performed on the Beckman Biomek 2000 automated robotics workstation as described in Chissoe et al., (supra. Briefly, each 96-deep well plate, containing the clones prepared above, was centrifuged to pellet the cells, the supernatant decanted, and the cells frozen (if necessary) at −20° C. Four 96-deep well blocks were placed on the Biomek table, and the liquid handling robot was used to prepare the template using an automated version of a typical SDS-NaOH lysis protocol as described in Chissoe et al., (supra.). The final ethanol-precipitated templates were each dissolved in 50 μl ddH 2 O, and used for DNA sequencing. 
         [0084]    Sequencing reactions were run by re-arraying the templates (from 96-well plates) into 384-well plates, using the Robbins Hydra 96 robot. Cycle-sequencing reactions were run using PE Big-Dye™ terminators and universal primers (M13 forward and reverse), cleaned up over Sephadex G50 columns, and analyzed on a PE Biosystems 3700 capillary electrophoresis DNA sequencer according to the manufacturer&#39;s instructions. Sequencing reads (8219) were assembled into 576 contigs (SEQ ID NOS: 1-576 herein). The statistics for the 3-fold sequencing are shown in Table 2A. The total unique sequence in assembly 17 is 1.74 Mb. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2A 
               
               
                   
               
               
                 Contig 
                   
                   
                   
               
               
                 Size 
                 Total Number 
                 Total Length 
                 % of Cumulative 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0-1 
                 kb 
                 65 
                 55961 
                 3.2% 
               
               
                 1-2 
                 kb 
                 228 
                 333665 
                 19.2% 
               
               
                 2-3 
                 kb 
                 101 
                 243059 
                 14.0% 
               
               
                 3-4 
                 kb 
                 49 
                 172385 
                 9.9% 
               
               
                 4-5 
                 kb 
                 45 
                 196699 
                 11.3% 
               
               
                 5-10 
                 kb 
                 74 
                 515152 
                 29.6% 
               
               
                 10-20 
                 kb 
                 11 
                 144591 
                 8.3% 
               
               
                 20-30 
                 kb 
                 3 
                 77352 
                 4.4% 
               
               
                   
               
             
          
         
       
     
         [0085]    Subsequently, 8-fold sequencing analysis of the NTHi genome was carried out. The 8-fold sequencing assembled the NTHi genome into 11 contigs. Contigs 5, 8, 9, 10, 12-18 are denoted as SEQ ID NOS: 675-685 herein. The statistics for the 8-fold sequencing are shown in Table 213. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2B 
               
               
                   
               
               
                 Contig Size 
                 Total Number 
                 Total Length 
                 % of Cumulative 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0-1 
                 kb 
                 5 
                 3950 
                 0.2% 
               
               
                 1-2 
                 kb 
                 3 
                 4316 
                 0.2% 
               
               
                 2-3 
                 kb 
                 0 
                 0 
                 0.0% 
               
               
                 3-4 
                 kb 
                 1 
                 3964 
                 0.2% 
               
               
                 4-5 
                 kb 
                 0 
                 0 
                 0.0% 
               
               
                 5-10 
                 kb 
                 0 
                 0 
                 0.0% 
               
               
                 10-20 
                 kb 
                 1 
                 15147 
                 0.8% 
               
               
                 20-30 
                 kb 
                 2 
                 51888 
                 2.7% 
               
               
                 30-40 
                 kb 
                 0 
                 0 
                 0.0% 
               
               
                 40-50 
                 kb 
                 0 
                 0 
                 0.0% 
               
               
                 50-100 
                 kb 
                 1 
                 85814 
                 4.5% 
               
               
                 &gt;100 
                 kb 
                 5 
                 1760339 
                 91.4% 
               
               
                   
               
             
          
         
       
     
       Example 2 
     Contig Description and Initial Gene Discovery 
       [0086]    Seventy-five of the 88 contigs with length ≧5000 bp, identified with the 3-fold sequence analysis, show significant similarity via BLASTN to genes in  H. influenzae  strain Rd. To visualize the potential relationship between the gene order in  H. influenzae  strain 86-028NP and  H. influenzae  strain Rd, the 86-028NP three-fold contig set and the Rd gene set were bidirectionally compared using BLASTN. The results were plotted in gene-order verses contig space by sorting the contigs based on gene coordinates of the Rd genes hit, anchoring each contig at the smallest coordinate found as described in Ray et al., ( Bioinformatics  17: 1105-12, 2001). Compared in this fashion, an incomplete assembly of a genome with identical gene order to a completely known genome would display a monotonically increasing stair-stepped form. 
         [0087]    BLASTX was used to identify hits to sequences with homology to genes in the strain Rd genome as well as genes not found in  H. influenzae  strain Rd. Hits to strain Rd sequences were removed from the data set and the other hits summarized in Table 3A. The data are presented as follows: contig #(=SEQ ID NO: #), column 1; E score for each hit, column 2; the name of the protein that had homology to a portion of the amino acid translation of the cited contig, column 3; the organism producing the homologue, column 4; and the Genbank protein identifier for each of the proteins cited in column 3, column 5; the corresponding nucleotides within the contig (referenced by SEQ ID NO:). In most instances, several homologues were identified but for clarity, the protein of greatest homology is cited in Table 3A. 
         [0088]    The sequences for some of the genes listed in Table 3A were identified within the 8-fold sequencing of the NTHi genome. Table 3B lists the location of these genes within the 11 contigs, the full length open reading frame sequence (identified by SEQ ID NO:), the derived amino acid sequence encoded by the open reading frame and the gene with high homology identified by BLASTX (as listed in Table 3A). 
         [0089]    To examine the relative short range gene arrangements in NTHi and the Rd strain, the gene order in two gene clusters that have been well-described were compared. First, the genes present in the hemagglutinating pilus (LKP) gene region were examined. (Mhlanga-Mutangadura et al.,  J Bacteriol.  180(17): 4693-703, 1998). The pilus gene cluster is located between the purE and pepN genes, only fragments of which are depicted in  FIG. 1 . The serotype b strain, Eagan, contains the hifABCDE gene cluster and produces hemagglutinating pili. Strain Rd lacks the hicAB genes as well as the hifABCDE gene cluster. In general, the nontypeable strains previously examined contained the hicAB genes but not the hif genes that encode the hemagglutinating pilus. The strain 86-028NP sequence (described herein) is identical in this region to the sequence in NTHi strain R3001 ( FIG. 1 ). The rfaD gene encodes an enzyme involved in the biosynthesis of endotoxin. In addition, the rfaD gene from NTHi strain 2019 has been characterized by Nichols et al. ( Infect Immunity  65(4): 1377-86, 1997). In strain 2019, the rfaD gene is immediately upstream of the rfaF gene that encodes another enzyme involved in endotoxin biosynthesis. The gene arrangement in strain Rd is different; the rfaD and rfaF genes are separated by approximately 11 kb of sequence. Most nontypeable strains examined contained the gene arrangement seen in strain 2019. In contrast, strain 86-028NP has a gene arrangement identical to that seen in strain Rd ( FIG. 2 ). 
         [0090]    A global analysis of the current assembly indicates that the gene content and order are similar to that in strain Rd. A more detailed analysis revealed that there are a substantial number of NTHi genes not previously seen in the Pasteurellaceae and some regions where the NTHi gene content and order is different than that seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features. 
         [0091]    The DFI strategy also identified novel NTHi sequences that had increased gene expression. A list of these novel contig sequences that contain genes or gene fragments that have homology to ORFs in other organisms (primarily gram-negative bacteria) is set out in Table 3A. For example, the nucleotide sequence of contig 442 (SEQ ID NO: 442), nucleotides 1498-1845 are highly homologous to the sequences encoding amino acids 1-116 of  H. influenzae  strain Rd lipoprotein B (LppB). The gene is positioned between the stationary phase survival gene, surE, and a gene encoding a 43 kD antigenic outer membrane lipoprotein that is highly homologous to the recently identified bacterial lipoprotein, LppB/1\11pD, which has been associated with virulence (Padmalayam et al.,  Infect. Immun.,  68: 4972-4979, 2000). Recently, Zhang and coworkers demonstrated that nlpD and surE gene expression was induced during stationary phase of bacterial growth in  Thermotoga maritima  (Zhang et al.,  Structure  ( Camb ), 9: 1095-1106, 2001). Therefore, under stress-induced conditions in the middle ear, this NTHi lipoprotein may be expressed. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3A 
               
               
                   
               
               
                   
                   
                   
                   
                 Genbank 
                   
               
               
                 Contig 
                 E score 
                 Hit Identity 
                 Organism 
                 Protein 
                 SEQ ID NO: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 104 
                 4.00E−59 
                 CpdB 
                 
                   Pasteurella 
                 
                 NP_246953.1 
                 nt. 204-659 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 104 
               
               
                 106 
                 9.00E−10 
                 hypothetical protein 
                 
                   Pyrococcus 
                 
                 G71244 
                 nt. 40-309 of 
               
               
                   
                   
                 PH0217- 
                 
                   horikoshii 
                 
                   
                 SEQ ID NO: 106 
               
               
                 106 
                 1.00E−08 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246871.1 
                 nt. 605-694 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 106 
               
               
                 106 
                 2.00E−20 
                 Orf122 
                 
                   Chlorobium 
                 
                 AAG12204.1 
                 nt. 7-210 of 
               
               
                   
                   
                   
                 
                   tepidum 
                 
                   
                 SEQ ID NO: 106 
               
               
                 110 
                 3.00E−05 
                 ArdC antirestriction 
                 IncW plasmid pSa 
                 AAD52160.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   
                   
                 nt. 959-1162 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 110 
               
               
                 110 
                 1.00E−33 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458676.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. nt. 181-825 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                 
                   Typhi 
                 
                   
                 110 
               
               
                 111 
                 5.00E−12 
                 putative membrane 
                 
                   Salmonella 
                 
                 NP_458664.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   enterica  subsp. 
                   
                 nt. 45-287 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 111 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 111 
                 6.00E−41 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458658.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 1091-1480 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 111 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 114 
                 7.00E−80 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245828.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 118-696 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 114 
               
               
                 115 
                 2.00E−09 
                 A111R 
                 Paramecium 
                 NP_048459.1 
                 nt. 555-869 of 
               
               
                   
                   
                   
                 bursaria Chlorella 
                   
                 SEQ ID NO: 115 
               
               
                   
                   
                   
                 virus 1 
               
               
                 118 
                 5.00E−45 
                 DNA methylase 
                 
                   Vibrio cholerae 
                 
                 NP_231404.1 
                 nt. 44-439 of 
               
               
                   
                   
                 HsdM, putative 
                   
                   
                 SEQ ID NO: 118 
               
               
                 122 
                 2.00E−18 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245314.1 
                 nt. 865-1302 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 122 
               
               
                 123 
                 4.00E−99 
                 RNA 
                 
                   Proteus mirabilis 
                 
                 P50509 
                 nt. 351-782 of 
               
               
                   
                   
                 POLYMERASE 
                   
                   
                 SEQ ID NO: 123 
               
               
                   
                   
                 SIGMA-32 
               
               
                   
                   
                 FACTOR 
               
               
                 124 
                 9.00E−58 
                 ACETOLACTATE 
                 
                   Spirulina platensis 
                 
                 P27868 
                 nt. 603-1025 of 
               
               
                   
                   
                 SYNTHASE 
                   
                   
                 SEQ ID NO: 124 
               
               
                   
                   
                 (ACETOHYDROXY- 
               
               
                   
                   
                 ACID SYNTHASE) 
               
               
                   
                   
                 (ALS) 
               
               
                 130 
                 0 
                 restriction 
                 
                   Neisseria 
                 
                 CAA09003.1 
                 nt. 495-1559 of 
               
               
                   
                   
                 modification 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 130 
               
               
                   
                   
                 system-R protein 
               
               
                 131 
                 6.00E−91 
                 uronate isomerase 
                 
                   Salmonella 
                 
                 NP_457532.1 
                 compliment of 
               
               
                   
                   
                 (glucuronate 
                   enterica  subsp. 
                   
                 nt. 661-1380 of 
               
               
                   
                   
                 isomerase) 
                   enterica  serovar 
                   
                 SEQ ID NO: 131 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 133 
                 3.00E−30 
                 GyrA 
                 
                   Pasteurella 
                 
                 NP_245778.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 1447-1626 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 133 
               
               
                 133 
                 1.00E−27 
                 DNA GYRASE 
                 
                   Pectobacterium 
                 
                 P41513 
                 compliment of 
               
               
                   
                   
                 SUBUNIT A 
                 
                   carotovorum 
                 
                   
                 nt. 1302-1442 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 133 
               
               
                 138 
                 7.00E−06 
                 KicA 
                 
                   Pasteurella 
                 
                 NP_245545.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 92-157 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 138 
               
               
                 138 
                 1.00E−148 
                 TYPE II 
                 
                   Haemophilus 
                 
                 O30869 
                 compliment of 
               
               
                   
                   
                 RESTRICTION 
                 
                   aegyptius 
                 
                   
                 nt. 164-1045 of 
               
               
                   
                   
                 ENZYME HAEII 
                   
                   
                 SEQ ID NO: 138 
               
               
                   
                   
                 (ENDONUCLEASE 
               
               
                   
                   
                 HAEII) ( R. HAEII ) 
               
               
                 143 
                 4.00E−06 
                 Gifsy-1 prophage 
                 
                   Salmonella 
                 
                 NP_461555.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   typhimurium  LT2 
                   
                 nt. 228-632 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 143 
               
               
                 143 
                 1.00E−14 
                 hypothetical protein 
                 Bacteriophage 
                 NP_050531.1 
                 compliment of 
               
               
                   
                   
                   
                 VT2-Sa 
                   
                 nt. 778-1248 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 143 
               
               
                 143 
                 5.00E−09 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 CAD09979.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 715-1026 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 143 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 143 
                 6.00E−10 
                 hypothetical 14.9 kd 
                 
                   Escherichia coli 
                 
                 NP_065324.1 
                 nt. 3-173 of 
               
               
                   
                   
                 protein 
                   
                   
                 SEQ ID NO: 143 
               
               
                 147 
                 1.00E−38 
                 GTP-binding 
                 
                   Escherichia coli 
                 
                 NP_289127.1 
                 compliment of 
               
               
                   
                   
                 elongation factor, 
                 O157:H7 EDL933 
                   
                 nt. 172-342 of 
               
               
                   
                   
                 may be inner 
                   
                   
                 SEQ ID NO: 147 
               
               
                   
                   
                 membrane protein 
               
               
                 147 
                 2.00E−14 
                 GTP-binding 
                 
                   Borrelia 
                 
                 NP_212222.1 
                 compliment of 
               
               
                   
                   
                 membrane protein 
                 
                   burgdorferi 
                 
                   
                 nt. 17-181 of 
               
               
                   
                   
                 (lepA) 
                   
                   
                 SEQ ID NO: 147 
               
               
                 148 
                 6.00E−17 
                 galactokinase 
                 
                   Homo sapiens 
                 
                 AAC35849.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 746-1246 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 148 
               
               
                 148 
                 7.00E−96 
                 GALACTOKINASE 
                 
                   Actinobacillus 
                 
                 P94169 
                 compliment of 
               
               
                   
                   
                 (GALACTOSE 
                 
                   pleuropneumoniae 
                 
                   
                 nt. 232-741 of 
               
               
                   
                   
                 KINASE) 
                   
                   
                 SEQ ID NO: 148 
               
               
                 149 
                 1.00E−92 
                 GTP-binding 
                   Buchnera  sp. 
                 NP_240245.1 
                 compliment of 
               
               
                   
                   
                 protein TypA/BipA 
                 APS 
                   
                 nt. 265-1077 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 149 
               
               
                 15 
                 2.00E−21 
                 ORF1 
                 
                   Escherichia coli 
                 
                 CAA39631.1 
                 nt. 665-850 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 15 
               
               
                 150 
                 6.00E−17 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245919.1 
                 nt. 171-665 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 150 
               
               
                 153 
                 7.00E−07 
                 cuter membrane 
                 
                   Rickettsia conorii 
                 
                 T30852 
                 nt. 51-623 of 
               
               
                   
                   
                 protein A 
                   
                   
                 SEQ ID NO: 153 
               
               
                 155 
                 7.00E−40 
                 cytochrome d 
                 
                   Vibrio cholerae 
                 
                 NP_233259.1 
                 nt. 583-1002 of 
               
               
                   
                   
                 ubiquinol oxidase, 
                   
                   
                 SEQ ID NO: 155 
               
               
                   
                   
                 subunit II 
               
               
                 157 
                 7.00E−13 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245490.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 1170-1367 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 157 
               
               
                 157 
                 2.00E−05 
                 glycosyl 
                 
                   Neisseria 
                 
                 AAA68012.1 
                 nt. 85-189 of 
               
               
                   
                   
                 transferase 
                 
                   gonorrhoeae 
                 
                   
                 SEQ ID NO: 157 
               
               
                 158 
                 1.00E−152 
                 MltC 
                 
                   Pasteurella 
                 
                 NP_246259.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 36-530 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 158 
               
               
                 161 
                 3.00E−25 
                 lipoprotein, putative 
                 
                   Vibrio cholerae 
                 
                 NP_230232.1 
                 nt. 870-1439 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 161 
               
               
                 163 
                 9.00E−53 
                 chorismate 
                 
                   Caulobacter 
                 
                 NP_421948.1 
                 nt. 1283-2029 of 
               
               
                   
                   
                 synthase 
                 
                   crescentus 
                 
                   
                 SEQ ID NO: 163 
               
               
                 168 
                 3.00E−13 
                 COPPER- 
                 
                   Mus musculus 
                 
                 Q64430 
                 nt. 66-995 of 
               
               
                   
                   
                 TRANSPORTING 
                   
                   
                 SEQ ID NO: 168 
               
               
                   
                   
                 ATPASE 1 
               
               
                   
                   
                 (COPPER PUMP 
               
               
                   
                   
                 1) 
               
               
                 168 
                 2.00E−22 
                 Cu transporting 
                 
                   Homo sapiens 
                 
                 2001422A 
                 nt. 135-989 of 
               
               
                   
                   
                 ATPase P 
                   
                   
                 SEQ ID NO: 168 
               
               
                 174 
                 8.00E−48 
                 magnesium/cobalt 
                 
                   Mesorhizobium 
                 
                 NP_103977.1 
                 nt. 918-1205 of 
               
               
                   
                   
                 transport protein 
                 
                   loti 
                 
                   
                 SEQ ID NO: 174 
               
               
                 175 
                 5.00E−26 
                 vacB protein 
                   Buchnera  sp. 
                 NP_240369.1 
                 compliment of 
               
               
                   
                   
                   
                 APS 
                   
                 nt. 1-1587 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 175 
               
               
                 176 
                 3.00E−21 
                 putative ABC 
                 
                   Campylobacter 
                 
                 NP_282774.1 
                 compliment of 
               
               
                   
                   
                 transport system 
                 
                   jejuni 
                 
                   
                 nt. 259-1089 of 
               
               
                   
                   
                 permease protein [ 
                   
                   
                 SEQ ID NO: 176 
               
               
                 183 
                 5.00E−29 
                 PROBABLE ATP 
                 
                   Ralstonia 
                 
                 NP_521442.1 
                 compliment of 
               
               
                   
                   
                 SYNTHASE A 
                 
                   solanacearum 
                 
                   
                 nt. 42-677 of 
               
               
                   
                   
                 CHAIN 
                   
                   
                 SEQ ID NO: 183 
               
               
                   
                   
                 TRANSMEMBRANE 
               
               
                   
                   
                 PROTEIN 
               
               
                 185 
                 6.00E−85 
                 putative exported 
                 
                   Salmonella 
                 
                 NP_458655.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   enterica  subsp. 
                   
                 nt. 162-1529 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 185 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 187 
                 2.00E−05 
                 transketolase 
                 
                   Homo sapiens 
                 
                 AAA61222.1 
                 nt. 709-819 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 187 
               
               
                 188 
                 1.00E−116 
                 ribonuclease E 
                 
                   Xylella fastidiosa 
                 
                 NP_299884.1 
                 compliment of 
               
               
                   
                   
                   
                 9a5c 
                   
                 nt. 280-1704 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 188 
               
               
                 192 
                 1.00E−38 
                 ImpA 
                 
                   Pasteurella 
                 
                 NP_245829.1 
                 nt. 35-448 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 192 
               
               
                 193 
                 3.00E−08 
                 Orf80 
                 
                   Enterobacteria 
                 
                 NP_052285.1 
                 nt. 1612-1818 of 
               
               
                   
                   
                   
                 phage 186 
                   
                 SEQ ID NO: 193 
               
               
                 193 
                 1.00E−06 
                 holin 
                 
                   Haemophilus 
                 
                 AAC45168.1 
                 nt. 370-576 of 
               
               
                   
                   
                   
                 
                   somnus 
                 
                   
                 SEQ ID NO: 193 
               
               
                 193 
                 0.007 
                 unknown 
                 
                   Enterobacteria 
                 
                 NP_052260.1 
                 nt. 1376-1609 of 
               
               
                   
                   
                   
                 phage 186 
                   
                 SEQ ID NO: 193 
               
               
                 193 
                 2.00E−48 
                 lysozyme 
                 
                   Haemophilus 
                 
                 AAC45169.1 
                 nt. 608-1093 of 
               
               
                   
                   
                   
                 
                   somnus 
                 
                   
                 SEQ ID NO: 193 
               
               
                 199 
                 4.00E−21 
                 unknown protein 
                 
                   Escherichia coli 
                 
                 NP_288675.1 
                 nt. 398-778 of 
               
               
                   
                   
                   
                 O157:H7 
                   
                 SEQ ID NO: 199 
               
               
                   
                   
                   
                 EDL933, 
               
               
                   
                   
                   
                 prophage CP- 
               
               
                   
                   
                   
                 933V 
               
               
                 199 
                 2.00E−49 
                 hypothetical protein 
                 Bacteriophage 
                 NP_049495.1 
                 compliment of 
               
               
                   
                   
                   
                 933W 
                   
                 nt. 1907-2392 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 199 
               
               
                 20 
                 1.00E−62 
                 RpL14 
                 
                   Pasteurella 
                 
                 NP_246344.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 233-601 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 20 
               
               
                 200 
                 2.00E−62 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458658.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 431-997 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 200 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 200 
                 3.00E−16 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458657.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 1028-1264 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 200 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 201 
                 2.00E−26 
                 TsaA 
                 
                   Pasteurella 
                 
                 NP_245732.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 1618-1809 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 201 
               
               
                 209 
                 6.00E−16 
                 TsaA 
                 
                   Pasteurella 
                 
                 NP_245732.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 2-136 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 209 
               
               
                 211 
                 2.00E−15 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245535.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 23-211 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 211 
               
               
                 211 
                 1.00E−70 
                 PUTATIVE 
                 
                   Ralstonia 
                 
                 NP_520082.1 
                 compliment of 
               
               
                   
                   
                 ATPASE PROTEIN 
                 
                   solanacearum 
                 
                   
                 nt. 475-915 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 211 
               
               
                 212 
                 3.00E−18 
                 hypothetical protein 
                 
                   Escherichia coli 
                 
                 NP_309775.1 
                 compliment of 
               
               
                   
                   
                   
                 O157:H7 
                   
                 nt. 895-1035 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 212 
               
               
                 216 
                 1.00E−173 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245069.1 
                 nt. 35-1543 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 216 
               
               
                 217 
                 9.00E−18 
                 diacylglycerol 
                 
                   Vibrio cholerae 
                 
                 NP_233101.1 
                 nt. 2083-2208 of 
               
               
                   
                   
                 kinase 
                   
                   
                 SEQ ID NO: 217 
               
               
                 221 
                 4.00E−34 
                 Tail-Specific 
                 
                   Chlamydia 
                 
                 NP_219953.1 
                 nt. 849-1421 of 
               
               
                   
                   
                 Protease 
                 
                   trachomatis 
                 
                   
                 SEQ ID NO: 221 
               
               
                 222 
                 4.00E−23 
                 AGR_C_3689p 
                 
                   Agrobacterium 
                 
                 NP_355005.1 
                 compliment of 
               
               
                   
                   
                   
                   tumefaciens  str. 
                   
                 nt. 940-1305 of 
               
               
                   
                   
                   
                 C58 (Cereon) 
                   
                 SEQ ID NO: 222 
               
               
                 224 
                 9.00E−19 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245536.1 
                 nt. 15-308 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 224 
               
               
                 225 
                 1.00E−89 
                 portal vector-like 
                 
                   Salmonella 
                 
                 NP_461651.1 
                 nt. 31-750 of of 
               
               
                   
                   
                 protein, in phage 
                 
                   typhimurium 
                 
                   
                 SEQ ID NO: 225 
               
               
                   
                   
                 P2 [ Salmonella   
                 LT2Fels-2 
               
               
                   
                   
                   typhimurium  LT2] 
                 prophage 
               
               
                 229 
                 2.00E−25 
                 anaerobic 
                 
                   Salmonella 
                 
                 CAB62266.1 
                 nt. 1806-2108 of 
               
               
                   
                   
                 ribonucleotide 
                 
                   typhimurium 
                 
                   
                 SEQ ID NO: 229 
               
               
                   
                   
                 reductase 
               
               
                 234 
                 3.00E−08 
                 conserved 
                 
                   Xylella fastidiosa 
                 
                 NP_299850.1 
                 nt. 1680-2048 of 
               
               
                   
                   
                 hypothetical protein 
                 9a5c 
                   
                 SEQ ID NO: 234 
               
               
                 234 
                 1.00E−42 
                 Methionine 
                 
                   Clostridium 
                 
                 NP_348177.1 
                 compliment of 
               
               
                   
                   
                 sulfoxide reductase 
                 
                   acetobutylicum 
                 
                   
                 nt. 415-654 of 
               
               
                   
                   
                 C-terminal domain 
                   
                   
                 SEQ ID NO: 234 
               
               
                   
                   
                 related protein, 
               
               
                   
                   
                 YPPQ ortholog 
               
               
                 235 
                 4.00E−16 
                 phage-related tail 
                 
                   Wolbachia 
                 
                 AAK85310.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   endosymbiont  of 
                   
                 nt. 931-1929 of 
               
               
                   
                   
                   
                 
                   Drosophila 
                 
                   
                 SEQ ID NO: 235 
               
               
                   
                   
                   
                 
                   melanogaster 
                 
               
               
                 235 
                 6.00E−56 
                 similar to orfG 
                 
                   Salmonella 
                 
                 NP_461625.1 
                 compliment of 
               
               
                   
                   
                 protein in phage 
                   typhimurium  LT2, 
                   
                 nt. 313-1863 of 
               
               
                   
                   
                 186,  Salmonella   
                 Fels-2 prophage 
                   
                 SEQ ID NO: 235 
               
               
                   
                   
                   typhimurium  LT2 
               
               
                 236 
                 6.00E−20 
                 conserved 
                 
                   Pseudomonas 
                 
                 NP_252693.1 
                 nt. 1572-1916 
               
               
                   
                   
                 hypothetical protein 
                 
                   aeruginosa 
                 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 236 
               
               
                 240 
                 5.00E−27 
                 MODIFICATION 
                 
                   Brevibacterium 
                 
                 P10283 
                 compliment of 
               
               
                   
                   
                 METHYLASE BEPI 
                 
                   epidermidis 
                 
                   
                 nt. 922-1305 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 240 
               
               
                 241 
                 2.00E−15 
                 phage-related 
                 
                   Xylella fastidiosa 
                 
                 NP_299573.1 
                 compliment of 
               
               
                   
                   
                 protein 
                 9a5c 
                   
                 nt. 865-1305 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 241 
               
               
                 241 
                 4.00E−08 
                 hypothetical protein 
                 phage SPP1 
                 T42296 
                 nt. 73-636 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 241 
               
               
                 241 
                 4.00E−07 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458686.1 
                 nt. 10-468 of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 SEQ ID NO: 241 
               
               
                   
                   
                   
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 242 
                 2.00E−29 
                 translation 
                 chloroplast- 
                 S35701 
                 compliment of 
               
               
                   
                   
                 elongation factor 
                 soybean 
                   
                 nt. 18-1085 of 
               
               
                   
                   
                 EF-G 
                   
                   
                 SEQ ID NO: 242 
               
               
                 247 
                 3.00E−23 
                 GTP 
                 
                   Synechococcus 
                 
                 Q54769 
                 compliment of 
               
               
                   
                   
                 CYCLOHYDROLASE 
                 sp. PCC 7942 
                   
                 nt. 1009-1257c 
               
               
                   
                   
                 I (GTP-CH-I) 
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 247 
               
               
                 248 
                 6.00E−05 
                 phospho-N- 
                 
                   Aquifex aeolicus 
                 
                 NP_213025.1 
                 nt. 830-1747 of 
               
               
                   
                   
                 acetylmuramoyl- 
                   
                   
                 SEQ ID NO: 248 
               
               
                   
                   
                 pentapeptide- 
               
               
                   
                   
                 transferase 
               
               
                 25 
                 2.00E−86 
                 PROBABLE 
                 
                   Ralstonia 
                 
                 NP_522358.1 
                 compliment of 
               
               
                   
                   
                 TRANSPORT 
                 
                   solanacearum 
                 
                   
                 nt. 309-854 of 
               
               
                   
                   
                 TRANSMEMBRANE 
                   
                   
                 SEQ ID NO: 25 
               
               
                   
                   
                 PROTEIN 
               
               
                 25 
                 7.00E−06 
                 major facilitator 
                 
                   Caulobacter 
                 
                 NP_419155.1 
                 compliment of 
               
               
                   
                   
                 family transporter 
                 
                   crescentus 
                 
                   
                 nt. 134-283 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 25 
               
               
                 250 
                 1.00E−150 
                 CpdB 
                 
                   Pasteurella 
                 
                 NP_246953.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 36-1016 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 250 
               
               
                 252 
                 3.00E−57 
                 alanyl-tRNA 
                 
                   Vibrio cholerae 
                 
                 AAA99922.1 
                 compliment of 
               
               
                   
                   
                 synthetase 
                   
                   
                 nt. 1418-1951 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 252 
               
               
                 253 
                 1.00E−108 
                 similar to 
                 
                   Listeria 
                 
                 NP_464432.1 
                 compliment of 
               
               
                   
                   
                 glutathione 
                 
                   monocytogenes 
                 
                   
                 nt. 411-1358 of 
               
               
                   
                   
                 Reductase 
                 EGD-e 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 253 
               
               
                 259 
                 3.00E−39 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458654.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 342-1037 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 259 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 259 
                 3.00E−17 
                 possible exported 
                 
                   Salmonella 
                 
                 NP_458653.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   enterica  subsp. 
                   
                 nt. 1251-1607 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                 
                   Typhi 
                 
                   
                 259 
               
               
                 261 
                 5.00E−74 
                 hypothetical protein 
                 
                   Haemophilus 
                 
                 S27582 
                 compliment of 
               
               
                   
                   
                 6- Haemophilus   
                 
                   influenzae 
                 
                   
                 nt. 3-422 of 
               
               
                   
                   
                 
                   influenzae 
                 
                   
                   
                 SEQ ID NO: 261 
               
               
                 263 
                 1.00E−94 
                 putative 
                 
                   Haemophilus 
                 
                 AAD01406.1 
                 nt. 2142-2672 of 
               
               
                   
                   
                 transposase 
                 
                   paragallinarum 
                 
                   
                 SEQ ID NO: 263 
               
               
                 264 
                 1.00E−126 
                 unknown 
                 
                   Actinobacillus 
                 
                 NP_067554.1 
                 nt. 40-714 of 
               
               
                   
                   
                   
                 
                   actinomycetemcomitans 
                 
                   
                 SEQ ID NO: 264 
               
               
                 264 
                 1.00E−103 
                 unknown 
                 
                   Actinobacillus 
                 
                 NP_067555.1 
                 nt. 695-1309 of 
               
               
                   
                   
                   
                 
                   actinomycetemcomitans 
                 
                   
                 SEQ ID NO: 264 
               
               
                 264 
                 2.00E−21 
                 unknown 
                 
                   Actinobacillus 
                 
                 NP_067556.1 
                 nt. 1302-1448 of 
               
               
                   
                   
                   
                 
                   actinomycetemcomitans 
                 
                   
                 SEQ ID NO: 264 
               
               
                 265 
                 6.00E−27 
                 Aminopeptidase 2 
                 chloroplast 
                 Q42876 
                 nt. 556-1539 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 265 
               
               
                 268 
                 1.00E−116 
                 MutY 
                 
                   Pasteurella 
                 
                 NP_246257.1 
                 nt. 1003-1581 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 268 
               
               
                 272 
                 1.00E−07 
                 hypothetical protein 
                 Bacteriophage 
                 NP_049495.1 
                 compliment of 
               
               
                   
                   
                   
                 933W 
                   
                 nt. 77-232 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 272 
               
               
                 274 
                 3.00E−13 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246952.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 1658-1975 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 274 
               
               
                 275 
                 3.00E−20 
                 CafA 
                 
                   Neisseria 
                 
                 AAG24267.1 
                 nt. 1299-1571 of 
               
               
                   
                   
                   
                 
                   gonorrhoeae 
                 
                   
                 SEQ ID NO: 275 
               
               
                 276 
                 1.00E−45 
                 mukE protein 
                 
                   Vibrio cholerae 
                 
                 NP_231351.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 650-1390 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 276 
               
               
                 276 
                 1.00E−69 
                 KicA 
                 
                   Pasteurella 
                 
                 NP_245545.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 647-1321 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 276 
               
               
                 278 
                 2.00E−56 
                 3-oxoacyl-[acyl- 
                 
                   Salmonella 
                 
                 NP_455686.1 
                 nt. 1366-1944 of 
               
               
                   
                   
                 carrier-protein] 
                   enterica  subsp. 
                   
                 SEQ ID NO: 278 
               
               
                   
                   
                 synthase III 
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 281 
                 5.00E−56 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246261.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 31-678 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 281 
               
               
                 282 
                 3.00E−09 
                 orf25; similar to T 
                 bacteriophage phi 
                 NP_490625.1 
                 compliment of 
               
               
                   
                   
                 gene of P2 
                 CTX 
                   
                 nt. 511-1032 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 282 
               
               
                 282 
                 1.00E−08 
                 orf11; similar to 
                 
                   Haemophilus 
                 
                 AAC45165.1 
                 compliment of 
               
               
                   
                   
                 phage P2 gene S- 
                 
                   somnus 
                 
                   
                 nt. 1450-1584 of 
               
               
                   
                   
                 like product, which 
                   
                   
                 SEQ ID NO: 282 
               
               
                   
                   
                 is involved in tail 
               
               
                   
                   
                 synthesis, 
               
               
                 282 
                 9.00E−27 
                 putative 
                 
                   Salmonella 
                 
                 NP_457167.1 
                 compliment of 
               
               
                   
                   
                 bacteriophage tail 
                   enterica  subsp. 
                   
                 nt. 3-509 of 
               
               
                   
                   
                 protein 
                   enterica  serovar 
                   
                 SEQ ID NO: 282 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 286 
                 5.00E−18 
                 plasmid-related 
                 
                   Listeria innocua 
                 
                 NP_471066.1 
                 compliment of 
               
               
                   
                   
                 protein 
                 plasmid 
                   
                 nt. 887-1501 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 286 
               
               
                 287 
                 8.00E−20 
                 GTP 
                 
                   Escherichia coli 
                 
                 NP_287920.1 
                 nt. 2-145 of 
               
               
                   
                   
                 cyclohydrolase II 
                 O157:H7 EDL933 
                   
                 SEQ ID NO: 287 
               
               
                 289 
                 1.00E−168 
                 MODIFICATION 
                 
                   Haemophilus 
                 
                 O30868 
                 compliment of 
               
               
                   
                   
                 METHYLASE 
                 
                   aegyptius 
                 
                   
                 nt. 138-1091 of 
               
               
                   
                   
                 HAEII 
                   
                   
                 SEQ ID NO: 289 
               
               
                 289 
                 5.00E−11 
                 TYPE II 
                 
                   Haemophilus 
                 
                 O30869 
                 compliment of 
               
               
                   
                   
                 RESTRICTION 
                 
                   aegyptius 
                 
                   
                 nt. 22-132 of 
               
               
                   
                   
                 ENZYME HAEII 
                   
                   
                 SEQ ID NO: 289 
               
               
                 289 
                 6.00E−47 
                 mukF homolog 
                 
                   Haemophilus 
                 
                 AAB70828.1 
                 compliment of 
               
               
                   
                   
                   
                   influenzae  biotype 
                   
                 nt. 1107-1385 
               
               
                   
                   
                   
                 
                   aegyptius 
                 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 289 
               
               
                 294 
                 1.00E−171 
                 LICA PROTEIN 
                 
                   Haemophilus 
                 
                 P14181 
                 compliment of 
               
               
                   
                   
                   
                 
                   influenzae 
                 
                   
                 nt. 677-1564 of 
               
               
                   
                   
                   
                 RM7004 
                   
                 SEQ ID NO: 294 
               
               
                 297 
                 1.00E−158 
                 DNA methylase 
                 
                   Vibrio cholerae 
                 
                 NP_231404.1 
                 compliment of 
               
               
                   
                   
                 HsdM, putative 
                   
                   
                 nt. 12-1136 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 297 
               
               
                 302 
                 0 
                 HEME-BINDING 
                 
                   Haemophilus 
                 
                 P33950 
                 nt.3-1316 of 
               
               
                   
                   
                 PROTEIN A 
                   influenzae  DL42 
                   
                 SEQ ID NO: 302 
               
               
                 304 
                 6.00E−19 
                 hypothetical protein 6 
                 
                   Haemophilus 
                 
                 S27582 
                 nt. 121-267 of 
               
               
                   
                   
                   
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 304 
               
               
                 305 
                 6.00E−40 
                 putative 
                 
                   Streptococcus 
                 
                 NP_269557.1 
                 nt. 65-805 of 
               
               
                   
                   
                 recombinase- 
                   pyogenes  M1 
                   
                 SEQ ID NO: 305 
               
               
                   
                   
                 phage associated 
                 GAS 
               
               
                 305 
                 7.00E−22 
                 single stranded 
                   Shewanella  sp. 
                 AAB57886.1 
                 nt. 1607-2014 of 
               
               
                   
                   
                 DNA-binding 
                 F1A 
                   
                 SEQ ID NO: 305 
               
               
                   
                   
                 protein 
               
               
                 305 
                 1.00E−43 
                 phage-related 
                 
                   Bacillus 
                 
                 NP_244410.1 
                 nt. 92-751 of 
               
               
                   
                   
                 protein 
                 
                   halodurans 
                 
                   
                 SEQ ID NO: 305 
               
               
                 312 
                 1.00E−28 
                 PUTATIVE 
                 
                   Ralstonia 
                 
                 NP_518994.1 
                 nt. 1819-2673 of 
               
               
                   
                   
                 BACTERIOPHAGE- 
                 
                   solanacearum 
                 
                   
                 SEQ ID NO: 312 
               
               
                   
                   
                 RELATED 
               
               
                   
                   
                 TRANSMEMBRANE 
               
               
                   
                   
                 PROTEIN 
               
               
                 312 
                 9.00E−25 
                 similar to 
                 
                   Homo sapiens 
                 
                 XP_068727.1 
                 nt. 27-1001 of 
               
               
                   
                   
                 BASEMENT 
                   
                   
                 SEQ ID NO: 312 
               
               
                   
                   
                 MEMBRANE- 
               
               
                   
                   
                 SPECIFIC 
               
               
                   
                   
                 HEPARAN 
               
               
                   
                   
                 SULFATE 
               
               
                   
                   
                 PROTEOGLYCAN 
               
               
                   
                   
                 CORE PROTEIN 
               
               
                   
                   
                 PRECURSOR 
               
               
                   
                   
                 (HSPG) 
               
               
                 315 
                 2.00E−45 
                 uracil permease 
                 
                   Deinococcus 
                 
                 NP_296001.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   radiodurans 
                 
                   
                 nt. 525-1592 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 315 
               
               
                 318 
                 7.00E−15 
                 CzcD 
                 
                   Pasteurella 
                 
                 NP_246276.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 3-227 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 318 
               
               
                 320 
                 2.00E−60 
                 orf3; similar to 
                 
                   Haemophilus 
                 
                 AAC45159.1 
                 compliment of 
               
               
                   
                   
                 
                   endonuclease 
                 
                 
                   somnus 
                 
                   
                 nt. 606-1241 of 
               
               
                   
                   
                 subunit of the 
                   
                   
                 SEQ ID NO: 320 
               
               
                   
                   
                 phage P2 
               
               
                   
                   
                 terminase (gene M) 
               
               
                 320 
                 2.00E−09 
                 orf4; similar to 
                 
                   Haemophilus 
                 
                 AAC45160.1 
                 compliment of 
               
               
                   
                   
                 head 
                 
                   somnus 
                 
                   
                 nt. 52-285 of 
               
               
                   
                   
                 completion/stabilization 
                   
                   
                 SEQ ID NO: 320 
               
               
                   
                   
                 protein (gene 
               
               
                   
                   
                 L) of phage P2 
               
               
                 320 
                 3.00E−35 
                 orf2; similar to 
                 
                   Haemophilus 
                 
                 AAC45158.1 
                 compliment of 
               
               
                   
                   
                 major capsid 
                 
                   somnus 
                 
                   
                 nt. 1271-1624 of 
               
               
                   
                   
                 protein precursor of 
                   
                   
                 SEQ ID NO: 320 
               
               
                   
                   
                 phage P2 (gene N) 
               
               
                 323 
                 4.00E−37 
                 dedC protein 
                 
                   Escherichia coli 
                 
                 AAA23966.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 74-463 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 323 
               
               
                 324 
                 1.00E−153 
                 conserved 
                 
                   Neisseria 
                 
                 NP_274972.1 
                 compliment of 
               
               
                   
                   
                 hypothetical protein 
                 
                   meningitidis 
                 
                   
                 nt. 930-1943 of 
               
               
                   
                   
                   
                 MC58 
                   
                 SEQ ID NO: 324 
               
               
                 326 
                 5.00E−52 
                 selenophosphate 
                 
                   Eubacterium 
                 
                 CAB53511.1 
                 compliment of 
               
               
                   
                   
                 synthetase 
                 
                   acidaminophilum 
                 
                   
                 nt. 1186-2292 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 326 
               
               
                 328 
                 1.00E−129 
                 secretion protein 
                 
                   Pseudomonas 
                 
                 NP_252510.1 
                 compliment of 
               
               
                   
                   
                 SecD 
                 
                   aeruginosa 
                 
                   
                 nt. 8-625 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 328 
               
               
                 333 
                 3.00E−08 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245489.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 5-418 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 333 
               
               
                 336 
                 6.00E−38 
                 probable methyl 
                 
                   Pseudomonas 
                 
                 NP_253353.1 
                 compliment of 
               
               
                   
                   
                 transferase 
                 
                   aeruginosa 
                 
                   
                 nt. 2547-2819 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 336 
               
               
                 338 
                 2.00E−98 
                 Pmi 
                 
                   Pasteurella 
                 
                 NP_245766.1 
                 nt. 144-842 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 338 
               
               
                 339 
                 2.00E−07 
                 tRNA 
                 
                   Escherichia coli 
                 
                 QQECPE 
                 nt. 2331-2540 of 
               
               
                   
                   
                 nucleotidyltransferase 
                   
                   
                 SEQ ID NO: 339 
               
               
                 340 
                 0 
                 DNA gyrase, 
                 
                   Salmonella 
                 
                 NP_461214.1 
                 compliment of 
               
               
                   
                   
                 subunit A, type II 
                 
                   typhimurium LT2 
                 
                   
                 nt. 93-1799 of 
               
               
                   
                   
                 topoisomerase 
                   
                   
                 SEQ ID NO: 340 
               
               
                 342 
                 4.00E−12 
                 tolA protein 
                 
                   Haemophilus 
                 
                 JC5212 
                 nt. 980-1318 of 
               
               
                   
                   
                   
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 342 
               
               
                 344 
                 1.00E−07 
                 MODIFICATION 
                 
                   Haemophilus 
                 
                 P50192 
                 compliment of 
               
               
                   
                   
                 METHYLASE 
                 
                   parahaemolyticus 
                 
                   
                 nt. 849-1034 of 
               
               
                   
                   
                 HPHIA 
                   
                   
                 SEQ ID NO: 344 
               
               
                 344 
                 8.00E−05 
                 ABC transporter 
                 
                   Leishmania major 
                 
                 AAF31030.1 
                 compliment of 
               
               
                   
                   
                 protein 1 
                   
                   
                 nt. 17-205 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 344 
               
               
                 349 
                 3.00E−44 
                 conserved 
                 
                   Neisseria 
                 
                 NP_273467.1 
                 compliment of 
               
               
                   
                   
                 hypothetical protein 
                 
                   meningitidis 
                 
                   
                 nt. 1397-1903 of 
               
               
                   
                   
                   
                 MC58 
                   
                 SEQ ID NO: 349 
               
               
                 349 
                 8.00E−09 
                 hypothetical protein 
                 
                   Pseudomonas 
                 
                 NP_252667.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   aeruginosa 
                 
                   
                 nt. 795-1121 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 349 
               
               
                 349 
                 9.00E−10 
                 conserved 
                 
                   Helicobacter 
                 
                 NP_207009.1 
                 compliment of 
               
               
                   
                   
                 hypothetical 
                   pylori  26695 
                   
                 nt. 1319-1816 of 
               
               
                   
                   
                 secreted protein 
                   
                   
                 SEQ ID NO: 349 
               
               
                 349 
                 2.00E−06 
                 putative TPR 
                 
                   Salmonella 
                 
                 NP_463149.1 
                 compliment of 
               
               
                   
                   
                 repeat protein 
                   typhimurium  LT2 
                   
                 nt. 2244-2558 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 349 
               
               
                 35 
                 1.00E−23 
                 type I restriction- 
                 
                   Xylella fastidiosa 
                 
                 NP_300003.1 
                 compliment of 
               
               
                   
                   
                 modification 
                 9a5c 
                   
                 nt. 29-388 of 
               
               
                   
                   
                 system specificity 
                   
                   
                 SEQ ID NO: 35 
               
               
                   
                   
                 determinant 
               
               
                 352 
                 1.00E−116 
                 putative peptidase 
                 
                   Escherichia coli 
                 
                 NP_416827.1 
                 compliment of 
               
               
                   
                   
                   
                 K12 
                   
                 nt. 951-1640 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 352 
               
               
                 352 
                 0 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245275.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 86-946 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 352 
               
               
                 354 
                 5.00E−86 
                 putative uronate 
                 
                   Salmonella 
                 
                 NP_462052.1 
                 compliment of 
               
               
                   
                   
                 isomerase 
                   typhimurium  LT2 
                   
                 nt. 168-914 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 354 
               
               
                 356 
                 1.00E−07 
                 isomerase-like 
                 
                   Escherichia coli 
                 
                 S57220 
                 nt. 5-73 of 
               
               
                   
                   
                 protein (DsbD)- 
                   
                   
                 SEQ ID NO: 356 
               
               
                 358 
                 1.00E−07 
                 USG protein 
                 
                   Pediococcus 
                 
                 CAC16793.1 
                 nt.534-1307 of 
               
               
                   
                   
                   
                 
                   pentosaceus 
                 
                   
                 SEQ ID NO: 358 
               
               
                 358 
                 0.005 
                 HsdS protein 
                 
                   Escherichia coli 
                 
                 CAA10700.1 
                 nt. 26-205 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 358 
               
               
                 361 
                 1.00E−152 
                 maltodextrin 
                 
                   Escherichia coli 
                 
                 NP_289957.1 
                 compliment of 
               
               
                   
                   
                 phosphorylase 
                 O157:H7 EDL933 
                   
                 nt. 77-922 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 361 
               
               
                 363 
                 6.00E−06 
                 BH2505~unknown 
                 
                   Bacillus 
                 
                 NP_243371.1 
                 nt. 554-844 of 
               
               
                   
                   
                 conserved protein 
                 
                   halodurans 
                 
                   
                 SEQ ID NO: 363 
               
               
                 368 
                 1.00E−12 
                 H02F09.3.p 
                 
                   Caenorhabditis 
                 
                 NP_508295.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   elegans 
                 
                   
                 nt. 1069-1977 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 368 
               
               
                 368 
                 6.00E−27 
                 hypothetical 
                 
                   Mesorhizobium 
                 
                 NP_102360.1 
                 compliment of 
               
               
                   
                   
                 glycine-rich protein 
                 
                   loti 
                 
                   
                 nt. 1201-1986 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 368 
               
               
                 37 
                 6.00E−09 
                 putative ATP- 
                 
                   Escherichia coli 
                 
                 NP_415469.1 
                 compliment of 
               
               
                   
                   
                 binding component 
                 K12 
                   
                 nt. 455-691 of 
               
               
                   
                   
                 of a transport 
                   
                   
                 SEQ ID NO: 37 
               
               
                   
                   
                 system 
               
               
                 372 
                 7.00E−18 
                 conserved 
                 
                   Clostridium 
                 
                 BAB80319.1 
                 compliment of 
               
               
                   
                   
                 hypothetical protein 
                 
                   perfringens 
                 
                   
                 nt. 1763-1924 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 372 
               
               
                 376 
                 7.00E−24 
                 putative 
                 
                   Salmonella 
                 
                 NP_456379.1 
                 compliment of 
               
               
                   
                   
                 bacteriophage 
                   enterica  subsp. 
                   
                 nt. 158-808 of 
               
               
                   
                   
                 protein 
                   enterica  serovar 
                   
                 SEQ ID NO: 376 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 376 
                 8.00E−10 
                 hypothetical protein 
                 
                   Xylella fastidiosa 
                 
                 NP_298882.1 
                 compliment of 
               
               
                   
                   
                   
                 9a5c 
                   
                 nt. 1129-1671 
               
               
                   
                   
                   
                   
                   
                 of SEQ ID 
               
               
                   
                   
                   
                   
                   
                 NO: 376 
               
               
                 376 
                 9.00E−06 
                 lin1713 
                 
                   Listeria innocua 
                 
                 NP_471049.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt 913-1557 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 376 
               
               
                 377 
                 6.00E−05 
                 Vng1732c 
                   Halobacterium  sp. 
                 NP_280487.1 
                 nt. 2378-2587 of 
               
               
                   
                   
                   
                 NRC-1 
                   
                 SEQ ID NO: 377 
               
               
                 377 
                 1.00E−11 
                 INVASIN 
                 
                   Yersinia 
                 
                 P31489 
                 compliment of 
               
               
                   
                   
                 PRECURSOR 
                 
                   enterocolitica 
                 
                   
                 nt. 127-345 of 
               
               
                   
                   
                 (OUTER 
                   
                   
                 SEQ ID NO: 377 
               
               
                   
                   
                 MEMBRANE 
               
               
                   
                   
                 ADHESIN) 
               
               
                 382 
                 4.00E−16 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246871.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 967-1068 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 382 
               
               
                 383 
                 4.00E−36 
                 putative 
                 
                   Streptomyces 
                 
                 BAB69302.1 
                 nt. 488-1162 of 
               
               
                   
                   
                 transposase 
                 
                   avermitilis 
                 
                   
                 SEQ ID NO: 383 
               
               
                 383 
                 3.00E−58 
                 recombinase 
                 IncN plasmid R46 
                 NP_511241.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 1-393 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 383 
               
               
                 383 
                 4.00E−24 
                 transposase 
                 
                   Escherichia coli 
                 
                 I69674 
                 nt. 1294-1740 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 383 
               
               
                 383 
                 0 
                 tnpA 
                 
                   Yersinia 
                 
                 CAA73750.1 
                 nt. 1782-2834 of 
               
               
                   
                   
                   
                 
                   enterocolitica 
                 
                   
                 SEQ ID NO: 383 
               
               
                 385 
                 2.00E−31 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246065.1 
                 nt. 1515-1772 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 385 
               
               
                 386 
                 5.00E−65 
                 cydC [ 
                 
                   Escherichia coli 
                 
                 AAA66172.1 
                 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 3438-4115 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 386 
               
               
                 386 
                 4.00E−33 
                 ABC transporter, 
                 
                   Mesorhizobium 
                 
                 NP_105463.1 
                 compliment of 
               
               
                   
                   
                 ATP-binding 
                 
                   loti 
                 
                   
                 nt. 2569-3390 of 
               
               
                   
                   
                 protein 
                   
                   
                 SEQ ID NO: 386 
               
               
                 388 
                 3.00E−45 
                 60 KDA INNER- 
                 
                   Coxiella burnetii 
                 
                 P45650 
                 compliment of 
               
               
                   
                   
                 MEMBRANE 
                   
                   
                 nt. 3211-3759 
               
               
                   
                   
                 PROTEIN 
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                 HOMOLOG 
                   
                   
                 388 
               
               
                 390 
                 4.00E−25 
                 putative DNA- 
                 
                   Salmonella 
                 
                 NP_458175.1 
                 nt. 1051-1416 of 
               
               
                   
                   
                 binding protein 
                   enterica  subsp. 
                   
                 SEQ ID NO: 390 
               
               
                   
                   
                   
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 390 
                 3.00E−13 
                 transcriptional 
                 
                   Bacillus 
                 
                 NP_241773.1 
                 compliment of 
               
               
                   
                   
                 regulator 
                 
                   halodurans 
                 
                   
                 nt. 84-578 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 390 
               
               
                 390 
                 3.00E−06 
                 DNA translocase 
                 
                   Staphylococcus 
                 
                 NP_372265.1 
                 compliment of 
               
               
                   
                   
                 stage III sporulation 
                   aureus  subsp. 
                   
                 nt. 620-871 of 
               
               
                   
                   
                 prot homolog 
                   aureus  Mu50 
                   
                 SEQ ID NO: 390 
               
               
                 395 
                 7.00E−31 
                 ATPase, Cu++ 
                 
                   Homo sapiens 
                 
                 NP_000044.1 
                 compliment of 
               
               
                   
                   
                 transporting, beta 
                   
                   
                 nt. 615-1406 of 
               
               
                   
                   
                 polypeptide 
                   
                   
                 SEQ ID NO: 395 
               
               
                 397 
                 3.00E−23 
                 terminase large 
                 Bacteriophage 
                 NP_112076.1 
                 compliment of 
               
               
                   
                   
                 subunit 
                 HK620 
                   
                 nt. 2363-2725 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 397 
               
               
                 397 
                 3.00E−16 
                 hypothetical protein 
                 
                   Xylella fastidiosa 
                 
                 NP_297824.1 
                 compliment of 
               
               
                   
                   
                   
                 9a5c 
                   
                 nt. 1517-1744 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 397 
               
               
                 398 
                 4.00E−67 
                 orf32 
                 
                   Haemophilus 
                 
                 NP_536839.1 
                 compliment of 
               
               
                   
                   
                   
                 phage HP2 
                   
                 nt. 1288-1866 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 398 
               
               
                 398 
                 8.00E−24 
                 putative 
                 
                   Salmonella 
                 
                 NP_463063.1 
                 compliment of 
               
               
                   
                   
                 cytoplasmic protein 
                   typhimurium  LT2 
                   
                 nt. 798-1220 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 398 
               
               
                 398 
                 2.00E−83 
                 orf31 
                 
                   Haemophilus 
                 
                 NP_043502.1 
                 compliment of 
               
               
                   
                   
                   
                 phage HP1 
                   
                 nt. 1881-2510 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 398 
               
               
                 399 
                 1.00E−94 
                 HEME/HEMOPEXIN- 
                 
                   Haemophilus 
                 
                 P45355 
                 nt. 88-774 of 
               
               
                   
                   
                 BINDING 
                   influenzae  N182 
                   
                 SEQ ID NO: 399 
               
               
                   
                   
                 PROTEIN 
               
               
                 401 
                 3.00E−63 
                 Sty SBLI 
                 
                   Salmonella 
                 
                 CAA68058.1 
                 nt. 1690-2742 of 
               
               
                   
                   
                   
                 
                   enterica 
                 
                   
                 SEQ ID NO: 401 
               
               
                 401 
                 3.00E−06 
                 RESTRICTION- 
                 
                   Mycoplasma 
                 
                 NP_325912.1 
                 nt. 79-489 of 
               
               
                   
                   
                 MODIFICATION 
                 
                   pulmonis 
                 
                   
                 SEQ ID NO: 401 
               
               
                   
                   
                 ENZYME 
               
               
                   
                   
                 SUBUNIT M3 
               
               
                 402 
                 2.00E−13 
                 OPACITY 
                 
                   Neisseria 
                 
                 Q05033 
                 compliment of 
               
               
                   
                   
                 PROTEIN OPA66 
                 
                   gonorrhoeae 
                 
                   
                 nt. 2634-2915 of 
               
               
                   
                   
                 PRECURSOR 
                   
                   
                 SEQ ID NO: 402 
               
               
                 406 
                 8.00E−13 
                 type I restriction 
                 
                   Neisseria 
                 
                 NP_273876.1 
                 nt. 281-520 of 
               
               
                   
                   
                 enzyme EcoR124IIR 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 406 
               
               
                   
                   
                   
                 MC58 
               
               
                 407 
                 6.00E−65 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246237.1 
                 nt. 938-2450 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 407 
               
               
                 407 
                 5.00E−99 
                 PepE 
                 
                   Pasteurella 
                 
                 NP_245391.1 
                 nt. 1216-1917 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 407 
               
               
                 407 
                 1.00E−16 
                 Hemoglobin- 
                 
                   Haemophilus 
                 
                 Q48153 
                 nt. 1-141 of 
               
               
                   
                   
                 haptoglobin binding 
                   influenzae  Tn106 
                   
                 SEQ ID NO: 407 
               
               
                   
                   
                 protein A 
               
               
                 409 
                 1.00E−106 
                 hypothetical protein 1 
                 
                   Haemophilus 
                 
                 S27577 
                 compliment of 
               
               
                   
                   
                   
                 
                   influenzae 
                 
                   
                 nt. 2524-3159 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 409 
               
               
                 411 
                 4.00E−29 
                 heme-repressible 
                 
                   Haemophilus 
                 
                 AAB46794.1 
                 nt. 391-615 of 
               
               
                   
                   
                 hemoglobin-binding 
                   influenzae , type b, 
                   
                 SEQ ID NO: 411 
               
               
                   
                   
                 protein 
                 strain HI689 
               
               
                 411 
                 0 
                 Hemoglobin- 
                 
                   Haemophilus 
                 
                 Q48153 
                 nt. 651-3263 of 
               
               
                   
                   
                 haptoglobin binding 
                   influenzae  Tn106 
                   
                 SEQ ID NO: 411 
               
               
                   
                   
                 protein A 
               
               
                 412 
                 2.00E−07 
                 REGULATORY 
                 bacteriophage 
                 P03036 
                 compliment of 
               
               
                   
                   
                 PROTEIN CRO 
                 434 
                   
                 nt. 59-259 of 
               
               
                   
                   
                 (ANTIREPRESSOR) 
                   
                   
                 SEQ ID NO: 412 
               
               
                 412 
                 4.00E−06 
                 hypothetical protein 
                 Bacteriophage 
                 CAC83535.1 
                 nt. 1436-1654 of 
               
               
                   
                   
                   
                 P27 
                   
                 SEQ ID NO: 412 
               
               
                 413 
                 8.00E−07 
                 hypothetical protein 
                 
                   Deinococcus 
                 
                 NP_294301.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   radiodurans 
                 
                   
                 nt. 791-1012 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 413 
               
               
                 414 
                 9.00E−65 
                 conserved 
                 
                   Vibrio cholerae 
                 
                 NP_230092.1 
                 nt. 1696-2103 of 
               
               
                   
                   
                 hypothetical protein 
                   
                   
                 SEQ ID NO: 414 
               
               
                 414 
                 3.00E−93 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246834.1 
                 nt. 1777-2109 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 414 
               
               
                 416 
                 2.00E−17 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246629.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 2565-2831 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 416 
               
               
                 416 
                 4.00E−26 
                 hypothetical protein 
                 
                   Escherichia coli 
                 
                 S30728 
                 compliment of 
               
               
                   
                   
                 o154 
                   
                   
                 nt. 1928-2254 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 416 
               
               
                 416 
                 3.00E−37 
                 transport protein 
                 
                   Pseudomonas 
                 
                 NP_253757.1 
                 compliment of 
               
               
                   
                   
                 TatC 
                 
                   aeruginosa 
                 
                   
                 nt. 1494-2018 of 
               
               
                   
                   
                   
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 416 
               
               
                 417 
                 1.00E−66 
                 weakly similar to 
                 
                   Listeria innocua 
                 
                 NP_471073.1 
                 compliment of 
               
               
                   
                   
                 methyltransferases 
                   
                   
                 nt. 999-1928 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 417 
               
               
                 417 
                 5.00E−05 
                 DNA-BINDING 
                 
                   Pectobacterium 
                 
                 Q47587 
                 compliment of 
               
               
                   
                   
                 PROTEIN RDGA 
                 
                   carotovorum 
                 
                   
                 nt. 3526-4212 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 417 
               
               
                 417 
                 2.00E−29 
                 putative phage- 
                 
                   Yersinia pestis 
                 
                 NP_407132.1 
                 compliment of 
               
               
                   
                   
                 related protein 
                   
                   
                 nt. 2546-2938 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 417 
               
               
                 417 
                 3.00E−06 
                 Adenine-specific 
                 
                   Thermoplasma 
                 
                 NP_393798.1 
                 compliment of 
               
               
                   
                   
                 DNA methylase 
                 
                   acidophilum 
                 
                   
                 nt. 826-1020 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 417 
               
               
                 43 
                 9.00E−16 
                 PcnB 
                 
                   Pasteurella 
                 
                 NP_245801.1 
                 nt. 511-870 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 43 
               
               
                 434 
                 2.00E−97 
                 beta′ subunit of 
                 
                   Nephroselmis 
                 
                 NP_050840.1 
                 compliment of 
               
               
                   
                   
                 RNA polymerase 
                 
                   olivacea 
                 
                   
                 nt. 32-1534 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 434 
               
               
                 435 
                 4.00E−52 
                 MODIFICATION 
                 
                   Brevibacterium 
                 
                 P10283 
                 compliment of 
               
               
                   
                   
                 METHYLASE BEPI 
                 
                   epidermidis 
                 
                   
                 nt. 11-565 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 435 
               
               
                 435 
                 4.00E−57 
                 pentafunctional 
                 
                   Saccharomyces 
                 
                 NP_010412.1 
                 compliment of 
               
               
                   
                   
                 arom polypeptide 
                 
                   cerevisiae 
                 
                   
                 nt. 757-2064 of 
               
               
                   
                   
                 (contains: 3- 
                   
                   
                 SEQ ID NO: 435 
               
               
                   
                   
                 dehydroquinate 
               
               
                   
                   
                 synthase, 3- 
               
               
                   
                   
                 dehydroquinate 
               
               
                   
                   
                 dehydratase (3- 
               
               
                   
                   
                 dehydroquinase), 
               
               
                   
                   
                 shikimate 5- 
               
               
                   
                   
                 dehydrogenase, 
               
               
                   
                   
                 shikimate kinase, 
               
               
                   
                   
                 and epsp synthase) 
               
               
                 437 
                 5.00E−70 
                 dihydrofolate 
                 
                   Haemophilus 
                 
                 S52336 
                 nt. 2393-2767 of 
               
               
                   
                   
                 reductase 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 437 
               
               
                   
                   
                   
                 (clinical isolate 
               
               
                   
                   
                   
                 R1042) 
               
               
                 438 
                 1.00E−106 
                 polyA polymerase 
                 
                   Vibrio cholerae 
                 
                 NP_230244.1 
                 nt. 3-1124 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 438 
               
               
                 439 
                 6.00E−60 
                 Porphyrin 
                 
                   Salmonella 
                 
                 NP_457816.1 
                 nt. 2343-2783 of 
               
               
                   
                   
                 biosynthetic protein 
                   enterica  subsp. 
                   
                 SEQ ID NO: 439 
               
               
                   
                   
                   
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 441 
                 5.00E−73 
                 RimM 
                 
                   Pasteurella 
                 
                 NP_246234.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 151-441 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 441 
               
               
                 442 
                 9.00E−31 
                 LIPOPROTEIN 
                 
                   Salmonella 
                 
                 P40827 
                 compliment of 
               
               
                   
                   
                 NLPD 
                 
                   typhimurium 
                 
                   
                 nt. 3362-3520 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 442 
               
               
                 444 
                 6.00E−24 
                 glycine betaine 
                 
                   Staphylococcus 
                 
                 NP_371872.1 
                 compliment of 
               
               
                   
                   
                 transporter 
                   aureus  subsp. 
                   
                 nt. 2242-2514 of 
               
               
                   
                   
                   
                   aureus  Mu50 
                   
                 SEQ ID NO: 444 
               
               
                 452 
                 6.00E−28 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245616.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 533-883 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 452 
               
               
                 452 
                 0 
                 Type I restriction 
                 
                   Escherichia coli 
                 
                 Q47163 
                 nt. 3291-4154 of 
               
               
                   
                   
                 enzyme Ecoprrl M 
                   
                   
                 SEQ ID NO: 452 
               
               
                   
                   
                 protein 
               
               
                 452 
                 2.00E−75 
                 type I restriction 
                 
                   Ureaplasma 
                 
                 NP_077929.1 
                 nt. 4156-4562 of 
               
               
                   
                   
                 enzyme M protein 
                 
                   urealyticum 
                 
                   
                 SEQ ID NO: 452 
               
               
                 455 
                 9.00E−56 
                 PROBABLE 
                 
                   Ralstonia 
                 
                 NP_520059.1 
                 nt. 2028-2774 of 
               
               
                   
                   
                 BACTERIOPHAGE 
                 
                   solanacearum 
                 
                   
                 SEQ ID NO: 455 
               
               
                   
                   
                 PROTEIN 
               
               
                 455 
                 2.00E−55 
                 orf2; similar to 
                 
                   Haemophilus 
                 
                 AAC45158.1 
                 nt. 2864-3490 of 
               
               
                   
                   
                 major capsid 
                 
                   somnus 
                 
                   
                 SEQ ID NO: 455 
               
               
                   
                   
                 protein precursor of 
               
               
                   
                   
                 phage P2 (gene N), 
               
               
                 455 
                 1.00E−175 
                 gpP 
                 
                   Enterobacteria 
                 
                 NP_046758.1 
                 compliment of 
               
               
                   
                   
                   
                 phage P2 
                   
                 nt. 127-1812 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 455 
               
               
                 456 
                 1.00E−38 
                 hypothetical protein 
                 
                   Pseudomonas 
                 
                 NP_542872.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   putida 
                 
                   
                 nt. 1010-1282 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 456 
               
               
                 456 
                 1.00E−172 
                 hypothetical protein 
                 
                   Pseudomonas 
                 
                 NP_542873.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   putida 
                 
                   
                 nt. 1443-2006 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 546 
               
               
                 457 
                 1.00E−116 
                 hypothetical protein 
                 
                   Haemophilus 
                 
                 S15287 
                 compliment of 
               
               
                   
                   
                 (galE 5′ region)- 
                 
                   influenzae 
                 
                   
                 nt. 62-961 of 
               
               
                   
                   
                 
                   Haemophilus 
                 
                   
                   
                 SEQ ID NO: 457 
               
               
                   
                   
                 
                   influenzae 
                 
               
               
                 457 
                 1.00E−134 
                 dTDPglucose 4,6- 
                 
                   Actinobacillus 
                 
                 T00102 
                 nt. 2637-3656 of 
               
               
                   
                   
                 dehydratase 
                 
                   actinomycetemco 
                 
                   
                 SEQ ID NO: 457 
               
               
                   
                   
                   
                 
                   mitans 
                 
               
               
                 459 
                 2.00E−10 
                 RNA polymerase 
                   Synechocystis  sp. 
                 NP_441586.1 
                 nt. 25-117 of 
               
               
                   
                   
                 gamma-subunit 
                 PCC 6803 
                   
                 SEQ ID NO: 459 
               
               
                 461 
                 9.00E−51 
                 conserved 
                 
                   Staphylococcus 
                 
                 NP_370593.1 
                 nt. 4124-4624 of 
               
               
                   
                   
                 hypothetical protein 
                   aureus  subsp. 
                   
                 SEQ ID NO: 461 
               
               
                   
                   
                   
                   aureus  Mu50 
               
               
                 462 
                 9.00E−06 
                 NADH 
                 
                   Burkholderia 
                 
                 AAG01016.1 
                 nt. 703-828 of 
               
               
                   
                   
                 dehydrogenase 
                 
                   pseudomallei 
                 
                   
                 SEQ ID NO: 462 
               
               
                 465 
                 3.00E−41 
                 GTP-binding 
                   Synechocystis  sp. 
                 NP_441951.1 
                 compliment of 
               
               
                   
                   
                 protein Era 
                 PCC 6803 
                   
                 nt. 2470-2787 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 465 
               
               
                 466 
                 1.00E−15 
                 putative 
                 
                   Salmonella 
                 
                 NP_455548.1 
                 nt. 837-1478 of 
               
               
                   
                   
                 bacteriophage 
                   enterica  subsp. 
                   
                 SEQ ID NO: 466 
               
               
                   
                   
                 protein 
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 466 
                 1.00E−90 
                 orf31 
                 
                   Haemophilus 
                 
                 NP_043502.1 
                 nt. 2396-3199 of 
               
               
                   
                   
                   
                 phage HP1 
                   
                 SEQ ID NO: 466 
               
               
                 469 
                 0 
                 Hemoglobin and 
                 
                   Haemophilus 
                 
                 Q9X442 
                 compliment of 
               
               
                   
                   
                 hemoglobin- 
                   influenzae  HI689 
                   
                 nt. 427-3459 of 
               
               
                   
                   
                 haptoglobin binding 
                   
                   
                 SEQ ID NO: 469 
               
               
                   
                   
                 protein C precursor 
               
               
                 471 
                 8.00E−05 
                 transposase, 
                 
                   Neisseria 
                 
                 NP_274608.1 
                 nt. 2957-3217 of 
               
               
                   
                   
                 putative 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 471 
               
               
                   
                   
                   
                 MC58 
               
               
                 472 
                 6.00E−08 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458660.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 2881-3270 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 472 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 472 
                 5.00E−23 
                 antirestriction 
                 
                   Mesorhizobium 
                 
                 NP_106707.1 
                 nt. 4908-5324 of 
               
               
                   
                   
                 protein 
                 
                   loti 
                 
                   
                 SEQ ID NO: 472 
               
               
                 472 
                 1.00E−75 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458661.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 1931-2776 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 472 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 472 
                 9.00E−72 
                 hypothetical protein 
                 
                   Salmonella 
                 
                 NP_458662.1 
                 compliment of 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 nt. 544-1689 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 472 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 475 
                 3.00E−25 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_244952.1 
                 nt. 3207-3626 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 475 
               
               
                 476 
                 8.00E−73 
                 putative DNA- 
                 
                   Salmonella 
                 
                 NP_458175.1 
                 compliment of 
               
               
                   
                   
                 binding protein 
                   enterica  subsp. 
                   
                 nt. 3339-4310 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 476 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 476 
                 6.00E−47 
                 anticodon nuclease 
                 
                   Neisseria 
                 
                 NP_273873.1| 
                 compliment of 
               
               
                   
                   
                   
                 
                   meningitidis 
                 
                   
                 nt. 4397-4885 of 
               
               
                   
                   
                   
                 MC58 
                   
                 SEQ ID NO: 476 
               
               
                 478 
                 3.00E−06 
                 methionin 
                 
                   Arabidopsis 
                 
                 CAB38313.1 
                 compliment of 
               
               
                   
                   
                 synthase-like 
                 
                   thaliana 
                 
                   
                 nt. 3554-3679 of 
               
               
                   
                   
                 enzyme 
                   
                   
                 SEQ ID NO: 478 
               
               
                 478 
                 3.00E−05 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245444.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 164-250 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 478 
               
               
                 479 
                 1.00E−18 
                 conserved 
                 
                   Xylella fastidiosa 
                 
                 NP_298841.1 
                 nt. 2302-2658 of 
               
               
                   
                   
                 hypothetical protein 
                 9a5c 
                   
                 SEQ ID NO: 479 
               
               
                 48 
                 3.00E−19 
                 Dca 
                 
                   Neisseria 
                 
                 AAF12796.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   gonorrhoeae 
                 
                   
                 nt. 225-746 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 48 
               
               
                 482 
                 1.00E−06 
                 hypothetical protein 
                 
                   Neisseria 
                 
                 NP_275122.1 
                 nt. 2055-2189 of 
               
               
                   
                   
                   
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 482 
               
               
                   
                   
                   
                 MC58 
               
               
                 482 
                 9.00E−28 
                 conserved 
                 
                   Neisseria 
                 
                 NP_274383.1 
                 nt. 1689-1898 of 
               
               
                   
                   
                 hypothetical protein 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 482 
               
               
                   
                   
                   
                 MC58 
               
               
                 487 
                 5.00E−75 
                 conserved 
                 
                   Neisseria 
                 
                 NP_284304.1 
                 nt. 2541-2978 of 
               
               
                   
                   
                 hypothetical protein 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 487 
               
               
                   
                   
                   
                 Z2491 
               
               
                 488 
                 2.00E−64 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246617.1 
                 nt. 2983-3540 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 488 
               
               
                 488 
                 8.00E−93 
                 1-deoxy-D-xylulose 
                 
                   Zymomonas 
                 
                 AAD29659.1 
                 nt. 1344-1880 of 
               
               
                   
                   
                 5-phosphate 
                 
                   mobilis 
                 
                   
                 SEQ ID NO: 488 
               
               
                   
                   
                 reductoisomerase 
               
               
                 491 
                 5.00E−51 
                 rubredoxin 
                 
                   Clostridium 
                 
                 AAB50346.1 
                 compliment of 
               
               
                   
                   
                 oxidoreductase 
                 
                   acetobutylicum 
                 
                   
                 nt. 1690-2439 of 
               
               
                   
                   
                 homolog 
                   
                   
                 SEQ ID NO: 491 
               
               
                 492 
                 1.00E−27 
                 phosphotransferase 
                 
                   Staphylococcus 
                 
                 AAK83253.1 
                 compliment of 
               
               
                   
                   
                 system enzyme 
                 
                   aureus 
                 
                   
                 nt. 755-970 of 
               
               
                   
                   
                 IIA-like protein 
                   
                   
                 SEQ ID NO: 492 
               
               
                 493 
                 2.00E−84 
                 unknown 
                 
                   Actinobacillus 
                 
                 AAC70895.1 
                 nt. 3333-3935 of 
               
               
                   
                   
                   
                 
                   actinomycetemcomitans 
                 
                   
                 SEQ ID NO: 493 
               
               
                 493 
                 4.00E−49 
                 unknown 
                 
                   Helicobacter 
                 
                 NP_223898.1 
                 nt. 3345-4010 of 
               
               
                   
                   
                   
                   pylori  J99 
                   
                 SEQ ID NO: 493 
               
               
                 493 
                 9.00E−31 
                 transcriptional 
                 
                   Acinetobacter 
                 
                 AAF20290.1 
                 nt. 1885-2793 of 
               
               
                   
                   
                 factor MdcH 
                 
                   calcoaceticus 
                 
                   
                 SEQ ID NO: 493 
               
               
                 493 
                 6.00E−30 
                 HimA 
                 
                   Pasteurella 
                 
                 NP_245565.1 
                 nt. 1129-1260 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 493 
               
               
                 494 
                 4.00E−85 
                 putative prophage 
                 
                   Yersinia pestis 
                 
                 NP_404712.1 
                 nt. 900-2099 of 
               
               
                   
                   
                 integrase 
                   
                   
                 SEQ ID NO: 494 
               
               
                 494 
                 4.00E−63 
                 DNA 
                 
                   Xylella fastidiosa 
                 
                 NP_299063.1 
                 compliment of 
               
               
                   
                   
                 methyltransferase 
                 9a5c 
                   
                 nt. 5544-6170 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 494 
               
               
                 494 
                 6.00E−19 
                 MODIFICATION 
                 
                   Lactococcus lactis 
                 
                 P34877 
                 compliment of 
               
               
                   
                   
                 METHYLASE 
                 subsp.  cremoris   
                   
                 nt. 5019-6113 of 
               
               
                   
                   
                 SCRFIA 
                   
                   
                 SEQ ID NO: 494 
               
               
                 497 
                 0 
                 transferrin-binding 
                 
                   Haemophilus 
                 
                 S70906 
                 nt. 3251-4999 of 
               
               
                   
                   
                 protein 1 
                   influenzae  (strain 
                   
                 SEQ ID NO: 497 
               
               
                   
                   
                   
                 PAK 12085) 
               
               
                 50 
                 5.00E−07 
                 AcpP 
                 
                   Pasteurella 
                 
                 NP_246856.1 
                 nt. 2-136 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 50 
               
               
                 501 
                 7.00E−50 
                 conserved 
                 
                   Vibrio cholerae 
                 
                 NP_231403.1 
                 compliment of 
               
               
                   
                   
                 hypothetical protein 
                   
                   
                 nt. 3649-4872 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 501 
               
               
                 501 
                 0 
                 type I restriction 
                 
                   Vibrio cholerae 
                 
                 NP_231400.1 
                 compliment of 
               
               
                   
                   
                 enzyme HsdR, 
                   
                   
                 nt. 1551-3440 of 
               
               
                   
                   
                 putative 
                   
                   
                 SEQ ID NO: 501 
               
               
                 501 
                 4.00E−13 
                 ATP-dependent 
                 
                   Deinococcus 
                 
                 NP_295921.1 
                 compliment of 
               
               
                   
                   
                 DNA helicase 
                 
                   radiodurans 
                 
                   
                 nt. 5317-5844 of 
               
               
                   
                   
                 RecG-related 
                   
                   
                 SEQ ID NO: 501 
               
               
                   
                   
                 protein 
               
               
                 501 
                 5.00E−11 
                 conserved 
                 
                   Ureaplasma 
                 
                 NP_077868.1 
                 compliment of 
               
               
                   
                   
                 hypothetical 
                 
                   urealyticum 
                 
                   
                 nt. 5098-5769 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 501 
               
               
                 504 
                 2.00E−44 
                 OUTER 
                 
                   Haemophilus 
                 
                 Q48218 
                 compliment of 
               
               
                   
                   
                 MEMBRANE 
                 
                   influenzae 
                 
                   
                 nt. 4681-5019 of 
               
               
                   
                   
                 PROTEIN P2 
                 AG30010 
                   
                 SEQ ID NO: 504 
               
               
                   
                   
                 PRECURSOR 
               
               
                   
                   
                 (OMP P2) 
               
               
                 507 
                 0 
                 SpoT 
                 
                   Pasteurella 
                 
                 NP_245857.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 3685-5316 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 507 
               
               
                 51 
                 6.00E−87 
                 glucosamine-- 
                 
                   Vibrio cholerae 
                 
                 NP_230141.1 
                 nt. 30-470 of 
               
               
                   
                   
                 fructose-6- 
                   
                   
                 SEQ ID NO: 51 
               
               
                   
                   
                 phosphate 
               
               
                   
                   
                 aminotransferase 
               
               
                   
                   
                 (isomerizing) 
               
               
                 512 
                 2.00E−28 
                 dipeptide transport 
                 
                   Yersinia pestis 
                 
                 NP_407439.1 
                 compliment of 
               
               
                   
                   
                 system permease 
                   
                   
                 nt. 1095-1580 of 
               
               
                   
                   
                 protein 
                   
                   
                 SEQ ID NO: 512 
               
               
                 512 
                 3.00E−82 
                 SapC 
                 
                   Pasteurella 
                 
                 NP_245850.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 730-1095 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 512 
               
               
                 514 
                 9.00E−06 
                 putative integral 
                 
                   Campylobacter 
                 
                 NP_281236.1 
                 compliment of 
               
               
                   
                   
                 membrane protein 
                 
                   jejuni 
                 
                   
                 nt. 577-684 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 514 
               
               
                 514 
                 3.00E−11 
                 orf, hypothetical 
                 
                   Escherichia coli 
                 
                 NP_286004.1 
                 compliment of 
               
               
                   
                   
                 protein 
                 O157:H7 EDL933 
                   
                 nt. 449-568 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 514 
               
               
                 518 
                 0 
                 putative inner 
                 
                   Neisseria 
                 
                 NP_284893.1 
                 nt. 92-1927 of 
               
               
                   
                   
                 membrane transacylase 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 518 
               
               
                   
                   
                 protein 
                 Z2491 
               
               
                 519 
                 4.00E−30 
                 hypothetical protein 
                 
                   Mesorhizobium 
                 
                 NP_108196.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   loti 
                 
                   
                 nt. 2221-3159 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 519 
               
               
                 519 
                 2.00E−12 
                 conserved 
                 
                   Listeria innocua 
                 
                 NP_471067.1 
                 compliment of 
               
               
                   
                   
                 hypothetical protein 
                   
                   
                 nt. 3994-5241 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 519 
               
               
                 519 
                 6.00E−20 
                 hypothetical protein 
                 
                   Mesorhizobium 
                 
                 NP_108198.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   loti 
                 
                   
                 nt. 707-1552 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 519 
               
               
                 519 
                 4.00E−26 
                 putative 
                 
                   Salmonella 
                 
                 NP_455526.1 
                 compliment of 
               
               
                   
                   
                 bacteriophage 
                   enterica  subsp. 
                   
                 nt. 3982-5163 of 
               
               
                   
                   
                 protein 
                   enterica  serovar 
                   
                 SEQ ID NO: 519 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 52 
                 3.00E−94 
                 OUTER 
                 
                   Haemophilus 
                 
                 Q48218 
                 nt. 45-788 of 
               
               
                   
                   
                 MEMBRANE 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 52 
               
               
                   
                   
                 PROTEIN P2 
               
               
                   
                   
                 PRECURSOR 
               
               
                   
                   
                 (OMP P2) 
               
               
                 520 
                 0 
                 excision nuclease 
                 
                   Escherichia coli 
                 
                 NP_418482.1 
                 compliment of 
               
               
                   
                   
                 subunit A 
                 K12 
                   
                 nt. 6309-7745 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 520 
               
               
                 521 
                 5.00E−08 
                 zinc/manganese 
                 
                   Rickettsia conorii 
                 
                 NP_359651.1 
                 nt. 2236-2652 of 
               
               
                   
                   
                 ABC transporter 
                   
                   
                 SEQ ID NO: 521 
               
               
                   
                   
                 substrate binding 
               
               
                   
                   
                 protein 
               
               
                 521 
                 1.00E−140 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245865.1| 
                 nt. 338-1390 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 521 
               
               
                 521 
                 1.00E−86 
                 ORF_f432 
                 
                   Escherichia coli 
                 
                 AAB40463.1 
                 nt. 203-1390 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 521 
               
               
                 522 
                 3.00E−22 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246093.1 
                 nt. 670-885 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 522 
               
               
                 526 
                 5.00E−33 
                 exodeoxyribonuclease 
                 
                   Yersinia pestis 
                 
                 NP_404635.1 
                 nt. 5582-6202 of 
               
               
                   
                   
                 V alpha chain 
                   
                   
                 SEQ ID NO: 526 
               
               
                 526 
                 1.00E−62 
                 exodeoxyribonuclease 
                 
                   Vibrio cholerae 
                 
                 NP_231950.1 
                 nt. 5675-6193 of 
               
               
                   
                   
                 V, 67 kDa 
                   
                   
                 SEQ ID NO: 526 
               
               
                   
                   
                 subunit 
               
               
                 527 
                 1.00E−147 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245980.1 
                 nt. 4283-5203 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 527 
               
               
                 527 
                 0 
                 Mfd 
                 
                   Pasteurella 
                 
                 NP_245978.1 
                 nt. 7545-8759 of 
               
               
                   
                   
                   
                 multocida 
                   
                 SEQ ID NO: 527 
               
               
                 527 
                 0 
                 transcription-repair 
                 
                   Salmonella 
                 
                 NP_455708.1 
                 nt. 7611-8762 of 
               
               
                   
                   
                 coupling factor 
                   enterica  subsp. 
                   
                 SEQ ID NO: 527 
               
               
                   
                   
                 (TrcF) 
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 527 
                 0 
                 PROBABLE 
                 
                   Ralstonia 
                 
                 NP_519763.1 
                 nt. 7611-8870 of 
               
               
                   
                   
                 TRANSCRIPTION- 
                 
                   solanacearum 
                 
                   
                 SEQ ID NO: 527 
               
               
                   
                   
                 REPAIR 
               
               
                   
                   
                 COUPLING 
               
               
                   
                   
                 FACTOR 
               
               
                   
                   
                 PROTEIN 
               
               
                 528 
                 1.00E−48 
                 undecaprenyl 
                 
                   Chlamydia 
                 
                 NP_297109.1 
                 nt. 2918-3712 of 
               
               
                   
                   
                 pyrophosphate 
                 
                   muridarum 
                 
                   
                 SEQ ID NO: 528 
               
               
                   
                   
                 synthetase 
               
               
                 528 
                 0 
                 leucyl-tRNA 
                 
                   Vibrio cholerae 
                 
                 NP_230603.1 
                 compliment of 
               
               
                   
                   
                 synthetase 
                   
                   
                 nt. 180-2822 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 528 
               
               
                 529 
                 1.00E−104 
                 DNA PRIMASE 
                 
                   Legionella 
                 
                 P71481 
                 compliment of 
               
               
                   
                   
                   
                 
                   pneumophila 
                 
                   
                 nt. 3316-3960 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 529 
               
               
                 534 
                 9.00E−29 
                 putative integrase 
                 
                   Salmonella 
                 
                 NP_461690.1 
                 nt. 4668-5009 of 
               
               
                   
                   
                   
                   typhimurium  LT2 
                   
                 SEQ ID NO: 534 
               
               
                 534 
                 6.00E−18 
                 hypothetical protein 
                 
                   Neisseria 
                 
                 NP_283002.1 
                 compliment of 
               
               
                   
                   
                 NMA0153 
                 
                   meningitidis 
                 
                   
                 nt. 5933-6337 of 
               
               
                   
                   
                   
                 Z2491 
                   
                 SEQ ID NO: 534 
               
               
                 534 
                 2.00E−23 
                 hypothetical protein 
                 
                   Deinococcus 
                 
                 NP_294868.1 
                 nt. 6908-7654 of 
               
               
                   
                   
                   
                 
                   radiodurans 
                 
                   
                 SEQ ID NO: 534 
               
               
                 534 
                 1.00E−88 
                 prophage CP4-57 
                 
                   Escherichia coli 
                 
                 NP_417111.1 
                 nt. 5057-5875 of 
               
               
                   
                   
                 integrase 
                 K12 
                   
                 SEQ ID NO: 534 
               
               
                 535 
                 1.00E−115 
                 phosphate 
                   Buchnera  sp. 
                 NP_240007.1 
                 nt. 3385-4596 of 
               
               
                   
                   
                 acetyltransferase 
                 APS 
                   
                 SEQ ID NO: 535 
               
               
                 536 
                 3.00E−35 
                 cobalt membrane 
                 
                   Actinobacillus 
                 
                 AAD49727.1 
                 compliment of 
               
               
                   
                   
                 transport protein 
                 
                   pleuropneumoniae 
                 
                   
                 nt. 3531-4136 of 
               
               
                   
                   
                 CbiQ 
                   
                   
                 SEQ ID NO: 536 
               
               
                 536 
                 6.00E−37 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_245305.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 6478-6921 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 536 
               
               
                 539 
                 2.00E−26 
                 Orf122 
                 
                   Chlorobium 
                 
                 AAG12204.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   tepidum 
                 
                   
                 nt. 1778-2008 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 539 
               
               
                 540 
                 1.00E−77 
                 heat shock protein 
                 
                   Neisseria 
                 
                 NP_273864.1 
                 compliment of 
               
               
                   
                   
                 HtpX 
                 
                   meningitidis 
                 
                   
                 nt. 2567-3481 of 
               
               
                   
                   
                   
                 MC58 
                   
                 SEQ ID NO: 540 
               
               
                 541 
                 0 
                 IleS 
                 
                   Pasteurella 
                 
                 NP_246601.1 
                 nt. 3167-4549 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 541 
               
               
                 545 
                 2.00E−09 
                 DNA-BINDING 
                 
                   Pectobacterium 
                 
                 Q47588 
                 nt. 3816-3977 of 
               
               
                   
                   
                 PROTEIN RDGB 
                 
                   carotovorum 
                 
                   
                 SEQ ID NO: 545 
               
               
                 545 
                 2.00E−11 
                 putative 
                 
                   Sinorhizobium 
                 
                 NP_437741.1 
                 compliment of 
               
               
                   
                   
                 transposase 
                 
                   meliloti 
                 
                   
                 nt. 2786-3019 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 544 
               
               
                 545 
                 2.00E−07 
                 Hypothetical 42.5 kd 
                 
                   Escherichia coli 
                 
                 BAA77933.1 
                 compliment of 
               
               
                   
                   
                 protein in thrW- 
                   
                   
                 nt. 2614-2811 of 
               
               
                   
                   
                 argF intergenic 
                   
                   
                 SEQ ID NO: 545 
               
               
                   
                   
                 region 
               
               
                 545 
                 4.00E−18 
                 putative IS element 
                 
                   Salmonella 
                 
                 NP_454711.1 
                 nt. 1955-2230 of 
               
               
                   
                   
                 transposase 
                   enterica  subsp. 
                   
                 SEQ ID NO: 545 
               
               
                   
                   
                   
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 546 
                 0 
                 HEME/HEMOPEXIN- 
                 
                   Haemophilus 
                 
                 P45354 
                 nt. 5551-7809 of 
               
               
                   
                   
                 BINDING 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 546 
               
               
                   
                   
                 PROTEIN 
               
               
                 546 
                 0 
                 HEME/HEMOPEXIN 
                 
                   Haemophilus 
                 
                 P45356 
                 nt. 3842-5536 of 
               
               
                   
                   
                 UTILIZATION 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 546 
               
               
                   
                   
                 PROTEIN B 
               
               
                 546 
                 0 
                 HEME/HEMOPEXIN 
                 
                   Haemophilus 
                 
                 P45357 
                 nt. 1638-3176 of 
               
               
                   
                   
                 UTILIZATION 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 546 
               
               
                   
                   
                 PROTEIN C 
               
               
                 546 
                 2.00E−12 
                 HasR 
                 
                   Pasteurella 
                 
                 NP_246561.1 
                 nt. 3149-3763 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 546 
               
               
                 549 
                 0 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246821.1 
                 nt. 2526-3512 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 549 
               
               
                 549 
                 1.00E−121 
                 putative membrane 
                 
                   Yersinia pestis 
                 
                 NP_404859.1 
                 nt. 605-1108 of 
               
               
                   
                   
                 protein 
                   
                   
                 SEQ ID NO: 549 
               
               
                 549 
                 0 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246822.1 
                 nt. 1122-1664 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 549 
               
               
                 551 
                 1.00E−157 
                 type I restriction- 
                 
                   Xylella fastidiosa 
                 
                 NP_300016.1 
                 compliment of 
               
               
                   
                   
                 modification 
                 9a5c 
                   
                 nt. 7396-8322 of 
               
               
                   
                   
                 system 
                   
                   
                 SEQ ID NO: 551 
               
               
                   
                   
                 endonuclease 
               
               
                 552 
                 1.00E−100 
                 valyl-tRNA 
                 
                   Deinococcus 
                 
                 NP_293872.1 
                 compliment of 
               
               
                   
                   
                 synthetase 
                 
                   radiodurans 
                 
                   
                 nt. 6691-8688 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 552 
               
               
                 552 
                 0 
                 VALYL-TRNA 
                 
                   Haemophilus 
                 
                 P36432 
                 compliment of 
               
               
                   
                   
                 SYNTHETASE 
                 
                   parainfluenzae 
                 
                   
                 nt. 5850-6647 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 552 
               
               
                 553 
                 0 
                 DNA-directed RNA 
                 
                   Vibrio cholerae 
                 
                 NP_229982.1 
                 nt. 2668-6699 of 
               
               
                   
                   
                 polymerase, beta 
                   
                   
                 SEQ ID NO: 553 
               
               
                   
                   
                 subunit 
               
               
                 554 
                 0 
                 iron utilization 
                 
                   Haemophilus 
                 
                 T10887 
                 nt. 991-2508 of 
               
               
                   
                   
                 protein B 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 554 
               
               
                 559 
                 1.00E−100 
                 PREPROTEIN 
                 
                   Bacillus firmus 
                 
                 P96313 
                 nt. 3420-4472 of 
               
               
                   
                   
                 TRANSLOCASE 
                   
                   
                 SEQ ID NO: 559 
               
               
                   
                   
                 SECA SUBUNIT 
               
               
                 56 
                 2.00E−23 
                 RpL30 
                 
                   Pasteurella 
                 
                 NP_246336.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 656-832 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 56 
               
               
                 56 
                 9.00E−13 
                 RpS5 
                 
                   Pasteurella 
                 
                 NP_246337.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 nt. 843-1064 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 56 
               
               
                 560 
                 1.00E−157 
                 Na+/H+ antiporter 
                 
                   Vibrio cholerae 
                 
                 NP_231535.1 
                 2 compliment of 
               
               
                   
                   
                   
                   
                   
                 nt. 279-2989 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 560 
               
               
                 562 
                 1.00E−72 
                 putative biotin 
                 
                   Yersinia pestis 
                 
                 NP_404419.1 
                 nt. 7862-8878 of 
               
               
                   
                   
                 sulfoxide reductase 2 
                   
                   
                 SEQ ID NO: 562 
               
               
                 562 
                 1.00E−125 
                 restriction 
                 
                   Neisseria 
                 
                 CAA09003.1 
                 nt. 2-985 of 
               
               
                   
                   
                 modification 
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 562 
               
               
                   
                   
                 system-R protein 
               
               
                 563 
                 0 
                 IMMUNOGLOBULIN 
                 
                   Haemophilus 
                 
                 P45384 
                 compliment of 
               
               
                   
                   
                 A1 PROTEASE 
                   influenzae  HK715 
                   
                 nt. 4127-9508 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 563 
               
               
                 563 
                 0 
                 3- 
                 
                   Schizosaccharomyces 
                 
                 O14289 
                 nt. 1980-3983 of 
               
               
                   
                   
                 ISOPROPYLMALATE 
                 
                   pombe 
                 
                   
                 SEQ ID NO: 563 
               
               
                   
                   
                 DEHYDRATASE 
               
               
                   
                   
                 (IPMI) 
               
               
                 564 
                 2.00E−79 
                 orf32 
                 
                   Haemophilus 
                 
                 NP_536839.1 
                 nt. 6241-6831 of 
               
               
                   
                   
                   
                 phage HP2 
                   
                 SEQ ID NO: 564 
               
               
                 564 
                 7.00E−33 
                 probable variable 
                 
                   Salmonella 
                 
                 NP_457882.1 
                 nt. 3707-4177 of 
               
               
                   
                   
                 tail fibre protein 
                   enterica  subsp. 
                   
                 SEQ ID NO: 564 
               
               
                   
                   
                   
                   enterica  serovar 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 564 
                 2.00E−14 
                 M protein 
                 
                   Enterobacteria 
                 
                 NP_052264.1 
                 nt. 1905-2213 of 
               
               
                   
                   
                   
                 phage 186 
                   
                 SEQ ID NO: 564 
               
               
                 564 
                 4.00E−44 
                 similar to tail fiber 
                 
                   Salmonella 
                 
                 NP_461635.1 
                 nt. 3171-3692 of 
               
               
                   
                   
                 protein (gpH) in 
                   typhimurium  LT2, 
                   
                 SEQ ID NO: 564 
               
               
                   
                   
                 phage P2 
                 Fels-2 prophage 
               
               
                 564 
                 2.00E−85 
                 gpJ 
                 
                   Enterobacteria 
                 
                 NP_046773.1 
                 nt. 2267-3166 of 
               
               
                   
                   
                   
                 phage P2 
                   
                 SEQ ID NO: 564 
               
               
                 564 
                 1.00E−24 
                 hypothetical protein 
                 
                   Neisseria 
                 
                 NP_284534.1 
                 nt. 6852-7334 of 
               
               
                   
                   
                   
                 
                   meningitidis 
                 
                   
                 SEQ ID NO: 564 
               
               
                   
                   
                   
                 Z2491 
               
               
                 564 
                 4.00E−26 
                 gpV 
                 
                   Enterobacteria 
                 
                 NP_046771.1 
                 nt. 1337-1912 of 
               
               
                   
                   
                   
                 phage P2 
                   
                 SEQ ID NO: 564 
               
               
                 564 
                 2.00E−47 
                 similar to 
                 
                   Escherichia coli 
                 
                 BAA16182.1 
                 nt. 11383-11961 
               
               
                   
                   
                 [SwissProt P44255 
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 564 
               
               
                 564 
                 2.00E−51 
                 hypothetical protein 
                 
                   Neisseria 
                 
                 NP_284066.1 
                 nt. 10452-11180 
               
               
                   
                   
                 NMA1315 
                 
                   meningitidis 
                 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                 Z2491 
                   
                 564 
               
               
                 564 
                 0 
                 orf31 
                 
                   Haemophilus 
                 
                 NP_043502.1 
                 nt. 4160-6226 of 
               
               
                   
                   
                   
                 phage HP1 
                   
                 SEQ ID NO: 564 
               
               
                 564 
                 2.00E−09 
                 rep 
                 
                   Haemophilus 
                 
                 NP_536816.1 
                 compliment of 
               
               
                   
                   
                   
                 phage HP2 
                   
                 nt. 9986-10234 
               
               
                   
                   
                   
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 564 
               
               
                 565 
                 2.00E−57 
                 resolvase/integrase- 
                 
                   Haemophilus 
                 
                 AAL47097.1 
                 nt. 11885-12445 
               
               
                   
                   
                 like protein 
                   influenzae  biotype 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                 
                   aegyptius 
                 
                   
                 565 
               
               
                 565 
                 1.00E−93 
                 integrase 
                 
                   Actinobacillus 
                 
                 AAC70901.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   actinomycetemcomitans 
                 
                   
                 nt. 4118-4900 
               
               
                   
                   
                   
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 565 
               
               
                 565 
                 6.00E−35 
                 probable phage 
                 
                   Salmonella 
                 
                 NP_458745.1 
                 compliment of 
               
               
                   
                   
                 integrase 
                   enterica  subsp. 
                   
                 nt. 4148-4990 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 565 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 565 
                 1.00E−107 
                 hypothetical protein 
                 
                   Xylella fastidiosa 
                 
                 NP_299042.1 
                 compliment of 
               
               
                   
                   
                   
                 9a5c 
                   
                 nt. 5066-6817 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 565 
               
               
                 566 
                 1.00E−126 
                 hypothetical protein 
                 
                   Haemophilus 
                 
                 S15287 
                 compliment of 
               
               
                   
                   
                 (galE 5′ region)- 
                 
                   influenzae 
                 
                   
                 nt. 10726-11607 
               
               
                   
                   
                   
                   
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 566 
               
               
                 567 
                 0 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246387.1 
                 nt.5343-7688 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 567 
               
               
                 568 
                 1.00E−151 
                 multidrug 
                 
                   Escherichia coli 
                 
                 NP_311575.1 
                 nt. 6-1403 of 
               
               
                   
                   
                 resistance 
                 O157:H7 
                   
                 SEQ ID NO: 568 
               
               
                   
                   
                 membrane 
               
               
                   
                   
                 translocase 
               
               
                 568 
                 1.00E−141 
                 YhbX/YhjW/YijP/YjdB 
                 
                   Neisseria 
                 
                 |NP_275002.1 
                 compliment of 
               
               
                   
                   
                 family protein 
                 
                   meningitidis 
                 
                   
                 nt. 11213-12634 
               
               
                   
                   
                   
                 MC58 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 568 
               
               
                 570 
                 1.00E−180 
                 hypothetical protein 
                 
                   Haemophilus 
                 
                 S71024 
                 compliment of 
               
               
                   
                   
                 3 (ksgA-lic2B 
                   influenzae  (strain 
                   
                 nt. 12845-13720 
               
               
                   
                   
                 intergenic region) 
                 RM7004) 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                   
                 570 
               
               
                 571 
                 0 
                 glycerophosphodiester 
                 
                   Haemophilus 
                 
                 A43576 
                 nt. 1656-2693 of 
               
               
                   
                   
                 phosphodiesterase 
                   influenzae  (isolate 
                   
                 SEQ ID NO: 571 
               
               
                   
                   
                   
                 772) 
               
               
                 571 
                 1.00E−137 
                 outer membrane 
                 
                   Haemophilus 
                 
                 A43604 
                 nt. 6145-6909 of 
               
               
                   
                   
                 protein P4 
                 
                   influenzae 
                 
                   
                 SEQ ID NO: 571 
               
               
                   
                   
                 precursor- 
               
               
                   
                   
                 
                   Haemophilus 
                 
               
               
                   
                   
                 
                   influenzae 
                 
               
               
                 571 
                 2.00E−72 
                 CG8298 gene 
                 
                   Drosophila 
                 
                 AAF58597.1 
                 nt. 3813-5339 of 
               
               
                   
                   
                 product [alt 1] 
                 
                   melanogaster 
                 
                   
                 SEQ ID NO: 571 
               
               
                 572 
                 1.00E−40 
                 hypothetical protein 
                 
                   Chlamydia 
                 
                 G81737 
                 nt. 3734-4099 of 
               
               
                   
                   
                 TC0130 
                   muridarum  (strain 
                   
                 SEQ ID NO: 572 
               
               
                   
                   
                   
                 Nigg) 
               
               
                 572 
                 5.00E−10 
                 hypothetical protein 
                 
                   Pyrococcus 
                 
                 NP_142215.1 
                 nt. 4472-4888 of 
               
               
                   
                   
                   
                 
                   horikoshii 
                 
                   
                 SEQ ID NO: 572 
               
               
                 572 
                 3.00E−11 
                 109aa long 
                 
                   Sulfolobus 
                 
                 NP_377117.1 
                 nt. 7303-7470 of 
               
               
                   
                   
                 hypothetical protein 
                 
                   tokodaii 
                 
                   
                 SEQ ID NO: 572 
               
               
                 572 
                 8.00E−43 
                 hypothetical protein 
                 
                   Chlamydophila 
                 
                 NP_445524.1 
                 nt. 4289-4618 of 
               
               
                   
                   
                   
                 
                   pneumoniae 
                 
                   
                 SEQ ID NO: 572 
               
               
                   
                   
                   
                 AR39 
               
               
                 572 
                 9.00E−08 
                 CDH1-D 
                 
                   Gallus gallus 
                 
                 AAL31950.1 
                 nt. 7183-7521 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 572 
               
               
                 575 
                 1.00E−173 
                 topoisomerase B 
                 
                   Salmonella 
                 
                 NP_458624.1 
                 nt. 18980-20923 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 575 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 575 
                 1.00E−100 
                 DNA helicase 
                 
                   Salmonella 
                 
                 NP_458617.1 
                 nt. 10399-11706 
               
               
                   
                   
                   
                   enterica  subsp. 
                   
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 575 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 65 
                 2.00E−53 
                 Sufl 
                 
                   Pasteurella 
                 
                 NP_245041.1 
                 nt. 3-821 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 65 
               
               
                 67 
                 4.00E−39 
                 putative MFS 
                 
                   Salmonella 
                 
                 NP_462786.1 
                 compliment of 
               
               
                   
                   
                 family tranport 
                   typhimurium  LT2 
                   
                 nt. 125-1033 of 
               
               
                   
                   
                 protein (1st mdule) 
                   
                   
                 SEQ ID NO: 67 
               
               
                 7 
                 4.00E−29 
                 putative membrane 
                 
                   Salmonella 
                 
                 NP_458664.1 
                 compliment of 
               
               
                   
                   
                 protein 
                   enterica  subsp. 
                   
                 nt. 2-559 of 
               
               
                   
                   
                   
                   enterica  serovar 
                   
                 SEQ ID NO: 7 
               
               
                   
                   
                   
                 
                   Typhi 
                 
               
               
                 72 
                 2.00E−51 
                 serine transporter 
                 
                   Vibrio cholerae 
                 
                 NP_230946.1 
                 nt. 18-803 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 72 
               
               
                 74 
                 3.00E−90 
                 hypothetical 21.8K 
                 
                   Haemophilus 
                 
                 JH0436 
                 compliment of 
               
               
                   
                   
                 protein (in locus 
                 
                   influenzae 
                 
                   
                 nt. 248-766 of 
               
               
                   
                   
                 involved in 
                   
                   
                 SEQ ID NO: 74 
               
               
                   
                   
                 transformation)- 
               
               
                 77 
                 2.00E−18 
                 RecX protein 
                 
                   Legionella 
                 
                 CAC33485.1 
                 nt. 480-920 of 
               
               
                   
                   
                   
                 
                   pneumophila 
                 
                   
                 SEQ ID NO: 77 
               
               
                 82 
                 4.00E−95 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246414.1 
                 nt. 128-955 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 82 
               
               
                 83 
                 2.00E−66 
                 unknown 
                 
                   Pasteurella 
                 
                 NP_246777.1 
                 nt. 5-556 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 83 
               
               
                 83 
                 6.00E−33 
                 CTP SYNTHASE 
                 
                   Helicobacter 
                 
                 NP_223042.1 
                 compliment of 
               
               
                   
                   
                   
                   pylori  J99 
                   
                 nt. 1027-1338 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 83. 
               
               
                 83 
                 4.00E−34 
                 CTP synthase 
                 
                   Campylobacter 
                 
                 NP_281249.1 
                 compliment of 
               
               
                   
                   
                   
                 
                   jejuni 
                 
                   
                 nt. 1024-1275 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 83 
               
               
                 84 
                 1.00E−16 
                 REPRESSOR 
                 Bacteriophage 
                 P14819 
                 nt. 823-1233 of 
               
               
                   
                   
                 PROTEIN CI 
                 phi-80 
                   
                 SEQ ID NO: 84 
               
               
                 84 
                 2.00E−05 
                 orf, hypothetical 
                 
                   Escherichia coli 
                 
                 NP_415875.1 
                 compliment of 
               
               
                   
                   
                 protein 
                 K12 
                   
                 nt. 533-700 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 84 
               
               
                 84 
                 4.00E−11 
                 orf33 
                 bacteriophage phi 
                 NP_490633.1 
                 compliment of 
               
               
                   
                   
                   
                 CTX 
                   
                 nt. 32-466 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 84 
               
               
                 85 
                 3.00E−42 
                 SpoT 
                 
                   Pasteurella 
                 
                 NP_245857.1 
                 nt. 899-1261 of 
               
               
                   
                   
                   
                 
                   multocida 
                 
                   
                 SEQ ID NO: 85 
               
               
                 90 
                 1.00E−103 
                 putative methylase 
                 Bacteriophage 
                 NP_108695.1 
                 compliment of 
               
               
                   
                   
                   
                 Tuc2009 
                   
                 nt. 478-1206 of 
               
               
                   
                   
                   
                   
                   
                 SEQ ID NO: 90 
               
               
                 90 
                 4.00E−11 
                 probable adenine 
                 
                   Thermoplasma 
                 
                 NP_394624.1 
                 compliment of 
               
               
                   
                   
                 specific DNA 
                 
                   acidophilum 
                 
                   
                 nt. 397-1140 of 
               
               
                   
                   
                 methyltransferase 
                   
                   
                 SEQ ID NO: 90 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE 3B 
               
               
                   
               
               
                   
                 Full Length 
                   
                   
                   
               
               
                   
                 Nucleotide 
                 Amino Acid 
                   
                 Homology to Genbank 
               
               
                 Hit Identity 
                 Sequence 
                 Sequence 
                 Location in Contig 
                 Protein 
               
               
                   
               
             
             
               
                 CpdB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 38041-36068 of 
                 NP_246953.1 
               
               
                   
                 686 
                 687 
                 SEQ ID NO: 681 
               
               
                   
                   
                   
                 (contig 14) 
               
               
                 putative membrane 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 906601-908094 
                 NP_458664.1 
               
               
                 protein 
                 688 
                 689 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 GTP-binding 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 42557-40995 of 
                 NP_240245.1 
               
               
                 protein TypA/BipA 
                 690 
                 691 
                 SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 outer membrane 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 7000420-704187 
                 T30852 
               
               
                 protein A 
                 692 
                 693 
                 of SEQ ID 
               
               
                   
                   
                   
                 NO: 685 (contig 18) 
               
               
                 vacB protein 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 39184-36836 of 
                 NP_240369.1 
               
               
                   
                 694 
                 695 
                 SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 putative ABC 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 59155-58370 of 
                 NP_282774.1 
               
               
                 transport system 
                 696 
                 697 
                 SEQ ID NO: 685 
               
               
                 permease protein [ 
                   
                   
                 (contig 18) 
               
               
                 putative exported 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 901142-902542 
                 NP_458655.1 
               
               
                 protein 
                 698 
                 699 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 ImpA 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 348187-347747 
                 NP_245829.1 
               
               
                   
                 700 
                 701 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 TsaA 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 74941-75548 of 
                 NP_245732.1 
               
               
                   
                 702 
                 703 
                 SEQ ID NO: 684 
               
               
                   
                   
                   
                 (contig 17) 
               
               
                 PROBABLE 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 74436-75176 of 
                 NP_522358.1 
               
               
                 TRANSPORT 
                 704 
                 705 
                 SEQ ID NO: 685 
               
               
                 TRANSMEMBRANE 
                   
                   
                 (contig 18) 
               
               
                 PROTEIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 75160-75660 of 
               
               
                   
                 706 
                 707 
                 SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 possible exported 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 899618-900262 
                 NP_458653.1 
               
               
                 protein 
                 708 
                 709 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 LICA PROTEIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 356917-355958 
                 P14181 
               
               
                   
                 710 
                 711 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 HEME-BINDING 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 NT. 26114-27739 
                 P33950 
               
               
                 PROTEIN A 
                 712 
                 713 
                 of SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 similar to 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 311610-312683 
                 XP_068727.1 
               
               
                 BASEMENT 
                 714 
                 715 
                 of SEQ ID NO: 685 
               
               
                 MEMBRANE- 
                   
                   
                 (contig 18) 
               
               
                 SPECIFIC 
               
               
                 HEPARAN 
               
               
                 SULFATE 
               
               
                 PROTEOGLYCAN 
               
               
                 CORE PROTEIN 
               
               
                 PRECURSOR 
               
               
                 (HSPG) 
               
               
                 CzcD 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 34865-35542 of 
                 NP_246276.1 
               
               
                   
                 716 
                 717 
                 SEQ ID NO: 681 
               
               
                   
                   
                   
                 (contig 14) 
               
               
                 conserved 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 194993-193977 
                 NP_274972.1 
               
               
                 hypothetical protein 
                 718 
                 719 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 secretion protein 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 203707-201857 
                 NP_252510.1 
               
               
                 SecD 
                 720 
                 721 
                 of SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 17) 
               
               
                 ABC transporter 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 3943-5859 of 
                 AAF31030.1 
               
               
                 protein 1 
                 722 
                 723 
                 SEQ ID NO: 681 
               
               
                   
                   
                   
                 (contig 14) 
               
               
                 conserved 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 331090-331749 
                 NP_273467.1 
               
               
                 hypothetical protein 
                 724 
                 725 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                   
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 331938-332492 
               
               
                   
                 726 
                 727 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                   
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 332681-33232 
               
               
                   
                 728 
                 729 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 INVASIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 416757-417020 
                 P31489 
               
               
                 PRECURSOR 
                 730 
                 731 
                 of SEQ ID NO: 685 
               
               
                 (OUTER 
                   
                   
                 (contig 18) 
               
               
                 MEMBRANE 
               
               
                 ADHESIN) 
               
               
                 HEME/HEMOPEXIN- 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 229430-232195 
                 P45355 
               
               
                 BINDING 
                 732 
                 733 
                 of SEQ ID NO: 384 
               
               
                 PROTEIN 
                   
                   
                 (contig 17) 
               
               
                 OPACITY 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 375592-375879 
                 Q05033 
               
               
                 PROTEIN OPA66 
                 734 
                 735 
                 of SEQ ID NO: 384 
               
               
                 PRECURSOR 
                   
                   
                 (contig 17) 
               
               
                 Hemoglobin- 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 45709-42566 of 
                 Q48153 
               
               
                 haptoglobin binding 
                 736 
                 737 
                 SEQ ID NO: 681 
               
               
                 protein A 
                   
                   
                 (contig 14) 
               
               
                 transport protein 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 134452-135222 
                 NP_253757.1 
               
               
                 TatC 
                 738 
                 739 
                 of SEQ ID NO: 384 
               
               
                   
                   
                   
                 (contig 17) 
               
               
                 LIPOPROTEIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 18895-20112 of 
                 P40827 
               
               
                 NLPD 
                 740 
                 741 
                 SEQ ID NO: 682 
               
               
                   
                   
                   
                 (contig 15) 
               
               
                 Hemoglobin and 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 34181-31041 of 
                 Q9X442 
               
               
                 hemoglobin- 
                 742 
                 743 
                 SEQ ID NO: 682 
               
               
                 haptoglobin binding 
                   
                   
                 (contig 15) 
               
               
                 protein C precursor 
               
               
                 HimA 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 382795-383085 
                 NP_245565.1 
               
               
                   
                 744 
                 745 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 transferrin-binding 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 178537-175799 
                 S70906 
               
               
                 protein 1 
                 746 
                 747 
                 of SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 SapC 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 197754-196867 
                 NP_245850.1 
               
               
                   
                 748 
                 749 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 heat shock protein 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 40414-41265 of 
                 NP_273864.1 
               
               
                 HtpX 
                 750 
                 751 
                 SEQ ID NO: 682 
               
               
                   
                   
                   
                 (contig 15) 
               
               
                 HEME/HEMOPEXIN- 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 229430-232195 
                 P45354 
               
               
                 BINDING 
                 752 
                 753 
                 of SEQ ID NO: 684 
               
               
                 PROTEIN 
                   
                   
                 (contig 17) 
               
               
                 HEME/HEMOPEXIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 227721-229418 
                 P45356 
               
               
                 UTILIZATION 
                 754 
                 755 
                 of SEQ ID NO: 684 
               
               
                 PROTEIN B 
                   
                   
                 (contig 17) 
               
               
                 HEME/HEMOPEXIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 225516-227645 
                 P45357 
               
               
                 UTILIZATION 
                 756 
                 757 
                 of SEQ ID NO: 684 
                 NP_246561.1 
               
               
                 PROTEIN C 
                   
                   
                 (contig 17) 
               
               
                 iron utilization 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 32076-33611 of 
                 T10887 
               
               
                 protein B 
                 758 
                 759 
                 SEQ ID NO: 684 
               
               
                   
                   
                   
                 (contig 17) 
               
               
                 PREPROTEIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 82314-84785 of 
                 P96313 
               
               
                 TRANSLOCASE 
                 760 
                 761 
                 SEQ ID NO: 683 
               
               
                 SECA SUBUNIT 
                   
                   
                 (contig 16) 
               
               
                 IMMUNOGLOBULIN 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 171647-166263 
                 P45384 
               
               
                 A1 PROTEASE 
                 762 
                 763 
                 of SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 multidrug 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 74524-72992 of 
                 NP_311575.1 
               
               
                 resistance 
                 764 
                 765 
                 SEQ ID NO: 683 
               
               
                 membrane 
                   
                   
                 (contig 16) 
               
               
                 translocase 
               
               
                 YhbX/YhjW/YijP/YjdB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 61734-63200 of 
                 NP_275002.1 
               
               
                 family protein 
                 766 
                 767 
                 SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig 16) 
               
               
                 putative membrane 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 906601-908094 
                 NP_458664.1 
               
               
                 protein 
                 768 
                 769 
                 of SEQ ID NO: 685 
               
               
                   
                   
                   
                 (contig 18) 
               
               
                 putative membrane 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 16185-17942 of 
                 NP_404859.1 
               
               
                 protein 
                 770 
                 771 
                 SEQ ID NO: 683 
               
               
                   
                   
                   
                 (contig) 
               
               
                   
               
             
          
         
       
     
       Example 3 
     Construction of the NTHi Promoter Trap Library 
       [0092]    To identify potential virulence determinants of NTHi, bacterial gene expression was monitored by differential fluorescence induction (DFI) during early disease progression in one specific anatomical niche of a chinchilla model of NTHi-induced otitis media (OM). Genomic DNA fragments from NTHi strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene using a promoter trap library. Plasmid pGZRS39A, a derivative of pGZRS-1 isolated from  Actinobacillus pleuropneumoniae , is an  A. pleuropneumoniae - Escherichia coli  shuttle vector. This plasmid contains the origin of replication from  A. pleuropneumoniae , the lacZa gene from pUC19 and the kanamycin resistance gene from Tn903. (West et al.,  Genes,  160: 81-86, 1995). 
         [0093]    The promoter trap vector was constructed by cloning the GTP mutant gfpmut3 gene, as a BamHI to EcoRI fragment into pGZRS-39A to form pRSM2167. This mutant GTP gene contains two amino acid changes, S65G and S72A, that enhance fluorescence emission when excited at 488 nm. This mutant also has high solubility and fast kinetics of chromophore formation (Cormack et al.,  Gene,  173: 33-38, 1996). This plasmid was transformed by electroporation into NTHi strain 86-028NP, generating the parent-plasmid strain 86-028NP/pRSM2169. 
         [0094]    Random genomic DNA fragments (described in Example 1) were prepared for ligation into the promoter probe vector. Genomic DNA was isolated from strain 86-028NP using the Puregene DNA isolation kit (Gentra Systems, Minneapolis. MN) according to the manufacturer&#39;s protocol. Due to restriction barriers, it was necessary to isolate the plasmid DNA and use this for the library generation. The isolated DNA was partially digested with Sau3AI (NEB, Beverly, MA; 0.25 units/μg DNA) for 1 hour at 37° C., separated by gel electrophoresis and DNA fragments 0.5-1.5 kb in size were recovered using the Qiagen gel extraction kit. For vector preparation, pRSM2167 was isolated from an overnight culture using the Wizard Plus Maxiprep DNA purification system (Promega, Madison Wis.) according to the manufacturer&#39;s protocol. 
         [0095]    Plasmid DNA was linearized by BamHI digestion and 5′ phosphate groups removed by treatment with calf intestinal alkaline phosphatase (CIAP; GibcoBRL Life Technologies). Genomic DNA fragments were ligated with the linearized, phosphatase-treated vector and electroporated into competent NTHi strain 86-028NP prepared for electroporation according to a modified protocol (Mitchell et al.,  Nucleic Acids Res.,  19: 3625-3628,1991). When plasmid DNA was electroporated back into NTHi strain 86-028NP, transformation efficiency was improved by one-thousand fold. Briefly, cells were grown to an OD 600 =0.3 in sBHI (brain heart infusion) broth at 37° C., 220 rpm. Cells were chilled on ice for 30 minutes and subsequently washed with an equal volume of 0.5×SG (1×SG: 15% glycerol, 272 mM sucrose) at 4° C. Washes were repeated a total of three times. Subsequently, the cells were diluted in 1×SG to a 100× concentrated volume. The cells were electroporated using the BioRad Gene Pulser II set at 200 ohms, 2.5 kV and 25 μF and then diluted in 1 ml prewarmed sBHI, incubated for 2 hours at 37° C., 5% CO 2  and plated on chocolate agar for overnight growth of transformants. 
         [0096]    Transformants were selected and frozen in pools of 1000 clones in skim milk containing 20% glycerol (vol/vol). A 68,000 member gfp promoter probe library was (generated. Using the probability calculation of Clarke and Carbon (Cell, 9: 91-99, 1976), to achieve a 99% probability of having a given DNA sequence represented in a library of 300 bp fragments of strain 86-028NP DNA (1.8×10 6  bp/genome), a library of 27,629 clones was needed. Therefore the present library represents 2.5 fold coverage of the 86-028NP genome. 
         [0097]    In order to assess the quality of the library, fifty clones were selected at random, grown overnight on chocolate agar and the plasmids were isolated and insert DNA sequenced. A majority (64%) of the selected clones had insert sizes ranging between 200 and 500 bp while 32% exceeded 500 bp. The majority of inserts showed homology to unique  H. influenzae  strain Rd open reading frames (ORFs), and 15 clones had sequence unique to strain 86-028NP DNA. Of those clones with homology to strain Rd, 60% were in the correct orientation, 36% of which contained sequence upstream an ORF. Although a majority of clones had an insert size less than 500 bp, no correlation was found between small insert size and increased GFP expression. In fact four clones exhibited slight to moderate fluorescence in vitro, 3 of which had insert sizes between 200-500 base pairs and one had an insert that was greater than 700 base pairs. 
         [0098]    A fraction of the library (approximately 1000 clones) was grown on chocolate agar, harvested in PBS and analyzed by flow cytometry for GFP fluorescence. Compared to strain 86-028NP/pRSM2169 that contains the promoter trap vector without insert DNA, the pool of library clones displays an increased fluorescence intensity. Thus, the library contains clones with promoters at varying levels of activity. 
       Example 4 
     Analysis of 86-028NP Derivatives Expressing GFP 
       [0099]    In order to establish the FACS parameters necessary to identify and sort gfp-expressing bacteria, a panel of isolates demonstrating varying levels of gfp expression was utilized. Background fluorescence was assessed using strain 86-028NP/pRSM2169 (negative control), therefore any observed fluorescence would be due to the lacZ promoter driving gfp expression. However, this strain does not produce detectable levels of GFP and in fact, does not demonstrate increased fluorescence when compared to the parent strain 86-028NP. A high-level gfp-expressing isolate was generated by cloning a 500 bp fragment containing the strong promoter for outer membrane protein P2 expression into SalI-BamHI digested pRSM2167. This plasmid was transformed into 86-028NP by electroporation, generating the high-level gfp expressing strain 86-028NP/pRSM2211 (highly fluorescent control). This strain demonstrated an approximate 100 fold increase in GFP fluorescence compared to strain 86-028NP/pRSM2169. An intermediate fluorescent derivative clone. 86-028NP/pKMM4B5 (intermediate fluorescent control), was isolated by FACS analysis and used both in preliminary experiments and as a control for cell sorting. The DNA fragment containing a promoter driving gfp expression in vitro is unique to strain 86-028NP, having no known homology to DNA of other organisms. This clone exhibits an approximate 10 fold increase in fluorescence compared to strain 86-028NP/pRSM2169. 
         [0100]    The control strains were resuspended from growth on chocolate agar and labeled with cross-reactive Phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, bacteria were resuspended in 300 μl DPBS for FACS analysis. These control preparations were used to set the appropriate size and fluorescence gates using a Coulter Epics Elite flow cytometer (Coulter Corp.) equipped with an argon laser emitting at 488 nm. Bacteria were gated for size based on log forward angle and side scatter detection and for sorting by FITC/PE labeling of bacteria. Sorted cells were collected into cold sBHI and plated on chocolate agar. After overnight growth, cells were collected for a secondary round of infection or were individually selected and grown overnight, screened by individual clone for fluorescence when grown in vitro, and frozen in skim milk containing 20% (vol/vol) glycerol prior to plasmid isolation and sequencing of insert DNA. Sorting efficiency of control strains was confirmed using a Coulter EPICS flow cytometer (Coulter Corp.). 
         [0101]    Many plasmids were segregated rapidly in vitro in the absence of antibiotic selection. Thus, in order to assess whether the promoter trap vector used here was prone to this event, a single colony of strain 86-028NP/pRSM2211 (highly fluorescent control) was isolated on chocolate agar and passaged 20 times in the absence of antibiotic selection. No significant decrease in fluorescence intensity was observed when compared to bacteria grown in the presence of antibiotic. In addition, the plasmid is maintained in the absence of antibiotic selection in vivo. Similar bacterial counts were observed when bacteria-containing middle ear fluids collected from a chinchilla were plated on chocolate agar with or without kanamycin. These data demonstrate that the promoter trap vector was stably maintained in the absence of antibiotic selection. 
         [0102]    In addition to problems with plasmid stability, early studies on the use of GFP as a reporter to study host-pathogen interactions demonstrated that GFP could be continuously synthesized as a cytoplasmic protein with low toxicity, having minimal effects on the bacterial cell-surface dynamics (Chalfie et al.,  Science,  263: 802-805, 1994). The construction of a high level gfp-expressing derivative allowed the assessment of the GFP toxicity on NTHi. Growth curves of both the wild-type strain (86-028NP) and the high GFP producing strain 86-028NP/pRSM2211 were compared when grown under similar conditions. The growth rates were similar, indicating that GFP expression was not toxic to the cells. 
         [0103]    The 86-028NP gfp-expressing derivatives were used to define the parameters for efficient cell sorting. Strain 86-028NP/pRSM2169 was mixed with the intermediate gfp-expressing derivative, strain 86-028NP/pKMM4B5, at a 100:1 ratio, simulating the in vivo environment that is expected to contain a small percentage of gfp-expressing clones relative to the total bacterial population. This mixture was subjected to FACS analysis, collecting the 1.8% most fluorescent population and the 52% least fluorescent population. Flow cytometric analysis of the sorted populations revealed an enrichment of strain 86-028NP/pKMM4B5 to 65% of the bacterial population, a phenomenon that was not observed when sorting on the negative population. Subsequent rounds of sorting would be expected to further enrich for this intermediate fluorescent population. The inability to decrease the amount of fluorescent bacteria in the negative sort was attributed to the size of the gate set for negative sorting. GFP-negative cells were enriched by gating on the 10% least fluorescent population. 
       Example 5 
     Direct Labeling of Bacteria from Middle Ear Fluids 
       [0104]    A similar strategy (as described in Example 5) was applied to sort fluorescent clones from effusions obtained from the chinchilla middle ear during AOM. Our ability to use differential fluorescence induction (DFI) in vivo was dependent upon our ability to sort gfp-expressing bacteria from non-fluorescent bacteria, fluorescent and non-fluorescent cellular debris, and eukaryotic cells. 
         [0105]    Healthy adult chinchillas ( Chinchilla lanigera ) with no evidence of middle ear infection by either otoscopy or tympanometry were used to screen the library for promoter activity in vivo. Two pools of the NTHi/pRSM2169 library (1000 clones each) were grown overnight on chocolate agar containing kanamycin. The library was combined and diluted in cold 10 mM sterile PBS to 3.3×10 6  CFU/ml and 300 μl (1.0×10 6  CFU; 500 CFU/clone) was used to inoculate the left and the right chinchilla transbullar cavity (2000 clones/ear). OM development was monitored by video otoscopy and tympanometry at 24 and 48 hours. The bacteria multiplied in the middle ear cavity, reaching a concentration 500 times the inoculum dose by 48 hours as expected (Bakaletz et al.,  Infect. Immunity  67: 2746-62, 1999). This bacterial adaptation to the host environment results in an inflammatory response, indicated by erythema, vessel dilation and bulging of the tympanic membrane, infiltration of polymorphonuclear cells (PMN&#39;s), and accumulation of fluid in the middle ear cavity as observed by otoscopy and microscopic examination of recovered effusions. Twenty-four and 48 hours later, middle ear fluids were retrieved by epitympanic tap, and prepared for FACS. 
         [0106]    It is important to note that this analysis was limited to those bacteria recoverable in the middle ear fluid. In some cases it was necessary to lavage the middle ear cavity to collect the bacteria for FACS analysis. Thus, this analysis includes genes up-regulated when NTHi are loosely adherent to mucosae. NTHi has been observed to form a biofilm in the middle ear cavity in a chinchilla model of OM (Erhlich et al.,  JAMA,  287: 1710-5, 2002). Since the protocols described herein select for clones recovered from the planktonic population, it is not expected to recover those clones in which genes are up-regulated when the bacteria are associated with mucosal biofilms. Homogenization of middle ear mucosae and subsequent bacterial cell isolation however, would enable us to recover these clones. It is also possible that some GFP-expressing clones were recovered in the effusion, yet were adherent to eukaryotic cells present in the effusion as exfoliated cells, or in aggregates. These bacteria are difficult to recover from the effusion without compromising the sorting efficiency. Therefore the middle ear fluids were treated with a mucolytic agent, then centrifuged to remove large aggregates and eukaryotic cells and prior to labeling. 
         [0107]    Chinchilla middle ear fluids were diluted, if necessary, to 250 μl with sterile saline. An equal volume of N-acetyl-L-cysteine (0.5%; w/v) in DPBS (pH 7.4) was added for 5 minutes at room temperature as a mucolytic agent (Miyamoto and Bakaletz,  Microb. Pathog.,  21: 343-356 1996). Fluids were centrifuged (300×g, 5 min) to remove cellular debris, red blood cells and inflammatory cells, and supernatants containing bacteria were transferred to a fresh tube. Bacteria were incubated with chinchilla antiserum (1:50 dilution) directed against a whole OMP preparation, derived from NTHi strain 86-028NP, for 45 minutes at 4° C., pelleted by centrifugation (2000×g, 5 min) and washed twice with cold DPBS containing 0.05% bovine serum albumin. Bacteria were subsequently labeled with cross-reactive phycoprobe R-PE anti-human IgG (H+L) antibody (10 μg/ml in 100 μl PBS; Biomeda Corp) for 30 minutes at 4° C. Following three successive washes to remove unbound antibody, cells were resuspended in 300 μl DPBS for FACS analysis. 
       Example 6 
     Identification of Promoters Induced In Vivo in Acute Otitis Media 
       [0108]      H. influenzae  86-028NP transformed with the promoter trap library was grown overnight on chocolate agar. To select against those clones containing promoters that expressed gfp in vitro, the library was subjected to one round of FACS analysis (as described in Example 6), collecting only those clones expressing low-level amounts of GFP. These clones were pooled and used to inoculate the chinchilla middle ear transbullarly. Following 24 and 48 hours of infection, bacteria-containing effusions were removed by epitympanic tap. Bacteria were indirectly labeled with R-PE-labeled antibody and subjected to FACS analysis by gating on fluorescently tagged bacteria but sorting for those that were also expressing. These clones were used to reinfect animals for further enrichment. Following the final round of sorting, single colony isolates were screened in vitro for lack of fluorescence. 
         [0109]    Those clones isolated by FACS analysis (positive for GFP fluorescence in vivo), which did not emit fluorescence in vitro were prepared for plasmid isolation and identification of insert DNA sequence. These clones were grown overnight on chocolate agar plates containing kanamycin and prepared for plasmid isolation using the Qiaprep Miniprep Kit (Qiagen) according to the manufacturer&#39;s protocol. Plasmid insert DNA was sequenced using the primer 5′-TGCCCATTAACATCACCATCTA-3′ (SEQ ID NO: 588) that is complementary to the gfpmut3 gene and downstream of the insert DNA. Sequencing reactions were performed using the ABI prism BigDye® terminator cycle sequencing ready reaction kit (Applied Biosystems) according to manufacturer&#39;s protocol using a GeneAmp PCR System 9700 (Applied Biosystems). The sequences were then purified by passage through sephadex G-50 in a 96-well multiscreen HV plate (Millipore) and subsequently analyzed on an ABI Prism 3100 DNA analyzer (Applied Biosystems). 
         [0110]    Insert sequences were compared to the complete annotated sequence of  H. influenzae  strain Rd Those inserts with no nucleotide homology to strain Rd were subsequently analyzed using the BLASTN and BLASTX algorithms. Further sequence analysis was performed with DNASTAR (Madison, Wis.). Inserts in the correct orientation and containing sequence 5′ to a predicted ORF contained a putative promoter that was preferentially active when the NTHi bacteria were in the chinchilla middle ear. 
         [0111]    Fifty-two clones with putative promoters that were regulated in vivo were isolated. Of the 44 candidate clones containing sequence similar to that identified in  H. influenzae  strain Rd, quantitative comparison of gene expression in vitro and in vivo conformed up-regulated gene expression for twenty-six genes (60%) when NTHi respond to environmental cues present in the chinchilla middle ear and these genes are summarized in Table 4A below. The in vivo-regulated promoters driving expression of genes are predicted to be involved in membrane transport, environmental informational processing, cellular metabolism, gene regulation, as well as hypothetical proteins with unknown function. 
         [0112]    In order to confirm the induction of putative promoter candidates in vivo, the relative amount of messenger RNA expression was compared when NTHi strain 86-028NP was grown in vitro to mid-log phase or in vivo for 48 hours. The RNA was isolated using TRIzol LS reagent (Gibco Life Technologies) according to the manufacturer&#39;s protocol. DNA was removed from the RNA preparation using DNA-free kit (Ambion) according to the manufacturer&#39;s protocol. DNase I treated RNA samples were purified by passage through a Qiagen RNeasy column. RNA purity and integrity was assessed by 260/280 nm spectrophotometer readings and on the Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. 
         [0113]    In order to independently confirm the FACS data, we determined the relative expression of candidate genes by quantitative RT-PCR. The parent strain 86-028NP, was used for these studies. Real-time quantitative RT-PCR using the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) assessed transcription levels according to the manufacture&#39;s instructions. Briefly, using primers generated to an open reading frame downstream of the putative in vivo-induced promoters identified by FACS analysis, gene-specific mRNA was reverse transcribed and amplified by RT-PCR on the ABI Prism 7700 sequence detection system (Applied Biosystems). The amount of product was calculated using a standard curve generated to known amounts of bacterial genomic DNA (10 2 -10 7  genomic copies DNA) by amplifying a fragment of the gyrase (gyr) gene. Controls were analyzed in parallel to verify the absence of DNA in the RNA preparation (-RT control) as well as the absence of primer dimers in control samples lacking template RNA. In addition, RT-PCR products were analyzed by gel electrophoresis and, in all cases, a single product was observed at the appropriate base pair size. Amounts of bacterial RNA between samples were normalized relative to gyr expression, shown to be constitutively expressed under various growth conditions that we tested in vitro. Known amounts of bacterial genomic DNA (10 2 -10 7  genomic copies DNA) were used to generate a standard curve for RT-PCR quantitation by amplifying a fragment of the gyrase (gyr) gene. Gyrase is constitutively expressed in vitro under various growth conditions and was therefore used to normalize total bacterial RNA levels between samples. Relative gene expression in vivo was compared to that of gene expression in vitro and data expressed as fold-increase are summarized in Table 4A. 
         [0114]    The 8-fold sequencing of the genome identified the full length open reading frames for the majority of genes listed in Table 4A. Table 4B provides the full length nucleotide sequence within the NTHi genome and the corresponding amino acid sequence. The fold induction of the gene due to environmental cues present in the chinchilla middle ear and the product or function of the gene are repeated in Table 4B for convenience. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4A 
               
               
                   
               
               
                   
                   
                 SEQ 
                   
                   
                   
               
               
                   
                 Gene or 
                 ID 
                 GenBank 
                 Fold 
               
               
                 Category 
                 ORF 
                 NO: 
                 Protein ID 
                 Induction 
                 Product or Function 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Amino acid 
                 hisB 
                 589 
                 NP_438632 
                 2.9 
                 Histidine biosynthesis 
               
               
                 metabolism 
                   
                   
                   
                   
                 bifunctional protein 
               
               
                 Lipoprotein 
                 lppB 
                 590 
                 NP_438862.1 
                 2.6 
                 Lipoprotein B homologue 
               
               
                 Membrane transport 
                 sapA 
                 591 
                 NP_439780.1 
                 2.8 
                 Peptide ABC transporter; 
               
               
                   
                   
                   
                   
                   
                 periplasmic SapA precursor 
               
               
                   
                 lolA 
                 592 
                 NP_439736.1 
                 2.4 
                 Outer membrane lipoproteins 
               
               
                   
                   
                   
                   
                   
                 carrier protein precursor 
               
               
                   
                 rbsC 
                 593 
                 NP_438661.1 
                 5.1 
                 Ribose transport system 
               
               
                   
                   
                   
                   
                   
                 permease protein 
               
               
                 Purine synthesis 
                 purE 
                 594 
                 NP_439757.1 
                 51.7 
                 Phosphoribosylaminoimidazole 
               
               
                   
                   
                   
                   
                   
                 carboxylase catalytic 
               
               
                   
                   
                   
                   
                   
                 subunit; PurE 
               
               
                 Biosynthetic and 
                 ribB 
                 595 
                 NP_438923.1 
                 8.3 
                 3,4-dihydroxy-2-butanone 4- 
               
               
                 metabolic functions 
                   
                   
                   
                   
                 phosphate synthase; 
               
               
                   
                   
                   
                   
                   
                 riboflavin biosynthesis 
               
               
                   
                 arcB 
                 596 
                 NP_438753.1 
                 10 
                 Ornithine 
               
               
                   
                   
                   
                   
                   
                 carbamolytransferase; 
               
               
                   
                   
                   
                   
                   
                 arginine degradation 
               
               
                   
                 uxuA 
                 597 
                 NP_438228.1 
                 3.1 
                 Mannonate dehydratase; 
               
               
                   
                   
                   
                   
                   
                 production of glyceraldehyde 
               
               
                   
                   
                   
                   
                   
                 3-phosphate 
               
               
                   
                 dsbB 
                 598 
                 NP_438589.1 
                 2.6 
                 Disulfide oxidoreductase; 
               
               
                   
                   
                   
                   
                   
                 disulfide bond formation 
               
               
                   
                   
                   
                   
                   
                 protein B 
               
               
                   
                 ureH 
                 599 
                 NP_438693.1 
                 3.9 
                 Urease accessory protein 
               
               
                   
                 licC 
                 600 
                 NP_439688.1 
                 2.3 
                 Phosphocholine (ChoP) 
               
               
                   
                   
                   
                   
                   
                 cytidylyltransferase 
               
               
                   
                 HI1647 
                 601 
                 NP_439789.1 
                 2.0 
                 Putative pyridoxin 
               
               
                   
                   
                   
                   
                   
                 biosynthesis protein; singlet 
               
               
                   
                   
                   
                   
                   
                 oxygen resistance protein 
               
               
                 DNA replication, 
                 ispZ 
                 602 
                 P43810 
                 2.5 
                 Probable intracellular 
               
               
                 repair 
                   
                   
                   
                   
                 septation protein 
               
               
                   
                 radC 
                 603 
                 NP_439113.1 
                 2.1 
                 DNA repair protein 
               
               
                   
                 mukF 
                 604 
                 P45185 
                 2.0 
                 MukF protein homologue; 
               
               
                   
                   
                   
                   
                   
                 remodeling of nucleiod 
               
               
                   
                   
                   
                   
                   
                 structure 
               
               
                 Gene regulation 
                 glpR 
                 605 
                 NP_438777.1, 
                 2.8 
                 Glycerol-3-phosphate regulon 
               
               
                   
                   
                   
                 NP_439170.1 
                   
                 repressor 
               
               
                   
                 ihfB 
                 606 
                 P43724 
                 2.5 
                 Integration host factor beta 
               
               
                   
                   
                   
                   
                   
                 subunit 
               
               
                   
                 argR 
                 607 
                 NP_439365.1 
                 2.7 
                 Arginine repressor 
               
               
                   
                 cspD 
                 608 
                 NP_439584.1 
                 2.1 
                 Cold shock like protein; 
               
               
                   
                   
                   
                   
                   
                 stress response protein 
               
               
                 Hypothetical or 
                 HI0094 
                 609 
                 NP_438267.1 
                 8.3 
                 Hypothetical protein 
               
               
                 unknown 
                 HI1163 
                 610 
                 NP_439321.1 
                 2.3 
                 Conserved hypothetical 
               
               
                 proteins 
                   
                   
                   
                   
                 protein; putative oxidase 
               
               
                   
                 HI1063 
                 611 
                 NP_439221.1 
                 2.7 
                 Hypothetical protein 
               
               
                   
                 HI0665 
                 612 
                 NP_438824.1 
                 2.8 
                 Hypothetical protein 
               
               
                   
                 HI1292 
                 613 
                 NP_439444.1 
                 2.6 
                 Hypothetical protein 
               
               
                   
                 HI1064 
                 614 
                 NP_439222.1 
                 2.6 
                 Hypothetical protein 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4B 
               
               
                   
               
               
                   
                   
                 Full 
                   
                   
                   
                   
               
               
                   
                 Gene 
                 Length 
               
               
                   
                 or 
                 Nucleotide 
                 Amino Acid 
                   
                 Fold 
                 Product or 
               
               
                 Category 
                 ORF 
                 Sequence 
                 Sequence 
                 Location in Contig 
                 Induction 
                 Function 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Amino 
                 hisB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 68378-67290 
                 2.9 
                 Histidine 
               
               
                 acid 
                   
                 615 
                 616 
                 of SEQ ID NO: 
                   
                 biosynthesis 
               
               
                 metabolism 
                   
                   
                   
                 680 (contig 13) 
                   
                 bifunctional protein 
               
               
                 Membrane 
                 sapA 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 200403-198709 
                 2.8 
                 Peptide ABC 
               
               
                 transport 
                   
                 617 
                 618 
                 of SEQ 
                   
                 transporter; 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 periplasmic SapA 
               
               
                   
                   
                   
                   
                 (contig 18) 
                   
                 precursor 
               
               
                   
                 rbsC 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 42773-41802 
                 5.1 
                 Ribose transport 
               
               
                   
                   
                 619 
                 620 
                 of SEQ ID NO: 
                   
                 system permease 
               
               
                   
                   
                   
                   
                 680 (contig 13) 
                   
                 protein 
               
               
                 Purine 
                 purE 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 219625-219131 
                 51.7 
                 Phosphoribosylaminoimidazole 
               
               
                 synthesis 
                   
                 621 
                 622 
                 of SEQ 
                   
                 carboxylase catalytic 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 subunit; PurE 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                 Biosynthetic 
                 ribB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 131537-132184 
                 8.3 
                 3,4-dihydroxy-2- 
               
               
                 and 
                   
                 623 
                 624 
                 of SEQ 
                   
                 butanone 4- 
               
               
                 metabolic 
                   
                   
                   
                 ID NO: 682 
                   
                 phosphate synthase; 
               
               
                 functions 
                   
                   
                   
                 (contig 15) 
                   
                 riboflavin 
               
               
                   
                   
                   
                   
                   
                   
                 biosynthesis 
               
               
                   
                 arcB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 49710-48706 
                 10 
                 Ornithine 
               
               
                   
                   
                 625 
                 626 
                 of SEQ ID NO: 
                   
                 carbamolytransferase; 
               
               
                   
                   
                   
                   
                 681 (contig 14) 
                   
                 arginine 
               
               
                   
                   
                   
                   
                   
                   
                 degradation 
               
               
                   
                 uxuA 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 840671-841855 
                 3.1 
                 Mannonate 
               
               
                   
                   
                 627 
                 628 
                 of SEQ 
                   
                 dehydratase; 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 production of 
               
               
                   
                   
                   
                   
                 (contig 18) 
                   
                 glyceraldehyde 3- 
               
               
                   
                   
                   
                   
                   
                   
                 phosphate 
               
               
                   
                 dsbB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 388050-388583 
                 2.6 
                 Disulfide 
               
               
                   
                   
                 629 
                 630 
                 of SEQ 
                   
                 oxidoreductase; 
               
               
                   
                   
                   
                   
                 ID NO: 384 
                   
                 disulfide bond 
               
               
                   
                   
                   
                   
                 (contig 17) 
                   
                 formation protein B 
               
               
                   
                 ureH 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 4452-5267 of 
                 3.9 
                 Urease accessory 
               
               
                   
                   
                 631 
                 632 
                 SEQ ID NO: 680 
                   
                 protein 
               
               
                   
                   
                   
                   
                 (contig 13) 
               
               
                   
                 licC 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 355083-354382 
                 2.3 
                 Phosphocholine 
               
               
                   
                   
                 633 
                 634 
                 of SEQ 
                   
                 (ChoP) 
               
               
                   
                   
                   
                   
                 ID NO: 385 
                   
                 cytidylyltransferase 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                   
                 HI1647 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 664017-664892 
                 2.0 
                 Putative pyridoxin 
               
               
                   
                   
                 635 
                 636 
                 of SEQ 
                   
                 biosynthesis protein; 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 singlet oxygen 
               
               
                   
                   
                   
                   
                 (contig 18) 
                   
                 resistance protein 
               
               
                 DNA 
                 ispZ 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 4512-5069 of 
                 2.5 
                 Probable 
               
               
                 replication, 
                   
                 637 
                 638 
                 SEQ ID NO: 683 
                   
                 intracellular 
               
               
                 repair 
                   
                   
                   
                 (contig 16) 
                   
                 septation protein 
               
               
                   
                 radC 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 132695-132030 
                 2.1 
                 DNA repair protein 
               
               
                   
                   
                 639 
                 640 
                 of SEQ 
               
               
                   
                   
                   
                   
                 ID NO: 683 
               
               
                   
                   
                   
                   
                 (contig 16) 
               
               
                   
                 mukF 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 504549-503215 
                 2.0 
                 MukF protein 
               
               
                   
                   
                 641 
                 642 
                 of SEQ 
                   
                 homologue; 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 remodeling of 
               
               
                   
                   
                   
                   
                 (contig 18) 
                   
                 nucleiod structure 
               
               
                 Gene 
                 glpR 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 72716-73483 
                 2.8 
                 Glycerol-3- 
               
               
                 regulation 
                   
                 643 
                 644 
                 of SEQ ID NO: 
                   
                 phosphate regulon 
               
               
                   
                   
                   
                   
                 682 (contig 15) 
                   
                 repressor 
               
               
                   
                 ihfB 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 661004-660720 
                 2.5 
                 Integration host 
               
               
                   
                   
                 645 
                 646 
                 of SEQ 
                   
                 factor beta subunit 
               
               
                   
                   
                   
                   
                 ID NO: 685 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                   
                 argR 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 178540-178085 
                 2.7 
                 Arginine repressor 
               
               
                   
                   
                 647 
                 648 
                 of SEQ 
               
               
                   
                   
                   
                   
                 ID NO: 685 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                   
                 cspD 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 435310-435528 
                 2.1 
                 Cold shock like 
               
               
                   
                   
                 649 
                 650 
                 of SEQ 
                   
                 protein; stress 
               
               
                   
                   
                   
                   
                 ID NO: 685 
                   
                 response protein 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                 Hypothetical 
                 HI1163 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 137202-134119 
                 2.3 
                 Conserved 
               
               
                 or 
                   
                 651 
                 652 
                 of SEQ 
                   
                 hypothetical protein; 
               
               
                 unknown 
                   
                   
                   
                 ID NO: 685 
                   
                 putative oxidase 
               
               
                 proteins 
                   
                   
                   
                 (contig 18) 
               
               
                   
                 HI1063 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 35158-34937 
                 2.7 
                 Hypothetical protein 
               
               
                   
                   
                 653 
                 654 
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                 685 (contig 18) 
               
               
                   
                 HI0665 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 17949-18980 
                 2.8 
                 Hypothetical protein 
               
               
                   
                   
                 655 
                 656 
                 of SEQ ID NO: 
               
               
                   
                   
                   
                   
                 679 (contig 12) 
               
               
                   
                 HI1292 
                 SEQ ID NO: 
                 SEQ ID NO: 
                 nt. 555002-555799 
                 2.6 
                 Hypothetical protein 
               
               
                   
                   
                 657 
                 658 
                 of SEQ 
               
               
                   
                   
                   
                   
                 ID NO: 685 
               
               
                   
                   
                   
                   
                 (contig 18) 
               
               
                   
               
             
          
         
       
     
       Example 7 
     Identification of Virulence-Associated Genes 
       [0115]    In many bacterial species, a subset of virulence-associated genes is regulated by errors in replication of short repeats. These repeats may be 5′ to a gene or in the coding sequence, and their presence is an indication of controlled expression of the gene, which indicates association with virulence. Addition or deletion of a repeat results in the expression or of lack of expression of the particular virulence determinant. 
         [0116]    The NTHi  H. influenzae  strain 86-028NP contig set was queried for short oligonucleotide repeats. The region surrounding the repeats was analyzed to identify the gene(s) associated with the repeat. Table 5 lists the identified repeats and the ORF (identified by BLAST) associated with each repeat. 
         [0117]    Further sequence analysis has identified the full length nucleotide sequence of the virulence-associated genes and the corresponding amino acid sequences encoded by the ORF. The derived amino acid sequences are highly homologous to the listed Genbank sequence. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                 Location in 
                 Location in 
                 Full Length 
                 Amino 
                   
               
               
                   
                 3-fold 
                 8-fold 
                 Nucleotide 
                 Acid 
                 Genebank 
               
               
                 Repeat 
                 Contigs 
                 Contigs 
                 Sequence 
                 Sequence 
                 Accession No. 
               
               
                   
               
             
             
               
                 SEQ ID 
                 115 
                 nt. 484533-483643 
                 SEQ ID 
                 SEQ ID 
                 NP_439538.1 
               
               
                 NO: 581 
                 nt. 473-540 
                 of 
                 NO: 659 
                 NO: 660 
               
               
                   
                 of 
                 SEQ ID 
               
               
                   
                 SEQ ID 
                 NO: 685 
               
               
                   
                 NO: 115 
                 (contig 18) 
               
               
                 SEQ ID 
                 377 
                 nt. 416274-414910 
                 SEQ ID 
                 SEQ ID 
                 P45217 
               
               
                 NO: 582 
                 nt. 546-597 
                 of 
                 NO: 661 
                 NO: 662 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 685 (contig 
               
               
                   
                 NO: 337 
                 18) 
               
               
                 SEQ ID 
                 505 
                 nt. 414500-416614 
                 SEQ ID 
                 SEQ ID 
                 AAK76425 
               
               
                 NO: 583 
                 nt. 310-393 
                 of 
                 NO: 663 
                 NO: 664 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 684 (contig 
               
               
                   
                 NO: 505 
                 17) 
               
               
                 SEQ ID 
                 508 
                 nt. 506516-507913 
                 SEQ ID 
                 SEQ ID 
                 NP_439520 
               
               
                 NO: 584 
                 nt. 2079-2120 
                 of 
                 NO: 665 
                 NO: 666 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 685 (contig 
               
               
                   
                 NO: 508 
                 18) 
               
               
                 SEQ ID 
                 518 
                 nt. 354274-352406 
                 SEQ ID 
                 SEQ ID 
                 NP_284893 
               
               
                 NO: 585 
                 nt. 758-789 
                 of 
                 NO: 667 
                 NO: 668 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 684 (contig 
               
               
                   
                 NO: 518 
                 17) 
               
               
                 SEQ ID 
                 543 
                 nt. 347864-243236 
                 SEQ ID 
                 SEQ ID 
                 AAA20524 
               
               
                 NO: 586 
                 nt. 1814-196 
                 of 
                 NO: 669 
                 NO: 670 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 685 (contig 
               
               
                   
                 NO: 543 
                 18) 
               
               
                 SEQ ID 
                 543 
                 nt. 699709-704187 
                 SEQ ID 
                 SEQ ID 
                 AAD56660 
               
               
                 NO: 586 
                 nt. 1814-196 
                 of 
                 NO: 671 
                 NO: 672 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 685 (contig 
               
               
                   
                 NO: 543 
                 18) 
               
               
                 SEQ ID 
                 567 
                 nt. 85546-84689 
                 SEQ ID 
                 SEQ ID 
                 ZP_00053190 
               
               
                 NO: 587 
                 nt. 13309-13320 
                 of 
                 NO: 673 
                 NO: 674 
               
               
                   
                 of 
                 SEQ ID NO: 
               
               
                   
                 SEQ ID 
                 681 (contig 
               
               
                   
                 NO: 567 
                 14) 
               
               
                   
               
             
          
         
       
     
       Example 8 
     Identification of Unique NTHi Gene Sequences 
       [0118]    Genes associated with NTHi virulence were also identified by comparing the level of expression of the gene when the NTHi bacterium was infecting a tissue verses the level of expression of the same gene when the NTHi was grown on artificial laboratory media. These novel genes were identified using the promoter trap techniques described above in Examples 4-6, and subsequently comparisons with the known Rd genome demonstrated these genes are unique to NTHi strain 86-028NP. 
         [0119]    The DNA sequence identified using this screening procedure are set forth as SEQ ID NOS: 577-580. These sequences did not contain genes or gene fragments that have homologues in the  H. influenzae  Rd. genome sequence. Even though these are completely novel sequences, due to their expression level during NTHi infection in the chinchilla middle ear, it is likely that expression of these genes are involved in NTHi virulence.