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. J. Med.  344: 403-409, 2001; Snow et 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. Pediatr. 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 lav 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.  
         [0000]     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 contig 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 4B 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 nonnative 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.  
                             TABLE 1                           Amino Acid Substitutions            Original       Preferred       Residues   Exemplary Substitutions   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, Pseudomonas, 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.  
         [0000]     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 carbamolytransferase, mannonate dehydratase, disulfide oxidoreductase, urease accessory protein, phosphocholine 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, HI1064, 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 alt 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.  
         [0000]     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, HI10352 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 NTHi 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, diolcoyl phosplhatidylethanolamine (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 Inmunodiffussion 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.  
         [0059]     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.  
         [0060]     Nontypeable  Haemophilus influenzae  (NTHi) p  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.  
         [0061]     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. NTHi 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.  
         [0062]     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.  
         [0063]     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. Immun.,  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.  
         [0000]     DFI Strategy  
         [0064]     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.  
         [0065]     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 vitro 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).  
         [0066]     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).  
         [0000]     Animal Model  
         [0067]     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,  Infest. 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).  
         [0068]     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).  
         [0000]     Genes Upregulated In Vivo in Response to NTHi Infection of the Middle Ear  
         [0069]     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.  
         [0070]     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.  
         [0071]     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).  
         [0072]     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.  
         [0073]     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 is 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.  
         [0074]     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. Immun.,  69: 472-478, 2000; Baltes et al.,  Infect. Immun.,  69: 472-478, 2001; Bosse and MacInnes,  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).  
         [0075]     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.    
         [0076]     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, 2,002). 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.  
         [0077]     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  
       [0078]      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.  
         [0079]      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.  
         [0080]      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  
       [0081]     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  
       [0082]     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.  
         [0083]     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.  
         [0084]     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.  
         [0085]     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.  
                                             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%                  
 
         [0086]     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 2B.  
                                                 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  
       [0087]     Seventy-five of the 88 contigs with length≧5000bp, 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.  
         [0088]     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.  
         [0089]     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).  
         [0090]     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 hifgenes 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 ).  
         [0091]     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.  
         [0092]     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/NlpD, 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.  
                                                             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   200E−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               (IepA)           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   ORF 1     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   outer 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                     actinomycetemco         SEQ ID NO: 264                     mitans         264   1.00E−103   unknown     Actinobacillus     NP_067555.1   nt. 695-1309 of                     actinomycetemco         SEQ ID NO: 264                     mitans         264   2.00E−21   unknown     Actinobacillus     NP_067556.1   nt. 1302-1448 of                     actinomycetemco         SEQ ID NO: 264                     mitans         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&#39; 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                     actinomycetemco         SEQ ID NO: 493                     mitans         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                     actinomycetemco         nt. 4118-4900                     mitans         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                  
 
         [0093]    
       
         
               
               
               
               
               
             
           
               
                 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  
       [0094]     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).  
         [0095]     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.  
         [0096]     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, Minn.) 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, Mass.; 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.  
         [0097]     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.  
         [0098]     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.  
         [0099]     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.  
         [0100]     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  
       [0101]     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.  
         [0102]     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.).  
         [0103]     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.  
         [0104]     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.  
         [0105]     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  
       [0106]     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.  
         [0107]     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  7: 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.  
         [0108]     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.  
         [0109]     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 μ/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  
       [0110]      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.  
         [0111]     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 gfpmut 3  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).  
         [0112]     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.  
         [0113]     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 confirmed 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.  
         [0114]     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.  
         [0115]     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.  
         [0116]     The 8-fold sequencing of the NTHi 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.  
                                                             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                  
 
         [0117]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 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: 
                 SEO 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  
       [0118]     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.  
         [0119]     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.  
         [0120]     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.  
                                   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  
       [0121]     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.  
         [0122]     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.