# Patent

## Title
Nucleotide sequence of the Haemophilus influenzae Rd genome, fragments thereof, and uses thereof

## Abstract

The present invention provides the sequencing of the entire genome of Haemophilus influenzae Rd, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies over 1700 protein encoding fragments of the genome and identifies, by position relative to a unique Not I restriction endonuclease site, any regulatory elements which modulate the expression of the protein encoding fragments of the Haemophilus genome.

## Background
This appln is a DIV of Ser. No. 09/557,884, filed Apr. 25, 2000, now U.S. Pat. No. 6,506,881 which is a con of Ser. No. 08/476,102 filed Jun. 7, 1995, now U.S. Pat. No. 6,355,450 which is a CIP of Ser. No. 08/426,787 filed Apr. 21, 1995, abandoned.


STATEMENT REGARDING FED SPONSORED R & D
Part of the work performed during development of this invention utilized U.S. Government funds. The government may have certain rights in this invention. NIH-5R01GM48251


REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER LISTING APPENDIX
This application refers to a “Sequence Listing” listed below, which is provided as an electronic document on two identical compact discs (CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each contain the file “PB186P2C1D1.ST25.txt” (2,385,030 bytes, created on May 31, 2002), which is hereby incorporated in its entirety herein.
1. Field of the Invention
The present invention relates to the field of molecular biology. The present invention discloses compositions comprising the nucleotide sequence of Haemophilus influenzae, fragments thereof and usage in industrial fermentation and pharmaceutical development.
2. Background of the Invention
The complete genome sequence from a free living cellular organism has never been determined. The first mycobacterium sequence should be completed by 1996, while E. coli and S. cerevisae are expected to be completed before 1998. These are being done by random and/or directed sequencing of overlapping cosmid clones. No one has attempted to determine sequences of the order of a megabase or more by a random shotgun approach.
H. influenzae is a small (approximately 0.4×1 micron) non-motile, non-spore forming germ-negative bacterium whose only natural host is human. It is a resident of the upper respiratory mucosa of children and adults and causes otitis media and respiratory tract infections mostly in children. The most serious complication is meningitis, which produces neurological sequelae in up to 50% of affected children. Six H. influenzae serotypes (a through f) have been identified based on immunologically distinct capsular polysaccharide antigens. A number of non-typeable strains are also known. Serotype b accounts for the majority of human disease.
Interest in the medically important aspects of H. influenzae biology has focused particularly on those genes which determine virulence characteristics of the organism. A number of the genes responsible for the capsular polysaccharide have been mapped and sequenced (Kroll et al., Mol. Microbiol. 5(6):1549-1560 (1991)). Several outer membrane protein (OMP) genes have been identified and sequenced (Langford et al., J. Gen. Microbiol. 138:155-159 (1992)). The lipoligosaccharide (LOS) component of the outer membrane and the genes of its synthetic pathway are under intensive study (Weiser et al., J. Bacteriol. 172:3304-3309 (1990)). While a vaccine has been available since 1984, the study of outer membrane components is motivated to some extent by the need for improved vaccines. Recently, the catalase gene was characterized and sequenced as a possible virulence-related gene (Bishni et al., in press). Elucidation of the H. influenzae genome will enhance the understanding of how H. influenzae causes invasive disease and how best to combat infection.
H. influenzae possesses a highly efficient natural DNA transformation system which has been intensively studied in the non-encapsulated (R), serotype d strain (Kahn and Smith, J. Membrane Biology 81:89-103 (1984)). At least 16 transformation-specific genes have been identified and sequenced. Of these, four are regulatory (Redfield, J. Bacteriol. 173:5612-5618 (1991), and Chandler, Proc. Natl. Acad. Sci. USA 89:1626-1630 (1992)), at least two are involved in recombination processes (Barouki and Smith, J. Bacteriol 163(2):629-634 (1985)), and at least seven are targeted to the membranes and periplasmic space (Tomb et al., Gene 104:1-10 (1991), and Tomb, Proc. Natl. Acad. Sci. USA 89:10252-10256 (1992)), where they appear to function as structural components or in the assembly of the DNA transport machinery. H. influenzae Rd transformation shows a number of interesting features including sequence-specific DNA uptake, rapid uptake of several double-stranded DNA molecules per competent cell into a membrane compartment called the transformasome, linear translocation of a single strand of the donor DNA into the cytoplasm, and synapsis and recombination of the strand with the chromosome by a single-strand displacement mechanism. The H. influenzae Rd transformation system is the most thoroughly studied of the gram-negative systems and distinct in a number of ways from the gram-positive systems.
The size of H. influenzae Rd genome has been determined by pulsed-field agarose gel electrophoresis of restriction digests to be approximately 1.9 Mb, making its genome approximately 40% the size of E. coli (Lee and Smith, J. Bacterol. 170:4402-4405 (1988)). The restriction map of H. influenzae is circular (Lee et al., J. Bacteriol. 171:3016-3024 (1989), and Redfield and Lee, “Haemophilus influenzae Rd”, pp. 2110-2112, In O'Brien, S. J. (ed), Genetic Maps: Locus Maps of Complex Genomes, Cold Spring Harbor Press, New York). Various genes have been mapped to restriction fragments by Southern hybridization probing of restriction digest DNA bands. This map will be valuable in verification of the assembly of a complete genome sequence from randomly sequenced fragments. GenBank currently contains about 100 kb of non-redundant H. influenzae DNA sequences. About half are from serotype b and half from Rd.
SUMMARY OF THE INVENTION
The present invention is based on the sequencing of the Haemophilus influenzae Rd genome. The primary nucleotide sequence which was generated is provided in SEQ ID NO:1.
The present invention provides the generated nucleotide sequence of the Haemophilus influenzae Rd genome, or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan. In one embodiment, present invention is provided as a contiguous string of primary sequence information corresponding to the nucleotide sequence depicted in SEQ ID NO:1.
The present invention further provides nucleotide sequences which are at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1.
The nucleotide sequence of SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence which is at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1 may be provided in a variety of mediums to facilitate its use. In one application of this embodiment, the sequences of the present invention are recorded on computer readable media. Such media includes, but is not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
The present invention further provides systems, particularly computer-based systems which contain the sequence information herein described stored in a data storage means. Such systems are designed to identify commercially important fragments of the Haemophilus influenzae Rd genome.
Another embodiment of the present invention is directed to isolated fragments of the Haemophilus influenzae Rd genome. The fragments of the Haemophilus influenzae Rd genome of the present invention-include, but are not limited to, fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in a sample, hereinafter, diagnostic fragments (DFs).
Each of the ORF fragments of the Haemophilus influenzae Rd genome disclosed in Tables 1(a) and 2, and the EMF found 5′ to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers for the presence of a specific microbe in a sample, for the production of commercially important pharmaceutical agents, and to selectively control gene expression.
The present invention further includes recombinant constructs comprising one or more fragments of the Haemophilus influenzae Rd genome of the present invention. The recombinant constructs of the present invention comprise vectors, such as a plasmid or viral vector, into which a fragment of the Haemophilus influenzae Rd has been inserted.
The present invention further provides host cells containing any one of the isolated fragments of the Haemophilus influenzae Rd genome of the present invention. The host cells can be a higher eukaryotic host such as a mammalian cell, a lower eukaryotic cell such as a yeast cell, or can be a procaryotic cell such as a bacterial cell.
The present invention is further directed to isolated proteins encoded by the ORFs of the present invention. A variety of methodologies known in the art can be utilized to obtain any one of the proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. In an alternative method, the protein is purified from bacterial cells which naturally produce the protein. Lastly, the proteins of the present invention can alternatively be purified from cells which have been altered to express the desired protein.
The invention further provides methods of obtaining homologs of the fragments of the Haemophilus influenzae Rd genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention. Specifically, by using the nucleotide and amino acid sequences disclosed herein as a probe or as primers, and techniques such as PCR cloning and colony/plaque hybridization, one skilled in the art can obtain homologs.
The invention further provides antibodies which selectively bind one of the proteins of the present invention. Such antibodies include both monoclonal and polyclonal antibodies.
The invention further provides hybridomas which produce the above-described antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
The present invention further provides methods of identifying test samples derived from cells which express one of the ORF of the present invention, or homolog thereof. Such methods comprise incubating a test sample with one or more of the antibodies of the present invention, or one or more of the DFs of the present invention, under conditions which allow a skilled artisan to determine if the sample contains the ORF or product produced therefrom.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the above-described assays.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the antibodies, or one of the DFs of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of bound antibodies or hybridized DFs.
Using the isolated proteins of the present invention, the present invention further provides methods of obtaining and identifying agents capable of binding to a protein encoded by one of the ORFs of the present invention. Specifically, such agents include antibodies (described above), peptides, carbohydrates, pharmaceutical agents and the like. Such methods comprise the steps of:

    
    
        (a) contacting an agent with an isolated protein encoded by one of the ORFs of the present invention; and
        (b) determining whether the agent binds to said protein.
    
    


The complete genomic sequence of H. influenzae will be of great value to all laboratories working with this organism and for a variety of commercial purposes. Many fragments of the Haemophilus influenzae Rd genome will be immediately identified by similarity searches against GenBank or protein databases and will be of immediate value to Haemophilus researchers and for immediate commercial value for the production of proteins or to control gene expression. A specific example concerns PHA synthase. It has been reported that polyhydroxybutyrate is present in the membranes of H. influenzae Rd and that the amount correlates with the level of competence for transformation. The PHA synthase that synthesizes this polymer has been identified and sequenced in a number of bacteria, none of which are evolutionarily close to H. influenzae. This gene has yet to be isolated from H. influenzae by use of hybridization probes or PCR techniques. However, the genomic sequence of the present invention allows the identification of the gene by utilizing search means described below.
Developing the methodology and technology for elucidating the entire genomic sequence of bacterial and other small genomes has and will greatly enhance the ability to analyze and understand chromosomal organization. In particular, sequenced genomes will provide the models for developing tools for the analysis of chromosome structure and function, including the ability to identify genes within large segments of genomic DNA, the structure, position, and spacing of regulatory elements, the identification of genes with potential industrial applications, and the ability to do comparative genomic and molecular phylogeny.



DESCRIPTION OF THE FIGURES
FIG. 1—restriction map of the Haemophilus influenzae Rd genome.
FIG. 2—Block diagram of a computer system 102 that can be used to implement the computer-based systems of present invention.
FIG. 3—A comparison of experimental coverage of up to approximately 4000 random sequence fragments assembled with AutoAssembler (squares) as compared to lander-Waterman prediction for a 2.5 Mb genome (triangles) and a 1.6 Mb genome (circles) with a 460 bp average sequence length and a 25 bp overlap.
FIG. 4—Data flow and computer programs used to manage, assemble, edit, and annotate the H. influenzae genome. Both Macintosh and Unix platforms are used to handle the AB 373 sequence data files (Kerlavage et al., Proceedings of the Twenty-Sixth Annual Hawaii International Conference on System Sciences, IEEE Computer Society Press, Washington D.C., 585 (1993)). Factura (AB) is a Macintosh program designed for automatic vector sequence removal and end trimming of sequence files. The program esp runs on a Macintosh platform and parses the feature data extracted from the sequence files by Factura to the Unix based H. influenzae relational database. Assembly is accomplished by retrieving a specific set of sequence files and their associated features using stp, an X-windows graphical interface and control program which can retrieve sequences from the H. influenzae database using user-defined or standard SQL queries. The sequence files were assembled using TIGR Assembler, an assembly engine designed at TIGR for rapid and accurate assembly of thousands of sequence fragments. TIGR Editor is a graphical interface which can parse the aligned sequence files from TIGR Assembler output and display the alignment and associated electropherograms for contig editing. Identification of putative coding regions was performed with Genemark (Borodovsky and McIninch, Computers Chem. 17(2):123 (1993)), a Markov and Bayes modeled program for predicting gene locations, and trained on a H. influenzae sequence data set. Peptide searches were performed against the three reading frames of each Genemark predicted coding region using blaze (Brutlag et al., Computers Chem. 17:203 (1993)) run on a Maspar MP-2 massively parallel computer with 4096 microprocessors. Results from each frame were combined into a single output file by mblzt. Optimal protein alignments were obtained using the program praze which extends alignments across potential frameshifts. The output was inspected using a custom graphic viewing program, gbyob, that interacts directly with the H. influenzae database. The alignments were further used to identify potential frameshift errors and were targeted for additional editing.
FIG. 5—A circular representation of the H. influenzae Rd chromosome illustrating the location of each predicted coding region containing a database match as well as selected global features of the genome. Outer perimeter: The location of the unique NotI restriction site (designated as nucleotide 1), the RsrII sites, and the SmaI sites. Outer concentric circle: The location of each identified coding region for which a gene identification was made. Second concentric circle: Regions of high G/C content and high A/T content. High G/C content regions are specifically associated with the 6 ribosomal operons and the mu-like prophage. Third concentric circle: Coverage by lambda clones. Over 300 lambda clones were sequenced from each end to confirm the overall structure of the genome and identify the 6 ribosomal-operons. Fourth concentric circle: The locations of the 6 ribosomal operons, the tRNAs and the cryptic mu-like prophage. Fifth concentric circle: Simple tandem repeats. The locations of the following repeats are shown: CTGGCT, GTCT, ATT, AATGGC, TTGA, TTGG, TTTA, TTATC TGAC, TCGTC, AACC, TTGC, CAAT, CCAA. The putative origin of replication is illustrated by the outward pointing arrows originating near base 603,000. Two potential termination sequences are shown near the opposite midpoint of the circle.
FIGS. 6(A) to 6(AN) Complete map of the H. influenzae Rd genome. Predicted coding regions are shown on each strand. rRNA and tRNA genes are shown as lines and triangles, respectively. GeneID numbers correspond to those in Tables 1(a), 1(b) and 2. Where possible, three-letter designations are also provided.
FIG. 7—A comparison of the region of the H. influenzae chromosome containing the 8 genes of the fimbrial gene cluster present in H. influenzae type b and the same region in H. influenzae Rd. The region is flanked by the pepN and purE genes in both organisms. However in the non-infectious Rd strain the 8 genes of the fimbrial gene cluster have been excised. A 172 bp spacer region is located in this region in the Rd strain and continues to be flanked by the pepN and purE genes.
FIG. 8—Hydrophobicity analysis of five predicted channel-proteins. The amino acid sequences of five predicted coding regions that do not display homology with known peptide sequences (GenBank release 87), each exhibit multiple hydrophobic domains that are characteristic of channel-forming proteins. The predicted coding region sequences were analyzed by the Kyte-Doolittle algorithm (Kyte and Doolittle, J. Mol. Biol. 157:105 (1982)) (with a range of 11 residues) using the GeneWorks software package (Intelligenetics).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based on the sequencing of the Haemophilus influenzae Rd genome. The primary nucleotide sequence which was generated is provided in SEQ ID NO:1. As used herein, the “primary sequence” refers to the nucleotide sequence represented by the IUPAC nomenclature system.
The sequence provided in SEQ ID NO:1 is oriented relative to a unique Not I restriction endonuclease site found in the Haemophilus influenzae Rd genome. A skilled artisan will readily recognize that this start/stop point was chosen for convenience and does not reflect a structural significance.
The present invention provides the nucleotide sequence of SEQ ID NO:1, or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan. In one embodiment, the sequence is provided as a contiguous string of primary sequence information corresponding to the nucleotide sequence provided in SEQ ID NO:1.
As used herein, a “representative fragment of the nucleotide sequence depicted in SEQ ID NO:1” refers to any portion of SEQ ID NO:1 which is not presently represented within a publicly available database. Preferred representative fragments of the present invention are Haemophilus influenzae open reading frames, expression modulating fragments, uptake modulating fragments, and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in sample. A non-limiting identification of such preferred representative fragments is provided in Tables 1(a) and and 2.
The nucleotide sequence information provided in SEQ ID NO:1 was obtained by sequencing the Haemophilus influenzae Rd genome using a megabase shotgun sequencing method. Using three parameters of accuracy discussed in the Examples below, the present inventors have calculated that the sequence in SEQ ID NO:1 has a maximum accuracy of 99.98%. Thus, the nucleotide sequence provided in SEQ ID NO:1 is a highly accurate, although not necessarily a 100% perfect, representation of the nucleotide sequence of the Haemophilus influenzae Rd genome.
As discussed in detail below, using the information provided in SEQ ID NO:1 and in Tables 1(a) and 2 together with routine cloning and sequencing methods, one of ordinary skill in the art will be able to clone and sequence all “representative fragments” of interest including open reading frames (ORFs) encoding a large variety of Haemophilus influenzae proteins. In very rare instances, this may reveal a nucleotide sequence error present in the nucleotide sequence disclosed in SEQ ID NO: 1. Thus, once the present invention is made available (i.e., once the information in SEQ ID NO:1 and Tables 1(a) and 2 have been made available), resolving a rare sequencing error in SEQ ID NO:1 will be well within the skill of the art. Nucleotide sequence editing software is publicly available. For example, Applied Biosystem's (AB) AutoAssemblerTM can be used as an aid during visual inspection of nucleotide sequences.
Even if all of the very rare sequencing errors in SEQ ID NO:1 were corrected, the resulting nucleotide sequence would still beat least 99.9% identical to the nucleotide sequence in SEQ ID NO:1.
The nucleotide sequences of the genomes from different strains of Haemophilus influenzae differ slightly. However, the nucleotide sequence of the genomes of all Haemophilus influenzae strains will be at least 99.9% identical to the nucleotide sequence provided in SEQ ID NO:1.
Thus, the present invention further provides nucleotide sequences which are at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1 in a form which can be readily used, analyzed and interpreted by the skilled artisan. Methods for determining whether a nucleotide sequence is at least 99.9 % identical to the nucleotide sequence of SEQ ID NO:1 are routine and readily available to the skilled artisan. For example, the well known fasta algothrithm (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988)) can be used to generate the percent identity of nucleotide sequences.
Computer Related Embodiments
The nucleotide sequence provided in SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 may be “provided” in a variety of mediums to facilitate use thereof. As used herein, provided refers to a manufacture, other than an isolated nucleic acid molecule, which contains a nucleotide sequence of the present invention, i.e., the nucleotide sequence provided in SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1. Such a manufacture provides the Haemophilus influenzae Rd genome or a subset thereof (e.g., a Haemophilus Influenzae Rd open reading frame (ORF)) in a form which allows a skilled artisan to examine the manufacture using means not directly applicable to examining the Haemophilas influenzae Rd genome or a subset thereof as it exists in nature or in purified form.
In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.
As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.
A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.
By providing the nucleotide sequence of SEQ ID NO: 1, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system was used to identify open reading frames (ORFs) within the Haemophilus influenzae Rd genome which contain homology to ORFs or proteins from other organisms. Such ORFs are protein encoding fragments within the Haemophilus influenzae Rd genome and are useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.
The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. Such systems are designed to identify commercially important fragments of the Haemophilus influenzae Rd genome.
As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention.
As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.
As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the Haemophilus influenzae Rd genome which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.
As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that searches for commercially important fragments of the Haemophilus influenzae Rd genome, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.
As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzymic active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).
A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. A preferred format for an output means ranks fragments of the Haemophilus influenzae Rd genome possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.
A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of the Haemophilus influenzae Rd genome. In the present examples, implementing software which implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol. Biol. 215:403-410. (1990)) was used to identify open reading frames within the Haemophilus influenzae Rd genome. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer-based systems of the present invention.
One application of this embodiment is provided in FIG. 2. FIG. 2 provides a block diagram of a computer system 102 that can be used to implement the present invention. The computer system 102 includes a processor 106 connected to a bus 104. Also connected to the bus 104 are a main memory 108 (preferably implemented as random access memory, RAM) and a variety of secondary storage devices 110, such as a hard drive 112 and a removable medium storage device 114. The removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device 114. The computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114 once inserted in the removable medium storage device 114.
A nucleotide sequence of the present invention may be stored in a well known manner in the main memory 108, any of the secondary storage devices 110, and/or a removable storage medium 116. Software for accessing and processing the genomic sequence (such as search tools, comparing tools, etc.) reside in main memory 108 during execution.
Biochemical Embodiments
Another embodiment of the present invention is directed to isolated fragments of the Haemophilus influenzae Rd genome. The fragments of the Haemophilus influenzae Rd genome of the present invention include, but are not limited to fragments which encode peptides, hereinafter open reading frames (ORFs), fragments which modulate the expression of an operably linked ORF, hereinafter expression modulating fragments (EMFs), fragments which mediate the uptake of a linked DNA fragment into a cell, hereinafter uptake modulating fragments (UMFs), and fragments which can be used to diagnose the presence of Haemophilus influenzae Rd in a sample, hereinafter diagnostic fragments (DFs).
As used herein, an “isolated nucleic acid molecule” or an “isolated fragment of the Haemophilus influenzae Rd genome” refers to a nucleic acid molecule possessing a specific nucleotide sequence which has been subjected to purification means to reduce, from the composition, the number of compounds which are normally associated with the composition. A variety of purification means can be used to generated the isolated fragments of the present invention. These include, but are not limited to methods which separate constituents of a solution based on charge, solubility, or size.
In one embodiment, Haemophilus influenaze Rd DNA can be mechanically sheared to produce fragments of 15-20 kb in length. These fragments can then be used to generate an Haemophilus influenzae Rd library by inserting them into labda clones as described in the Examples below. Primers flanking, for example, an ORF provided in Table 1(a) can then be generated using nucleotide sequence information provided in SEQ ID NO:1. PCR cloning can then be used to isolate the ORF from the lambda DNA library. PCR cloning is well known in the art. Thus, given the availability of SEQ ID NO:1, Table 1(a) and Table 2, it would be routine to isolate any ORF or other nucleic acid fragment of the present invention.
The isolated nucleic acid molecules of the present invention include, but are not limited to single stranded and double stranded DNA, and single stranded RNA.
As used herein, an “open reading frame,” ORF, means a series of triplets coding for amino acids without any termination codons and is a sequence translatable into protein. Tables 1a, 1b and 2 identify ORFs in the Haemophilus influenzae Rd genome. In particular, Table 1a indicates the location of ORFs within the Haemophilus influenzae genome which encode the recited protein based on homology matching with protein sequences from the organism appearing in parentheticals (see the fourth column of Table 1(a)).
The first column of Table 1(a) provides the “GeneID” of a particular ORF. This information is useful for two reasons. First, the complete map of the Haemophilus influenzae Rd genome provided in FIGS. 6(A) 6(AN) refers to the ORFs according to their GeneID numbers. Second, Table 1(b) uses the GeneID numbers to indicate which ORFs were provided previously in a public database.
The second and third columns in Table 1(a) indicate an ORFs position in the nucleotide sequence provided in SEQ ID NO:1. One of ordinary skill will recognize that ORFs may be oriented in opposite directions in the Haemophilus influenae genome. This is reflected in columns 2 and 3.
The fifth column of Table 1(a) indicates the percent identity of the protein encoded for by an ORF to the corresponding protein from the orgaism appearing in parentheticals in the fourth column.
The sixth column of Table 1(a) indicates the percent similarity of the protein encoded for by an ORF to the corresponding protein from the organism appearing in parentheticals in the fourth column. The concepts of percent identity and percent similarity of two polypeptide sequences is well understood in the art. For example, two polypeptides 10 amino acids in length which differ at three amino acid positions (e.g., at positions 1, 3 and 5) are said to have a percent identity of 70%. However, the same two polypeptides would be deemed to have a percent similarity of 80% if, for example at position 5, the amino acids moieties, although not identical, were “similar” (i.e., possessed similar biochemical characteristics).
The seventh column in Table 1(a) indicates the length of the amino acid homology match.
Table 2 provides ORFs of the Haemophilus influenzae Rd genome which encode polypeptide sequences which did not elicit a “homology match” with a known protein sequence from another organism. Further details concerning the algorithms and criteria used for homology searches are provided in the Examples below.
A skilled artisan can readily identify ORFs in the Haemophilus influenzae Rd genome other than those listed in Tables 1(a), 1(b) and 2, such as ORFs which are overlapping or encoded by the opposite strand of an identified ORF in addition to those ascertainable using the computer-based systems of the present invention.
As used herein, an “expression modulating fragment,” EMF, means a series of nucleotide molecules which modulates the expression of an operably linked ORF or EMF.
As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments which induce the expression or an operably linked ORF in response to a specific regulatory factor or physiological event. A review of known EMFs from Haemophilus are described by (Tomb et al. Gene 104:1-10 (1991), Chandler, M. S., Proc. Natl. Acad. Sci. USA 89:1626-1630 (1992).
EMF sequences can be identified within the Haemophilus influenzae Rd genome by their proximity to the ORFs provided in Tables 1(a), 1(b) and 2. An intergenic segment, or a fragment of the intergenic segment, from about 10 to 200 nucleotides in length, taken 5′ from any one of the ORFs of Tables 1(a), 1(b), or 2 will modulate the expression of an operably linked 3′ ORF in a fashion similar to that found with the naturally linked ORF sequence. As used herein, an “intergenic segment” refers to the fragments of the Haemophilus genome which are between two ORF(s) herein described. Alternatively, EMFs can be identified using known EMFs as a target sequence or target motif in the computer-based systems of the present invention.
The presence and activity of an EMF can be confirmed using an EMF trap vector. An EMF trap vector contains a cloning site 5′ to a marker sequence. A marker sequence encodes an identifiable phenotype, such as antibiotic resistance or a complementing nutrition auxotrophic factor, which can be identified or assayed when the EMF trap vector is placed within an appropriate host under appropriate conditions. As described above, a EMF will modulate the expression of an operably linked marker sequence. A more detailed discussion of various marker sequences is provided below.
A sequence which is suspected as being a EMF is cloned in all three reading frames in one or more restriction sites upstream from the marker sequence in the EMF trap vector. The vector is then transformed into an appropriate host using known procedures and the phenotype of the transformed host in examined under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence.
As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotide molecules which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described above.
The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence. A review of DNA uptake in Haemophilus is provided by Goodgall, S. H., et al., J. Bact. 172:5924-5928 (1990).
As used herein, a “diagnostic fragment,” DF, means a series of nucleotide molecules which selectively hybridize to Haemophilus influenzae sequences. DFs can be readily identified by identifying unique sequences within the Haemophilus influenzae Rd genome, or by generating and testing probes or amplification primers consisting of the DF sequence in an appropriate diagnostic format which determines amplification or hybridization selectivity.
The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO:1, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another which encodes the same amino acid is expressly contemplated.
Any specific sequence disclosed herein can be readily screened for errors by resequencing a particular fragment, such as an ORF, in both directions (i.e., sequence both strands). Alternatively, error screening can be performed by sequencing corresponding polynucleotides of Haemophilus influenzae origin isolated by using part or all of the fragments in question as a probe or primer. Each of the ORFs of the Haemophilus influenzae Rd genome disclosed in Tables 1(a), 1(b) and 2, and the EMF found 5′ to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers to detect the presence of a specific microbe, such as Haemophilus influenzae RD, in a sample. This is especially the case with the fragments or ORFs of Table 2, which will be highly selective for Haemophilus influenzae. 
In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on-the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.
The present invention further provides recombinant constructs comprising one or more fragments of the Haemophilus influenzae Rd genome of the present invention. The recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a fragment of the Haemophilus influenzae Rd has been inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. For vectors comprising tie EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF or UMF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
The present invention further provides host cells containing any one of the isolated fragments of the Haemophilus influenzae Rd genome of the present invention, wherein the fragment has been introduced into the host cell using known transformulation methods. The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a procaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).
The host cells containing one of the fragments of the Haemophilus influenzae Rd genome of the present invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORP) or can be used to produce a heterologous protein under the control of the EMF.
The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By “degenerate variant” is intended 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 polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs depicted in Table 1(a) which encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.
The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.
Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis. The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.
“Recombinant,” as used herein, means that a polypeptide or protein is derived from recombinant (e.g., microbial or mammalian) expression systems. “Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern different from that expressed in mammalian cells.
“Nucleotide sequence” refers to a heteropolymer of deoxyribonucleotides. Generally, DNA segments encoding the polypeptides and proteins provided by this invention are assembled from fragments of the Haemophilus influenzae Rd genome and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
“Recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. The expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
“Recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extra chromosomally. The cells can be prokaryotic or eukaryotic. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryatic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may, also be employed as a matter of choice. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture is usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
The present invention further includes isolated polypeptides, proteins and nucleic acid molecules which are substantially equivalent to those herein described. As used herein, substantially equivalent can refer both to nucleic acid and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, sequences having equivalent biological activity, and equivalent expression characteristics are considered substantially equivalent. For purposes of determining equivalence, truncation of the mature sequence should be disregarded.
The invention further provides methods of obtaining homologs from other strains of Haemophilus influenzae, of the fragments of the Haemophilus influenzae Rd genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention. As used herein, a sequence or protein of Haemophilus influenzae is defined as a homolog of a fragment of the Haemophilus influenzae Rd genome or a protein encoded by one of the ORFs of the present invention, if it shares significant homology to one of the fragments of the Haemophilus influenzae Rd genome of the present invention or a protein encoded by one of the ORFs of the present invention. Specifically, by using the sequence disclosed herein as a probe or as primers, and techniques such as PCR cloning and colony/plaque hybridization, one skilled in the art can obtain homologs.
As used herein, two nucleic acid molecules or proteins are said to “share significant homology” if the two contain regions which process greater than 85% sequence (amino acid or nucleic acid) homology.
Region specific primers or probes derived from the nucleotide sequence provided in SEQ ID NO:1 or from a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 can be used to prime DNA synthesis and PCR amplification, as well as to identify colonies containing cloned DNA encoding a homolog using known methods (Innis et al., PCR Protocols, Academic Press, San Diego, Calif. (1990)).
When using primers derived from SEQ ID NO:1 or from a nucleotide sequence at least 99.9% identical to SEQ ID NO:1, one skilled in the art will recognize that by employing high stringency conditions (e.g., annealing at 50-60° C.) only sequences which are greater than 75% homologous to the primer will be amplified. By employing lower stringency conditions (e.g., annealing at 35-37° C.), sequences which are greater than 40-50% homologous to the primer will also be amplified.
When using DNA probes derived from SEQ ID NO:1 or from a nucleotide sequence at least 99.9% identical to SEQ ID NO:1 for colony/plaque hybridization, one skilled in the art will recognize that by employing high stringency conditions (e.g., hybridizing at 50-65° C. in 5×SSPC and 50% formamide, and washing at 50-65° C. in 0.5×SSPC), sequences having regions which are greater than 90% homologous to the probe can be obtained, and that by employing lower stringency conditions (e.g., hybridizing at 35-37° C. in 5×SSPC and 40-45% formamide, and washing at 42° C. in SSPC), sequences having regions which are greater than 35-45% homologous to the probe will be obtained.
Any organism can be used as the source for homologs of the present invention so long as the organism naturally expresses such a protein or contains genes encoding the same. The most preferred organism for isolating homologs are bacterias which are closely related to Haemophilus influenzae Rd.
Uses for the Compositions of the Invention
Each ORF provided in Table 1(a) was assigned to one of 102 biological role categories adapted from Riley, M., Microbiology Reviews 57(4):862 (1993)). This allows the skilled artisan to determine a use for each identified coding sequence. Tables 1(a) further provides an identification of the type of polypeptide which is encoded for by each ORF. As a result, one skilled in the art can use the polypeptides of the present invention for commercial, therapeutic and industrial purposes consistent with the type of putative identification of the polypeptide.
Such identifications permit one skilled in the art to use the Haemophilus influenzae ORFs in a manner similar to the known type of sequences for which the identification is made; for example, to ferment a particular sugar source or to produce a particular metabolite. (For a review of enzymes used within the commercial industry, see Biochemical Engineering and Biotechnology Handbook 2nd, eds. Macmillan Publ. Ltd., N.Y. (1991) and Biocatalysts in Organic Syntheses, ed. J. Tramper et al., Elsevier Science Publishers, Amsterdam, The Netherlands (1985)).
1. Biosynthetic Enzymes
Open reading frames encoding proteins involved in mediating the catalytic reactions involved in intermediary and macromolecular metabolism, the biosynthesis of small molecules, cellular processes and other functions includes enzymes involved in the degradation of the intermediary products of metabolism, enzymes involved in central intermediary metabolism, enzymes involved in respiration, both aerobic and anaerobic, enzymes involved in fermentation, enzymes involved in ATP proton motor force conversion, enzymes involved in broad regulatory function, enzymes involved in amino acid synthesis, enzymes involved in nucleotide synthesis, enzymes involved in cofactor and vitamin synthesis, can be used for industrial biosynthesis. The various metabolic pathways present in Haemophilus can be identified based on absolute nutritional requirements as well as by examining the various enzymes identified in Table 1(a).
Identified within the category of intermediary metabolism, a number of the proteins encoded by the identified ORFs in Tables 1(a) are particularly involved in the degradation of intermediary metabolites as well as non-macromolecular metabolism. Some of the enzymes identified include amylases, glucose oxidases, and catalase.
Proteolytic enzymes are another class of commercially important enzymes. Proteolytic enzymes find use in a number of industrial processes including the processing of flax and other vegetable fibers, in the extraction, clarification and depectinization of fruit juices, in the extraction of vegetables' oil and in the maceration of fruits and vegetables to give unicellular fruits. A detailed review of the proteolytic enzymes used in the food industry is provided by Rombouts et al., Symbiosis 21:79 (1986) and Voragen et al. in Biocatalyst in Agricultural Biotechnology, edited J. R. Whitaker et al., American Chemical Society Symposium Series 389:93 (1989)).
The metabolism of glucose, galactose, fructose and xylose are important parts of the primary metabolism of Haemophilus. Enzymes involved in the degradation of these sugars can be used in industrial fermentation. Some of the important sugar transforming enzymes, from a commercial viewpoint, include sugar isomerases such as glucose isomerase. Other metabolic enzymes have found commercial use such as glucose oxidases which produces ketogulonic acid (KGA). KGA is an intermediate in the commercial production of ascorbic acid using the Reichstein's procedure (see Krueger et al., Biotechnology 6(A), Rhine, H. J. et al., eds., Verlag Press, Weinheim, Germany (1984)).
Glucose oxidase (GOD) is commercially available and has been used in purified form as well as in an immobilized form for the deoxygenation of beer. See Hartmeir et al., Biotechnology Letters 1:21 (1979). The most important application of GOD is the industrial scale fermentation of gluconic acid. Market for gluconic acids which are used in the detergent, textile, leather, photographic, pharmaceutical, food, feed and concrete industry (see Bigelis in Gene Manipulations and Fungi, Benett, J. W. et al., eds., Academic Press, New York (1985), p. 357). In addition to industrial applications, GOD has found applications in medicine for quantitative determination of glucose in body fluids recently in biotechnology for analyzing syrups from starch and cellulose hydrosylates. See Owusu et al., Biochem. et Biophysica. Acta. 872:83 (1986).
The main sweetener used in the world today is sugar which comes from sugar beets and sugar cane. In the field of industrial enzymes, the glucose isomerase process shows the largest expansion in the market today. Initially, soluble enzymes were used and later immobilized enzymes were developed (Krueger et al., Biotechnology, The Textbook of Industrial Microbiology, Sinauer Associated Incorporated, Sunderland, Mass. (1990)). Today, the use of glucose-produced high fructose syrups is by far the largest industrial business using immobilized enzymes. A review of the industrial use of these enzymes is provided by Jorgensen, Starch 40:307 (1988).
Proteinases, such as alkaline serine proteinases, are used as detergent additives and thus represent one of the largest volumes of microbial enzymes used in the industrial sector. Because of their industrial importance, there is a large body of published and unpublished information regarding the use of these enzymes in industrial processes. (See Faultinan et al., Acid Proteases Structure Function and Biology, Tang, J., ed., Plenum Press, New York (1977) and Godfrey et al., Industrial Enzymes, MacMillan Publishers, Surrey, UK (1983) and Hepner et al., Report Industrial Enzymes by 1990, Hel Hepner & Associates, London (1986)).
Another class of commercially usable proteins of the present invention are the microbial lipases identified in Table 1 (see Macrae et al., Philosophical Transactions of the Chiral Society of London 310:227 (1985) and Poserke, Journal of the American Oil Chemist Society 61:1758 (1984). A major use of lipases is in the fat and oil industry for the production of neutral glycerides using lipase catalyzed inter-esterification of readily available triglycerides. Application of lipases include the use as a detergent additive to facilitate the removal of fats from fabrics in the course of the washing procedures.
The use of enzymes, and in particular microbial enzymes, as catalyst for key steps in the synthesis of complex organic molecules is gaining popularity at a great rate. One area of great interest is the preparation of chiral intermediates. Preparation of chiral intermediates is of interest to a wide range of synthetic chemists particularly those scientists involved with the preparation of new pharmaceuticals, agrochemicals, fragrances and flavors. (See Davies et al., Recent Advances in the Generation of Chiral Intermediates Using Enzymes, CRC Press, Boca Raton, Fla. (1990)). The following reactions catalyzed by enzymes are of interest to organic chemists: hydrolysis of carboxylic acid esters, phosphate esters, amides and nitriles, esterification reactions, trans-esterification reactions, synthesis of amides, reduction of alkanones and oxoalkanates, oxidation of alcohols to carbonyl compounds, oxidation of sulfides to sulfoxides, and carbon bond forming reactions such as the aldol reaction. When considering the use of an enzyme encoded by one of the ORFs of the present invention for biotransformation and organic synthesis it is sometimes necessary to consider the respective advantages and disadvantages of using a microorganism as opposed to an isolated enzyme. Pros and cons of using a whole cell system on the one hand or an isolated partially purified enzyme on the other hand, has been described in detail by Bud et al., Chemistry in Britain (1987), p. 127.
Amino transferases, enzymes involved in the biosynthesis and metabolism of amino acids, are useful in the catalytic production of amino acids. The advantages of using microbial based enzyme systems is that the amino transferase enzymes catalyze the stereo-selective synthesis of only l-amino acids and generally possess uniformly high catalytic rates. A description of the use of amino transferases for amino acid production is provided by Roselle-David, Methods of Enzymology 136:479 (1987).
Another category of useful proteins encoded by the ORFs of the present invention include enzymes involved in nucleic acid synthesis, repair, and recombination. A variety of commercially important enzymes have previously been isolated from members of Haemophilus sp. These include the Hinc II, Hind III, and Hinf I restriction endonucleases. Table 1(a) identifies a wide array of enzymes, such as restriction enzymes, ligases, gyrases and methylases, which have immediate use in the biotechnology industry.
2. Generation of Antibodies
As described here, the proteins of the present invention, as well as homologs thereof, can be used in a variety procedures and methods known in the art which are currently applied to other proteins. The proteins of the present invention can further be used to generate an antibody which selectively binds the protein. Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments of these antibodies, and humanized forms.
The invention further provides antibodies which selectively bind to one of the proteins of the present invention and hybridomas which produce these antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. Methods 35:1-21 (1980); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).
Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with the pseudogene polypeptide. Methods for immunization are well known in the art. Such methods include subcutaneous or interperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection.
The protein which is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization.
For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124 (1988)).
Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)).
Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention.
For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
The present invention further provides the above-described antibodies in detectably labelled form. Antibodies can be detectably labelled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labelling are well-known in the art, for example see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol 109:129 (1972); Goding, J. W. J. Immuol. Meth. 13:215 (1976)).
The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the Haemophilus influenzae Rd genome is expressed.
The present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immunoaffinity purification of the proteins of the present invention.
3. Diagnostic Assays and Kits
The present invention further provides methods to identify the expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using one of the DFs or antibodies of the present invention.
In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the DFs of the present invention and assaying for binding of the DFs or antibodies to components within the test sample.
Conditions for incubating a DF or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay. the detection methods employed, and the type and nature of the DF or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the DFs or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the DFs or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound DF or antibody.
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagent antibody or DF.
Types of detection reagents include labelled nucleic acid probes, labelled secondary antibodies, or in the alternative, if the primary antibody is labelled, the enzymatic, or antibody binding reagents which are capable of reacting with the labelled antibody. One skilled in the art will readily recognize that the disclosed DFs and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
4. Screening Assay for Binding Agents
Using the isolated proteins of the present invention, the present invention further provides methods of obtaining and identifying agents which bind to a protein encoded by one of the ORFs of the present invention or to one of the fragments and the Haemophilus genome herein described.
In detail, said method comprises the steps of:

    
    
        (a) contacting an agent with an isolated protein encoded by one of the ORFs of the present invention, or an isolated fragment of the Haemophilus genome; and
        (b) determining whether the agent binds to said protein or said fragment.
    
    


The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.
Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W. H. Freeman, N.Y. (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.
One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.
Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251: 1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.
Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent, in the control of bacterial infection by modulating the activity of the protein encoded by the ORF. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition for use in controlling Haemophilus growth and infection.
5. Vaccine and Pharmaceutical Composition
The present invention further provides pharmaceutical agents which can be used to modulate the growth of Haemophilus influenzae, or another related organism, in vivo or in vitro. As used herein, a “pharmaceutical agent” is defined as a composition of matter which can be formulated using known techniques to provide a pharmaceutical compositions. As used herein, the “pharmaceutical agents of the present invention” refers the pharmaceutical agents which are derived from the proteins encoded by the ORFs of the present invention or are agents which are identified using the herein described assays.
As used herein, a pharmaceutical agent is said to “modulated the growth of Haemophilus sp., or a related organism, in vivo or in vitro,” when the agent reduces the rate of growth, rate of division, or viability of the organism in question. The pharmaceutical agents of the present invention can modulate the growth of an organism in many fashions, although an understanding of the underlying mechanism of action is not needed to practice the use of the pharmaceutical agents of the present invention. Some agents will modulate the growth by binding to an important protein thus blocking the biological activity of the protein, while other agents may bind to a component of the outer surface of the organism blocking attachment or rendering the organism more prone to act the bodies nature immune system. Alternatively, the agent may be comprise a protein encoded by one of the ORFs of the present invention and serve as a vaccine. The development and use of a vaccine based on outer membrane components, such as the LPS, are well known in the art.
As used herein, a “related organism” is a broad term which refers to any organism whose growth can be modulated by one of the pharmaceutical agents of the present invention. In general, such an organism will contain a homolog of the protein which is the target of the pharmaceutical agent or the protein used as a vaccine. As such, related organism do not need to be bacterial but may be fungal or viral pathogens.
The pharmaceutical agents and compositions of the present invention may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
The agents of the present invention can be used in native form or can be modified to form a chemical derivative. As used herein, a molecule is said to be a “chemical derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).
For example, a change in the immunological character of the functional derivative, such as affinity for a given antibody, is measured by a competitive type immunoassay. Changes in immunomodulation activity are measured by the appropriate assay. Modifications of such protein properties as redox or thermal stability, biological half-life, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan.
The therapeutic effects of the agents of the present invention may be obtained by providing the agent to a patient by any suitable means (i.e., inhalation, intravenously, intramuscularly, subcutaneously, enterally, or parenterally). It is preferred to administer the agent of the present invention so as to achieve an effective concentration within the blood or tissue in which the growth of the organism is to be controlled.
To achieve an effective blood concentration, the preferred method is to administer the agent by injection. The administration may be by continuous infusion, or by single or multiple injections.
In providing a patient with one of the agents of the present invention, the dosage of the administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower or higher dosage may be administered. The therapeutically effective dose can be lowered by using combinations of the agents of the present invention or another agent.
As used herein, two or more compounds or agents are said to be administered “in combination” with each other when either (1) the physiological effects of each compound, or (2) the serum concentrations of each compound can be measured at the same time. The composition of the present invention can be administered concurrently with, prior to, or following the administration of the other agent.
The agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to decrease the rate of growth (as defined above) of the target organism.
The administration of the agent(s) of the invention may be for either a “prophylactic” or “therapeutic” purpose. When provided prophylactically, the agent(s) are provided in advance of any symptoms indicative of the organisms growth. The prophylactic administration of the agent(s) serves lo prevent, attenuate, or decrease the rate of onset of any subsequent infection. When provided therapeutically, the agent(s) are provided at (or shortly after) the onset of an indication of infection. The therapeutic administration of the compound(s) serves to attenuate the pathological symptoms of the infection and to increase the rate of recovery.
The agents of the present invention are administered to the mammal in a pharmaceutically acceptable form and in a therapeutically effective concentration. A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
The agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the agents of the present invention, together with a suitable amount of carrier vehicle.
Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb one or more of the agents of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate agents of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).
The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the agents of the present invention may be employed in conjunction with other therapeutic compounds.
6. Shot-Gun Approach to Megabase DNA Sequencing
The present invention further provides the first demonstration that a sequence of greater than one megabase can be sequenced using a random shotgun approach. This procedure, described in detail in the examples that follow, has eliminated the up front cost of isolating and ordering overlapping or contiguous subclones prior to the start of the sequencing protocols.
Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLES
Experimental Design and Methods
1. Shotgun Sequencing Strategy
The overall strategy for a shotgun approach to whole genome sequencing is outlined in Table 3. The theory of shotgun sequencing follows from the Lander and Waterman (Landerman and Waterman, Genomics 2: 231 (1988)) application of the equation for the Poisson distribution px=mxe−m/x!, where x is the number of occurrences of an event, m is the mean number of occurrences, and px is the probability that any given base is not sequenced after a certain amount of random sequence has been generated. If L is the genome length, n is the number of clone insert ends sequenced, and w is the sequencing read length, then m=nw/L, and the probability that no clone originates at any of the w bases preceding a given base, i.e., the probability that the base is not sequenced, is p0=e−m. Using the fold coverage as the unit for m, one sees that after 1.8 Mb of sequence has been randomly generated, m=1, representing 1× coverage. In this case, p0=e−1=0.37, thus approximately 37% is unsequenced. For example, 5× coverage (approximately 9500 clones sequenced from both insert ends and an average sequence read length of 460 bp) yields p0=e−5=0.0067, or 0.67% unsequenced. The total gap length is Le−m, and the average gap size is L/n. 5× coverage would leave about 128 gaps averaging about 100 bp in size. The treatment is essentially that of Lander and Waterman, Genomics 2:231 (1988). Table 4 illustrates the coverage for a 1.9 Mb genome with an average fragment size of 460 bp.
2. Random Library Construction
In order to approximate the random model described above during actual sequencing, a nearly ideal library of cloned genomic fragment is required. The following library construction procedure was developed to achieve this.
H. influenzae Rd KW20 DNA was prepared by phenol extraction. A mixture (3.3 ml) containing 600 μg DNA, 300 mM sodium acetate, 10 mM Tris-HCl, 1 mM Na-EDTA, 30% glycerol was sonicated (Branson Model 450 Sonicator) at the lowest energy setting for 1 min. at 0° using a 3 mm probe. The DNA was ethanol precipitated and redissolved in 500 μl TE buffer. To create blunt-ends, a 100 μl aliquot was digested for 10 min at 30° in 200 μl BAL31 buffer with 5 units BAL31 nuclease (New England BioLabs). The DNA was phenol-extracted, ethanol-precipitated, redissolved in 100 μl TE buffer, electrophoresed on a 1.0% low melting agarose gel, and the 1.6-2.0 kb size fraction was excised, phenol-extracted, and redissolved in 20 μl TE buffer. A two-step ligation procedure was used to produce a plasmid library with 97% insert of which >99% were single inserts. The first ligation mixture (50 μl) contained 2 μg of DNA fragments, 2 μg SmaI/BAP pUC18 DNA (Pharmacia), and 10 units T4 ligase (GIBCO/BRL), and incubation was at 14° for 4 hr. After phenol extraction and ethanol precipitation, the DNA was dissolved in 20 μl TE buffer and electrophoresed on a 1.0% low melting agarose gel. A ladder of ethidium bromide-stained linear bands, identified by size as insert (i), vector (v), v+i, v+2i, v+3i, . . . was visualized by 360 nm UV light, and the v+i DNA was excised and recovered in 20 μl TE. The v+i DNA was blunt-ended by T4 polymerase treatment for 5 min. at 37° in a reaction mixture (50 μl) containing the v+i linears, 500 μM each of the 4 dNTP's, and 9 units of T4 polymerase (New England BioLabs) under recommended buffer conditions. After phenol extraction and ethanol precipitation the repaired v+i linears were dissolved in 20 μl TE. The final ligation to produce circles was carried out in a 50 μl reaction containing 5 μl of v+i linears and 5 units of T4 ligase at 14° overnight. After 10 min. at 70° the reaction mixture was stored at −20°.
This two-stage procedure resulted in a molecularly random collection of single-insert plasmid recombinants with minimal contamination from double-insert chimeras (<1%) or free vector (<3%). Since deviation from randomness is most likely to occur during cloning, E. coli host cells deficient in all recombination and restriction functions (A. Greener, Strategies 3 (I):5 (1990)) were used to prevent rearrangements, deletions, and loss of clones by restriction. Transformed cells were plated directly on antibiotic diffusion plates to avoid the usual broth recovery phase which allows multiplication and selection of the most rapidly growing cells. Plating occured as follows:
A 100 μl aliquot of Epicurian Coli SURE II Supercompetent Cells (Stratagene 200152) was thawed on ice and transferred to a chilled Falcon 2059 tube on ice. A 1.7 μl aliquot of 1.42 M β-mercaptoethanol was added to the aliquot of cells to a final concentration of 25 mM. Cells were incubated on ice for 10 min. A 1 μl aliquot of the final ligation was added to the cells and incubated on ice for 30 min. The cells were heat pulsed for 30 sec. at 42° and placed back on ice for 2 min. The outgrowth period in liquid culture was eliminated from this protocol in order to minimize the preferential growth of any given transformed cell. Instead the transformation were plated directly on a nutrient rich SOB plate containing a 5 ml bottom layer of SOB agar (1.5% SOB agar: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 1.5% Difco Agar/L). The 5 ml bottom layer is supplemented with 0.4 ml ampicillin (50 mg/ml)/100 ml SOB agar. The 15 ml top layer of SOB agar is supplemented with 1 ml X-Gal (2%), 1 ml MgCl2 (1 M), and 1 ml MgSO4/100 ml SOB agar. The 15 ml top layer was poured just prior to plating. Our titer was approximately 100 colonies/10 μl aliquot of transformation.
All colonies were picked for template preparation regardless of size. Only clones lost due to “poison” DNA or deleterious gene products would be deleted from the library, resulting in a slight increase in gap number over that expected.
In order to evaluate the quality of the H. influenzae library, sequence data were obtained from approximately 4000 templates using the M13-21 primer. The random sequence fragments were assembled using the AutoAssemblerTM software (Applied Biosystems division of Perkin-Elmer (AB)) after obtaining 1300, 1800, 2500, 3200, and 3800 sequence fragments, and the number of unique assembled base pairs was determined. Based on the equations described above, an ideal plot of the number of base pairs remaining to be sequenced as a function of the # of sequenced fragments obtained with an average read length of 460 bp for a 2.5×106 and a 1.9×106 bp genome was determined (FIG. 3). The progression of assembly was plotted using the actual data obtained from the assembly of up to 3800 sequence fragments and compared the data that is provided in the ideal plot (FIG. 3). FIG. 3 illustrates that there was essentially no deviation of the actual assembly data from the ideal plot, indicating that we had constructed close to an ideal random library with minimal contamination from double insert chimeras and free of vector.
3. Random DNA Sequencing
High quality double stranded DNA plasmid templates (19,687) were prepared using a “boiling bead” method developed in collaboration with Advanced Genetic Technology Corp. (Gaithersburg, Md.) (Adams et al., Science 252:165:1 (1991); Adams et al., Nature 355:632 (1992)). Plamid preparation was performed in a 96-well format for all stages of DNA preparation from bacterial growth through final DNA purification. Template concentration was determined using Hoechst Dye and a Millipore Cytofluor. DNA concentrations were not adjusted, but low-yielding templates were identified where possible and not sequenced. Templates were also prepared from two H. influenzae lambda genomic libraries. An amplified library was constructed in vector Lambda GEM-12 (Promega) and an unamplified library was constructed in Lambda DASH II (Stratagene). In particular, for the unamplified lambda library, H. influenzae Rd KW20 DNA (>100 kb) was partially digested in a reaction mixture (200 μl) containing 50 μg DNA, 1× Sau3AI buffer, 20 units Sau3AI for 6 min. at 23°. The digested DNA was phenol-extracted and electrophoresed on a 0.5% low melting agarose gel at 2V/cm for 7 hours. Fragments from 15 to 25 kb were excised and recovered in a final volume of 6 μl. One μl of fragments was used with 1 μl of DASHII vector (Stratagene) in the recommended ligation reaction. One μl of the ligation mixture was used per packaging reaction following the recommended protocol with the Gigapack II XL Packaging Extract (Stratagene, #227711). Phage were plated directly without amplification from the packaging mixture (after dilution with 500 μl of recommended SM buffer and chloroform treatment). Yield was about 2.5×103 pfu/μl. The amplified library was prepared essentially as above except the lambda GEM-12 vector was used. After packaging, about 3.5×103 pfu were plated on the restrictive NM539 host. The lysate was harvested in 2 ml of SM buffer and stored frozen in 7% dimethylsulfoxide. The phage titer was approximately 1×109 pfu/ml.
Liquid lysates (10 ml) were prepared from randomly selected plaques and template was prepared on an anion-exchange resin (Qiagen). Sequencing reactions were carried out on plasmid templates using the AB Catalyst LabStation with Applied Biosystems PRISM Ready Reaction Dye Primer Cycle Sequencing Kits for the M13 forward (M13-21) and the M13 reverse (M13RP1) primers (Adams et al., Nature 368:474 (1994)). Dye terminator sequencing reactions were carried out on the lambda templates on a Perkin-Elmer 9600 Thermocycler using the Applied Biosystems Ready Reaction Dye Terminator Cycle Sequencing kits. T7 and SP6 primers were used to sequence the ends of the inserts from the Lambda GEM-12 library and T7 and T3 primers were used to sequence the ends of the inserts from the Lambda DASH II library. Sequencing reactions (28,643) were performed by eight individuals using an average of fourteen AB 373 DNA Sequencers per day over a 3 month period. All sequencing reactions were analyzed using the Stretch modification of the AB 373, primarily using a 34 cm well-to-read distance. The overall sequencing success rate was 84% for M13-21 sequences, 83% for M13RP1 sequences and 65% for dye-terminator reactions. The average usable read length was 485 bp for M13-21 sequences, 444 bp for M13RP1 sequences, and 375 bp for dye-terminator reactions. Table 5 summarizes the high-throughput sequencing phase of the invention.
Richards et al. (Richards et al., Automated DNA sequencing and Analysis, M. D. Adams, C. Fields, J. C. Venter, Eds. (Academic Press, London, 1994), Chap. 28.) described the value of using sequence from both ends of sequencing templates to facilitate ordering of contigs in shotgun assembly projects of lambda and cosmid clones. We balanced the desirability of both-end sequencing (including the reduced cost of lower total number of templates) against shorter read-lengths for sequencing reactions performed with the M13RP1 (reverse) primer compared to the M13-21 (forward) primer. Approximately one-half of the templates were sequenced from both ends. In total, 9,297 M13RP1 sequencing reactions were done. Random reverse sequencing reactions were done based on successful forward sequencing reactoins. Some M13RP1 sequences were obtained in a semi-directed fashion: M 13-21 sequences pointing outward at the ends of contigs were chosen for M13RP1 sequencing in an effort to specifically order contigs. The semi-directed strategy was effective, and clone-based ordering formed an integral part of assembly and gap closure (see below).
4. Protocol for Automated Cycle Sequencing
The sequencing consisted of using eight ABI Catalyst robots and fourteen AB 373 Automated DNA Sequencers. The Catalyst robot is a publicly available sophisticated pipetting and temperature control robot which has been developed specifically for DNA sequencing reactions. The Catalyst combines pre-aliquoted templates and reaction mixes consisting of deoxy- and dideoxynucleotides, the Taq thermostable DNA polymerase, fluorescently-labelled sequencing primers, and reaction buffer. Reaction mixes and templates were combined in the wells of an aluminum 96-well thermocycling plate. Thirty consecutive cycles of linear amplification (e.g., one primer synthesis) steps were performed including denaturation, annealing of primer and template, and extension of DNA synthesis. A heated lid with rubber gaskets on the thermocycling plate prevented evaporation without the need for an oil overlay.
Two sequencing protocols were used: dye-labelled primers and dye-labelled dideoxy chain terminators. The shotgun sequencing involves use of four dye-labelled sequencing primers, one for each of the four terminator nucleotide. Each dye-primer is labelled with a different fluorescent dye, permitting the four individual reactions to be combined into one lane of the 373 DNA Sequencer for electrophoresis, detection, and base-calling. AB currently supplies pre-mixed reaction mixes in bulk packages containing all the necessary non-template reagents for sequencing. Sequencing can be done with both plasmid and PCR-generated templates with both dye-primers and dye-terminators with approximately equal fidelity, although plasmid templates generally give longer usable sequences.
Thirty-two reactions were loaded per 373 Sequencer each day, for a total of 960 samples. Electrophoresis was run overnight following the manufacture's protocols, and the data was collected for twelve hours. Following electrophoresis and fluorescence detection, the AB 373 performs automatic lane tracking and base-calling. The lane-tracking was confirmed visually. Each sequence electropherogram (or fluorescence lane trace) was inspected visually and assessed for quality. Trailing sequences of low quality were removed and the sequence itself was loaded via software to a Sybase database (archived daily to a 8 mm tape). Leading vector polylinker sequence was removed automatically by software program. Average edited lengths of sequences from the standard ABI 373 were around 400 bp and depended mostly on the quality of the template used for the sequencing reaction. All of the ABI 373 Sequencers were converted to Stretch Liners, which provided a longer electrophoresis path prior to fluorescence detection, thus increasing the average number of usable bases to 500-600 bp.
Informatics
1. Data Management
A number of information management systems (LIMA) for a large-scale sequencing lab have been developed (Kerlavage et al., Proceedings of the twenty-Sixth Annual Hawaii International Conference on System Sciences, IEEE Computer Society Press, Washington D.C., 585 (1993)). The system used to collect and assemble the sequence data was developed using the Sybase relational data management system and was designed to automate data flow whereever possible and to reduce user error. The database stores and correlates all information collected during the entire operation from template preparation to final analysis of the genome. Because the raw output of the AB 373 Sequencers was based on a Macintosh platform and the data management system chosen was based on a Unix platform, it was necessary to design and implement a variety of multi-user, client server applications which allow the raw data as well as analysis results to flow seamlessly into the database with a minimum of user effort. A description of the software programs used for large sequence assembly and management is provided in FIG. 4.
2. Assembly
An assembly engine (TIGR Assembler) was developed for the rapid and accurate assembly of thousands of sequence fragments. The AB AutoAssemblerTM was modified (and named TIGR Editor) to provide a graphical interface to the electropherogram for the purpose of editing data associated with the aligned sequence file output of TIGR Assembler. TIGR Editor maintains synchrony between the electropherogram files on the Macintosh platform and the sequence data in the H. influenzae database on the Unix platform.
The TIGR assembler simultaneously clusters and assembles fragments of the genome. In order to obtain the speed necessary to assemble more than 104 fragments, the algorithm builds a hash table of 10 bp oligonucleotide subsequences to generate a list of potential sequence fragment overlaps. The number of potential overlaps for each fragment determines which fragments are likely to fall into repetitive elements. Beginning with a single seed sequence fragment, TIGR Assembler extends the current contig by attempting to add the best matching fragment based on oligonucleotide content. The current condg and candidate fragment are aligned using a modified version of the Smith-Waterman algorithm (Waterman, M. S., Methods in Enzymology 164:765 (1988)) which provides for optimal gapped alignments. The current contig is extended by the fragment only if strict criteria for the quality of the match are met. The match criteria include the minimum length of overlap, the maximum length of an unmatched end, and the minimum percentage match. These criteria are automatically lowered by the algorithm in regions of minimal coverage and raised in regions with a possible repetitive element. The number of potential overlaps for each fragment determines which fragments are likely to fall into repetitive elements. Fragments representing the boundaries of repetitive elements and potentially chimeric fragments are often rejected based on partial mismatches at the ends of alignments and excluded from the current contig. TIGR Assembler is designed to take advantage of clone size information coupled with sequencing from both ends of each template. It enforces the constraint that sequence fragments from two ends of the same template point toward one another in the contig and are located within a certain ranged of base pairs (definable for each clone based on the known clone size range for a given library). Assembly of 24,304 sequence fragments of H. influenzae required 30 hours of CPU time using one processor on a SPARCenter 2000 with 512 Mb of RAM. This process resulted in approximately 210 contigs. Because of the high stringency of the TIGR Assembler, all contigs were searched against each other using grasta (a modified fasta (Person and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988)). In this way, additional overlaps were detected which enabled compression oof the data set into 140 contigs. The location of each fragment in the contigs and extensive information about the consensus sequence itself were loaded into the H. influenzae relational database.
3. Ordering Assembled Contigs
After assembly the relative positions of the 140 contigs were unknown. The contigs were ordered by asm_align. Asm_align uses a number of relationships to identify and align contigs that are adjacent to each other. Using this algorithm, the 140 contigs were placed into 42 groups totaling 42 physical gaps (no template DNA for the region) and 98 sequence gaps (template available for gap closure).
Ordering Contigs Separated by Physical Gaps and Achieving Closure
Four integrated strategies were developed to order contigs separated by physical gaps. Oligonucleotide primers were designed and synthesized from the end of each contig group. These primers were then available for use in one or more of the strategies outlined below:
1. Southern analysis was done to develop a unique “fingerprint” for a subset of 72 of the above oligonucleotides. This procedure was based upon the supposition that labeled oligonucleotides homologous to the ends of adjacent contigs should hybridize to common DNA restriction fragments, and thus share a similar or identical hybridization pattern or “fingerprint”. Oligonucleotides were labeled using 50 pmoles of each 20 mer and 250 mCi of [γ-32P]ATP and T4 polynucleotide kinase. The labeled oligonucleotides were purified using Sephadex G-25 superfine (Pharmacia) and 107 cpm of each was used in a Southern hybridization analysis of H. influenzae Rd chromosomal DNA digested with one frequent cutters (Asel) and five less frequent cutters (BglII, EcoRI, PstI, XbaI, and PvuII). The DNA from each digest was fractionated on a 0.7% agarose gel and transferred to Nytran Plus nylon membranes (Schleicher & Schuell). Hybridization was carried out for 16 hours at 40°. To remove non-specific signals, each blot was sequentially washed at room temperature with increasingly stringent conditions up to 0.1×SSC+0.5% SDS. Blots were exposed to a PhosphorImager cassette (Molecular Dynamics) for several hours and hybridization patterns were visually compared.
Adjacent contigs identified in this manner were targeted for specific PCR reactions.
2. Peptide links were made by searching each contig end using blastx (Altschul et al., J. Mol. Biol. 215:403 (1990)) against a peptide database. If the ends of two contigs matched the same database sequence in an appropriate manner, then the two contigs were tentatively considered to be adjacent to each other.
3. The two lambda libraries constructed from H. influenaze genomic DNA were probed with oligonucleotides designed from the ends of contig groups (Kirkness et al., Genomics 10:985 (1991)). The positive plaques were then used to prepare templates and the sequence was determined from each end of the lambda clone insert. These sequence fragments were searched using grasta against a database of all contigs. Two contigs that matched the sequence from the opposite ends of the same lambda clone were ordered. The lambda clone then provided the template for closure of the sequence gap between the adjacent contigs. The lambda clones were especially valuable for solving repeat structures.
4. To confirm the order of contigs found by the other approaches and establish the order of non-ordered contigs, standard and long range (XL) PCR reactions were performed as follows.
Standard PCR was performed in the following manner. Each reaction contained a 37 μl cocktail; 16.5 μl H2O, 3 μl 25 mM MgCl2, 8 μl of a dNTP mix (1.25 mM each dNTP), 4.5 μl 10×PCR core buffer II (Perkin Elmer), 25 ng H. influenzae Rd KW20 genomic DNA. The appropriate two primers (4 μl, 3.2 pmole/μl) were added to each reaction. A hot start was performed at 95° for 5 min followed by a 75° hold. During the hold Amplitaq DNA polymerase (Perkin Elmer) 0.3 μl in 4.3 μl H2O, 0.5 μl 10×PCR core buffer II, was added to each reaction. The PCR profile was 25 cycles of 94°/45 sec., denature; 55°/1 min., anneal; 72°/3 min, extension. All reactions were performed in a 96 well format on a Perkin Elmer GeneAmp PCR System 9600.
Long range PCR (XL PCR) was performed as follows: Each reaction contained a 35.2 μl cocktail; 12.0 μl H2O, 2.2 μl 25 mM Mg(OAc)2, 4 μl of a dNTP mix (200 μM final concentration), 12.0 μl 3.3×PCR buffer, 25 ng H. influenzae Rd KW20 genomic DNA. The appropriate two primers (5 μl, 3.2 pmoles/μl) was added to each reaction. A hot start was performed at 94° for 1 minute. rTth polymerase, 2.0 μl (4 U/reaction) in 2.8 μl 3.3×PCR buffer II was added to each reaction. The PCR profile was 18 cycles of 94°/15 sec., denature; 62°/8 min., anneal and extend followed by 12 cycles 94°/15 sec., denature; 62°/8 min. (increase 15 sec./cycle), anneal and extend; 72°/10 min., final extension. All reactions were performed in a 96 well format on a Perkin Elmer GeneAmp PCR System 9600.
Although a PCR reaction was performed for essentially every combination of physical gap ends, techniques such as Southern fingerprinting, database matching, and the probing of large insert clones were particularly valuable in ordering contigs adjacent to each other and reducing the number of combinatorial PCR reactions necessary to achieve complete gap closure. Employing these strategies to an even greater extent in future genome projects will increase the overall efficiency of complete genome closure. The number of physical gaps ordered and closed by each of these techniques is summarized in Table 5.
Sequence information from the ends of 15-20 kb clones is particularly suitable for gap closure, solving repeat structures, and providing general confirmation of the overall genome assembly. We were also concerned that some fragments of the H. influenaze genome would be non-clonable in a high copy plasmid in E. coli. We reasoned that lytic lambda clones would provide the DNA for these segments. Approximately 100 random plaques were picked from the amplified lambda library, templates prepared, and sequence information obtained from each end. These sequences were searched (grasta) against the contigs and linked in the database to their appropriate contig, thus providing a scaffolding of lambda clones contributing additional support to the accuracy of the genome assembly (FIG. 5). In addition to confirmation of the contig structure, the lambda clones provided closure for 23 physical gaps. Approximately 78% of the genome is covered by lambda clones.
Lambda clones were also useful for solving repeat structures. Repeat structures identified in the genome were small enough to be spanned by a single clone from the random insert library, except for the six ribosomal RNA operons and one repeat (2 copies) which was 5,340 bp in length. Oligonucleotide probes were designed from the unique flanks at the beginning of each repeat and hybridized to the lambda libraries. Positive plaques were identified for each flank and the sequence fragments from the ends of each clone were used to correctly orient the repeats within the genome.
The ability to distinguish and assemble the six ribosomal RNA (rRNA) operons of H. influenaze (16S subunit-23S subunit-5S subunit) was a test of our overall strategy to sequence and assemble a complex genome which might contain a significant number of repeat regions. The high degree of sequence similarity and the length of the six operons caused the assembly process to cluster all the underlying sequences into a few indistinguishable contigs. To determine the correct placement of the operons in the sequence, a pair of unique flanking sequences was required for each. No unique flanking sequences could be found at the left (16S rRNA) ends. This region contains the ribosomal promoter and appeared to be non-clonable in the high copy number pUC18 plasmid. However, unique sequences could be identified at the right (5S) ends. Oligonucleotide primers were designed from these six flanking regions and used to probe the two lambda libraries. For each of the six rRNA operons at least one positive plaque was identified which completely spanned the rRNA operon and contained unique flanking sequence at the 16S and 5S ends. These plaques provided the templates for obtaining the unique sequence for each of the six rRNA operons.
An additional confirmation of the global structure of the assembled circular genome was obtained by comparing a computer generated restriction map based on the assembled sequence for the enzymes Apal, SmaI, and RsrII with the predicted physical map of Redfield and Lee (Genetic Maps: locus maps of complex genomes, S. J. O'Brien, Ed. Cold Spring Harbor Laboratory Press, New York, N.Y., 1990, 2110.). The restriction fragments from the sequence-derived map matched those from the physical map in size and relative order (FIG. 5).
Editing
Simultaneous with the final gap filling process, each contig was edited visually by reassembling overlapping 10 kb sections of contigs using the AB AutoAssemblerTM and the Fast Data FindersTM hardware. AutoAssemblerTM provides a graphical interface to electropherogram data for editing. The electropherogram data was used to assign the most likely base at each position. Where a discrepancy could not be resolved or a clear assignment made, the automatic base calls were left unchanged. Individual sequence changes were written to the electropherogram files and a replication protocol (crash) was used to maintain the synchrony of sequence data between the H. influenzae database and the electropherogram files. Following editing, contigs were reassembled with TIGR Assembler prior to annotation.
Potential frameshifts identified in the course of annotating the genome were saved as reports in the database. These reports include the coordinates in a contig which the alignment software (praze) predicts to be the most likely location of a missing or inserted base and a representation of the sequence alignment containing the frameshift. Apparent frameshifts were used to indicate areas of the sequence which may require further editing. Frameshifts were not corrected in cases where clear electropherogram data disagreed with a frameshift. Frameshift editing was performed with TIGR Editor.
The rRNA and other repeat regions precluded complete assembly of the circular genome with TIGR Assembler. Final assembly of the genome was accomplished using comb_asm which splices together contigs based on short overlaps.
Accuracy of the Genome Sequence
The accuracy of the H. influenaze genome sequence is difficult to quantitate because there is very little previously determined H. influenaze sequence and most of these sequences are from other strains. There are, however, three parameters of accuracy that can be applied to the data. First, the number of apparent frameshifts in predicted H. influenaze genes, based on database similarities, is 148. Some of these apparent frameshifts may be in the database sequences rather than in ours, particularly considering that 49 of the apparent frameshifts are based on matches to hypothetical proteins from other organisms. Second, there are 188 bases in the genome that remain as N ambiguities (1/9,735 bp). Combining these two types of “known” errors, we can calculate a maximum sequence accuracy of 99.98%. The average coverage is 6.5× and less than 1% of the genome is single-fold coverage.
Identifying Genes
An attempt was made to predict all of the coding regions of the H. influenzae Rd genome and identify genes, tRNAs and rRNAs, as well as other features of the DNA sequence (e.g., repeats, regulatory sites, replication origin sites, nucleotide composition). A description of some of the readily apparent sequence features is provided below.
The H. influenaze Rd genome is a circular chromosome of 1,830,121 bp. The overall G/C nucleotide content is approximately 38% (A=31%, C=19%, G=19%, T=31%, IUB=0.035%). The G/C content of the genome was examined with several window lengths to look for global structural features. With a window of 5,000 bp, the GIC content is relatively even except for 7 large G/C-rich regions and several A/T-rich regions (FIG. 5). The G/C rich regions correspond to six rRNA operons and the location of a cryptic mu-like prophage. Genes for several proteins with similarity to proteins encoded by bacteriophage mu are located at approximately position 1.56-1.59 Mbp of the genome. This area of the genome has a markedly higher G/C content than average for H. influenaze ( ̃50% G/C compared to  ̃38% for the rest of the genome). No significance has yet been ascertained for the source or importance of the A/T rich regions.
The minimal origin of replication (oriC) in E. coli is a 245 bp region defined by three copies of a thirteen base pair repeat containing a GATC core sequence at one end and four copies of a nine base pair repeat containing a TTAT core sequence at the other end. The GATC sites are methylation targets and control replication while the TTAT sites provide the binding sites for DnaA, the first step in the replication process (Genes V, B. Lewin Ed. (Oxford University Press, New York, 1994), chap. 18-19). An approximately 281 bp sequence (602,483-602,764) whose limits are defined by these same core sequences appears to define the origin of replication in H. influenaze Rd. These coordinates lie between sets of ribosomal operons rrnF, rrnE, rrnD and rrnA, rrnB, rrnC. These two groups of ribosomal operons are transcribed in opposite directions and the placement of the origin is consistent with their polarity for transcription. Termination of E. coli replication is marked by two 23 bp termination sequences located  ̃100 kb. on either side of the midway point at which the two replication forks meet. Two potential termination sequences sharing a 10 bp core sequence with the E. coli termination sequence were identified in H. influenaze at coordinates 1,375,949-1,375,958 and 1,558,759-1,558,768. These two sets of coordinates are offset approximately 100 kb from the point 180° opposite of the proposed origin of H. influenaze replication.
Six rRNA operons were identified. Each rRNA operon contains three rRNA subunits and a variable spacer region in the order: 16S subunit-spacer region-23S subunit-5S subunit. The subunit lengths are 1539 bp, 2653 bp, and 116 bp. respectively. The G/C content of the three ribosomal subunits (50%) is higher than the genome as a whole. The G/C content of the spacer region (38%) is consistent with the remainder of the genome. The nucleotide sequence of the three rRNA subunits is 100% identical in all six ribosomal operons. The rRNA operons can be grouped into two classes based on the spacer region between the 16S and 23S sequences. The shorter of the two spacer regions is 478 bp in length (rrnB, rrnE, and rrnF) and contains the gene for tRNA Glu. The longer spacer is 723 bp in length (rrnA, rrnC, and rrnD) and contains the genes for tRNA Ile and tRNA Ala. The two sets of spacer regions are also 100% identical across each group of three operons. tRNA genes are also present at the 16S and 5S ends of two of the rRNA operons. The genes for tRNA Arg, tRNA His, and tRNA Pro are located at the 16S end of rrnE while the genes for tRNA Trp, and tRNA Asp are located at the 5S end of rrnA.
The predicted coding regions of the H. influenaze genome were initially defined by evaluating their coding potential with the program Genemark (Borodovsky and McIninch, Computers Chem. 17(2):123 (1993)) using codon frequency matrices derived from 122 H. influenaze coding sequences in GenBank. The predicted coding region sequences (plus 300 bp of flanking sequence) were used in searches against a database of non-redundant bacterial proteins (NRBP) created specifically for the annotation. Redundancy was removed from NRBP at two stages. All DNA coding sequences were extracted from GenBank (release 85), and sequences from the same species were searched against each other. Sequences having >97% similarity over regions >100 nucleotides were combined. In addition, the sequences were translated and used in protein comparisons with all sequences in Swiss-Prot (release 30). Sequences belonging to the same species and having >98% similarity over 33 amino acids were combined. NRBP is composed of 21,445 sequences extracted from 23,751 GenBank sequences and 11,183 Swiss-Prot sequences from 1,099 different species.
A total of 1,749 predicted coding regions were identified. Searches of the H. influenzae predicted coding regions were performed using an algorithm that translates the query DNA sequence in the three plus-strand reading frames for searching against NRBP, identifies the protein sequences that match the query, and aligns the protein-protein matches using praze, a modified Smith-Waterman (Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444 (1988)) algorithm. In cases where insertion or deletions in the DNA sequence produced a frameshift error, the alignment algorithm started with protein regions of maximum similarity and extended the alignment tothe same database match in alternative frames using the 300 bp flanking region. Regions known to contain frameswft errors were saved in the database and evaluated for possible correction. Unidentified predicted coding regions and the remaining intergenic sequences were searched against a dataset of all available peptide sequences from Swiss-Prot, PIR, and GenBank. Identification of operon structures will be facilitated by experimental determination of transcription promoter and termination sites.
Each putatively identified H. influenaze gene was assigned to one of 102 biological role categories adapted from Riley (Riley, M., Microbiology Reviews 57(4):862 (1993)). Assignments were made by linking the protein sequence of the predicted coding regions with the Swiss-Prot sequences in the Riley database. Of the 1,749 predicted coding regions, 724 have no role assignment. Of these, no database match was found for 384, while340 matched “hypothetical proteins” in the database. Role assignments were made for 1,025 of the predicted coding regions. A compilation of all the predicted coding regions, their unique identifiers, a three letter gene identifier, percent identity, percent similarity, and amino acid match length are presented in Table 1(a).
An annotated complete genome map of H. influenaze Rd is presented in FIGS. 6(A) to 6(AN). The map places each predicted coding region on the H. influenaze chromosome and indicates its direction of transcription.
A survey of the genes and their chromosomal organization in H. influenaze Rd make possible a description of the metabolic processes H. influenaze requires for survival as a free living organism, the nutritional requirements for its growth in the laboratory, and the characteristics which make it unique from other organisms specifically as it relates to its pathogenicity and virulence. The genome would be expected to have complete complements of certain classes of genes known to be essential for life. For example, there is a one-to-one correspondence of published E. coli ribosomal protein sequences to potential homologs in the H. influenaze database. Likewise, as shown in Table 1(a), an aminoacyl tRNA-synthetase is present in the genome for each amino acid. Finally, the location of tRNA genes was mapped onto the genome. There are 54 identified tRNA genes, including representatives of all 20 amino acids.
In order to survive as a free living organism, H. influenaze must produce energy in the form of ATP via fermentation and/or electron transport. As a facultative anaerobe, H. influenaze Rd is known to ferment glucose, fructose, galactose, ribose, xylose and fucose (Dorocicz et al., J. Bacteriol. 175:7142 (1993)). The genes identified in Table 1(a) indicate that transport systems are available for the uptake of these sugars via the phosphoenolpyruvate-phosphotransferase system (PTS), and via non-PTS mechanisms. Genes that specify the common phosphatecarriers Enzyme I and Hpr (ptsI and ptsH) of the PTS system were identified as well as the glucose specific crr gene. The ptsH, ptsI, and crr genes constitute the pts operon. We have not however identified the gene encoding membrane-bound glucose specific Enzyme II. The latter enzyme is required for transport of glucose by the PTS system. A complete FTS system for fructose was identified.
Genes encoding the complete glycolytic pathway and for the production of fermentative end products were identified. Growth utilizing anaerobic respiratory mechanisms were found by identifying genes encoding functional electron transport systems using inorganic electron acceptors such as nitrates, nitrites, and dimethylsulfoxide. Genes encoding three enzymes of the tricarboxylic acid (TCA) cycle appear to be absent from the genome. Citrate synthase, isocitrate dehydrogenase, and acordtase were not found by searching the predicted coding regions or by using the E. coli enzymes as peptide queries against the entire genome in translation. This provides an explanation for the very high level of glutamate (lg/L) which is required in defined culture media (Klein and Luginbuhl, J. Gen. Microbiol. 113:409 (1979)). Glutamate can be directed into the TCA cycle via conversion to alpha-ketoglutarate by glutamate dehydrogenase. In the absence of a complete TCA cycle, glutamate presumably serves as the source of carbon for biosynthesis of amino acids using precursors which branch from the TCA cycle. Functional electron transport systems are available for the production of ATP using oxygen as a terminal electron acceptor.
Previously unanswered questions regarding pathogenicity and virulence can be addressed by examining certain classes of genes such as adhesions and the lipooligosaccharide biogenesis genes. Moxon and co-workers (Weiser et al., Cell 59:657 (1989)) have obtained evidence that a number of these virulence-related genes contain tandem tetramer repeats which undergo frequent addition and deletion of one or more repeat units during replication such that the reading frame of the gene is changed and its expression thereby altered. It is now possible, using the complete genome sequence, to locate all such tandem repeat tracts (FIG. 5) and to begin to determine their roles in phase variation of such potential virulence genes.
H. influenzae Rd possesses a highly efficient natural DNA transformation system (Kahn and Smith, J. Membrane Biol. 138:155 (1984). A unique DNA uptake sequence site, 5′ AAGTGCGGT, present in multiple copies in the genome, has been shown to be necessary for efficient DNA uptake. It is now possible to locate all of these sites and completely describe their distribution with respect to genic and intergenic regions. Fifteen genes involved in transformation have already been described and sequenced (Redfield, R., J. Bacteriol. 173:5612 (1991); Chandler, M., Proc. Natl. Acad. Sci. U.S.A 89:1616 (1992); Barouki and Smith, J. Bacterol. 163(2):629 (1985); Tomb et al., Gene 104:1 (1991); Tomb, J, Proc. Natl. Acad. Sci. U.S.A 89:10252 (1992)). Six of the genes, comA to comF, comprise an operon which is under positive control by a 22-bp palindromic competence regulatory element (CRE) about one helix turn upstream of the promoter. The rec-2 transformation gene is also controlled by this element. It is now possible to locate additional copies of CRE in the genome and discover potential transformation genes under CRE control. In addition, it may now be possible to discover other global regulatory elements with an ease not previously possible.
One well-described gene regulatory system in bacteria is the “two-component” system composed of a sensor molecule that detects some sort of environmental signal and a regulator molecule that is phosphorylated by the activated form of the sensor. The regulator protein is generally a transcription factor which, when activated by the sensor, turns on or off expression of a specific set of genes (for review, see Albright et al., Ann. Rev. Genet. 23:311 (1989); Parkinson and Kofoid, Ann. Rev. Genet. 26:71 (1992)). It has been estimated that E. coli harbors 40 sensor-regulator pairs (Albright et al., Ann. Rev. Genet. 23:311 (1989); Parkinson and Kofoid, Ann. Rev. Genet. 26:71 (1992)). The H. influenaze genome was searched with representative proteins from each family of sensor and regulator proteins using tblastn and tfasta. Four sensor and five regulator proteins were identified with similarity to proteins from other species (Table 6). There appears to be a corresponding sensor for each regulator protein except CpxR. Searches with the CpxA protein from E. coli identified three of the four sensors listed in Table 6, but no additional significant matches were found. It is possible that the level of sequence similarity is low enough to be undetectable with tfasta. No representatives of the NtrC-class of regulators were found. This class of proteins interacts directly with the sigma-54 subunit of RNA polymerase, which is not present in H. influenaze. All of the regulator proteins fall into the OmpR subclass (Albright et al., Ann. Rev. Genet. 23:311 (1989); Parkinson and Kofoid, Ann. Rev. Genet. 26:71 (1992)). The phoBR and basRS genes of H. influenaze are adjacent to one another and presumably form an operon. The nar and arc genes are not located adjacent to one another.
Some of the most interesting questions that can be answered by a complete genome sequence relate to what genes or pathways are absent. The non-pathogenic H. influenaze Rd strain varies significantly from the pathogenic serotype b strains. Many of the differences between these two strains appear in factors affecting infectivity. For example, the eight genes which make up the fimbrial gene cluster (vanHam et al., Mol. Microbiol. 13:673 (1994)) involved in adhesion of bacteria to host cells are now shown to be absent in the Rd strain. The pepN and purE genes which flank the fimbrial cluster in H. influenaze type b strains are adjacent to one another in the Rd strain (FIG. 7), suggesting that the entire fimbrial duster was excised. On a broader level, we determined which E coli proteins are not in H. influenzae by taking advantage of non-redundant set of protein coding genes from E. coli, namely the University of Wisconsin Genome Project contigs in GenBank: 1,216 predicted protein sequences from GenBank accessions D10483, L10328, U00006, U00039, U14003, and U18997 (Yura et al., Nucleic Acids Research 20:3305 (1992); Burland et al., Genomics 16:551 (1993)). The minimum threshold for matches was set so that even weak matches would be scored as positive, thereby giving a minimal estimate of the E. coli genes not present in H. influenaze. tblastn was used to search each of the E. coli proteins against the complete genome. All blast scores >100 were considered matches. Altogether 627 E. coli proteins matched at least one region of the H. influenaze genome and 589 proteins did not The 589 non-matching proteins were examined and found to contain a disproportionate number of hypothetical proteins from E. coli. Sixty-eight percent of the identified E. coli proteins were matched by an H. influenaze sequence whereas only 38% of the hypothetical proteins were matched. Proteins are annotated as hypothetical based on a lack of matches with any other known protein (Yura et al., Nucleic Acids Research 20:3305 (1992); Burland et al., Genomics 16:551 (1993)). At least two potential explanations can be offered for the over representation of hypothetical proteins among those without matches: some of the hypothetical proteins are not, in fact, translated (at least in the annotated frame), or these are E. coli-specific proteins that are unlikely to be found in any species except those most closely related to E. coli, for example Salmonella typhimurium. 
A total of 384 predicted coding regions did not display significant similarity with a six-frame translation of GenBank release 87. These unidentified coding regions were compared to one another with fasta. Several novel gene families were identified. For example, two predicted coding regions without database matches (H10591, H10852) share 75% identity over almost their entire lengths (139 and 143 amino acid residues respectively). Their similarity to each other but failure to match any protein available in the current databases suggest that they could represent a novel cellular function.
Other types of analyses can be applied to the unidentified coding regions, including hydropathy analysis, which indicates the patterns of potential membrane-spanning domains that are often conserved between members of receptor and transporter gene families, even in the absence of significant amino acid identity. Five examples of unidentified predicted coding regions that display potential transmembrane domains with a periodic pattern that is characteristic of membrane-bound channel proteins are shown in FIG. 8. Such information can be used to focus on specific aspects of cellular function that are affected by targeted deletion or mutation of these genes.
Interest in the medically important aspects of H. influenaze biology has focused particularly on those genes which determine virulence characteristics of the organism. Recently, the catalase gene was characterized and sequenced as a possible virulence-related gene (Bishai et al., J. Bacteriol. 176:2914 (1994)). A number of the genes responsible for the capsular polysaccharide have been mapped and sequenced (Kroll et al., Mol. Microbiol. 5(6):1549 (1991)). Several outer membrane protein genes have been identified and sequenced (Langford et al., J. Gen. Microbiol. 138:155 (1992)). The lipooligosaccharide component of the outer membrane and the genes of its synthetic pathway are under intensive study (Weiser et al., J. Bacteriol. 173:3304 (1990)). While a vaccine is available, the study of outer membrane components is motivated to some extent by the need for improved vaccines.
Data Availability
The H. influenaze genome sequence has been deposited in the Genome Sequence DataBase (GSDB) with the accession number LA2023. The nucleotide sequence and peptide translation of each predicted coding region with identified start and stop codons have also been accessioned by GSDB.
Production of an Antibody to a Haemophilus influenzae Protein
Substantially pure protein or polypeptide is isolated from the transfected or transformed cells using any one of the methods known in the art. The protein can also be produced in a recombinant prokaryotic expression system, such as E. coli, or can by chemically synthesized. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:
Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C., Nature 256:495 (1975) or modifications of the methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and modified methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 (1989).
Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al., J. Clin. Endocrinol. Metab. 33:988-991 (1971).
Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology, Wier, D., ed, Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, second edition, Rose and Friedman, eds., Amer. Soc. For Microbiology, Washington, D.C. (1980).
Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
Preparation of PCR Primers and Amplification of DNA
Various fragments of the Haemophilus influenzae Rd genome, such as those disclosed in Tables 1(a) and 2 can be used, in accordance with the present invention, to prepare PCR primers for a variety of uses. The PCR primers are preferably at least 15 bases, and more preferably at least 18 bases in length. When selecting a primer sequence, it is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same. The PCR primers and amplified DNA of this Example find use in the Examples that follow.
Gene Expression from DNA Sequences Corresponding to ORFs
A fragment of the Haemophilus influenzae Rd genome provided in Tables 1(a) or 2 is introduced into an expression vector using conventional technology. (Techniques to transfer cloned sequences into expression vectors that direct protein translation in mammalian, yeast, insect or bacterial expression systems are well known in the art.) Commercially available vectors and expression systems are available from a variety of suppliers including Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism, as explained by Hatfield et al., U.S. Pat. No. 5,082,767, incorporated herein by this reference.
The following is provided as one exemplary method to generate polypeptide(s) from cloned ORFs of the Haemophilus genome fragment. Since the ORF lacks a poly A sequence because of the bacterial origin of the ORF, this sequence can be added to the construct by, for example, splicing out the poly A sequence from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene) for use in eukaryotic expression systems. pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex thymidine kinase promoter and the selectable neomycin gene. The Haemophilus DNA is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the Haemophilus DNA and containing restriction endonuclease sequences for PstI incorporated into the 5′ primer and BglII at the 5′ end of the corresponding Haemophilus DNA 3′ primer, taking care to ensure that the Haemophilus DNA is positioned such that its followed with the poly A sequence. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with BglII, purified and ligated to pXT1, now containing a poly A sequence and digested BglII.
The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.). The protein is preferably released into the supernatant. However if the protein has membrane binding domains, the protein may additionally be retained within the cell or expression may be restricted to the cell surface.
Since it may be necessary to purify and locate the transfected product, synthetic 15-mer peptides synthesized from the predicted Haemophilus DNA sequence are injected into mice to generate antibody to the polypeptide encoded by the Haemophilus DNA.
If antibody production is not possible, the Haemophilus DNA sequence is additionally incorporated into eukaryotic expression vectors and expressed as a chimeric with, for example, β-globin. Antibody to β-globin is used to purify the chimeric. Corresponding protease cleavage sites engineered between the β-globin gene and the Haemophilus DNA are then used to separate the two polypeptide fragments from one another after translation. One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene). This vector encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al. and many of the methods are available from the technical assistance representatives from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from either construct using in vitro translation systems such as In vitro ExpressTM Translation Kit (Stratagene).
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.
All patents, patent applications and publications referred to above are hereby incorporated by reference.







TABLE 1(a)







Amino acid biosynthesis









Glutamate family













HI0190
202698
204044
glutamate dehydrogenase (gdhA) (Escherchia coli)
74.1
84.4
446


HI0867
915793
917833
glutamine synthetase (glnA) (Proteus vulgaris)
70.7
85.9
467


HI1725
1792409
1789821
uridylyl transferase (glnD) (Escherichia coli)
46.6
67.8
854


HI0813
861610
860240
argininosuccinate lyase (arginosuccinase) (asal) (argH)
73.5
84.5
457





(Escherichia coli)


HI1733
1799112
1800443
argininosuccinate synthetase (argG) (Escherichia coli)
78.6
87.5
438


HI0598
618753
617752
omithine carbamoyltransferase (arcB) (Pseudomonas aeruginosa)
82.3
90.7
334


HI1242
1313013
1311763
gamma-glutamyl phosphate reductase (proA) (Escherichia coli)
61.7
79.4
406


HI0902
955518
956621
glutamate 5-kinsse (gamma-glutamyl kinase) (proB) (Escherchia coli)
65.7
80.2
363









Aspartate family













HI0288
319209
320419
aspartate aminotransferase (aspC) (Bacillus sp.)
31.1
53.8
349


HI1623
1684147
1685334
aspartate aminotransferase (aspC) (Escherichia coli)
62.6
79.0
396


HI0566
582379
583368
asparagine synthetase A (asnA) (Escherchia coli)
63.3
77.0
330


HI0648
690744
689632
aspartate-semialdehyde dehydrogenase (asd) (Escherichis coli)
71.9
84.9
367


HI1311
1385700
1386509
dehydrodipicolinate reductase (dapB) (Escherichia coli)
70.3
82.5
269


HI0729
779456
778212
diaminopimelate decarboxylase (dap decarboxylase) (lysA)
57.6
78.8
413





(Pseudomomas aeruginosa)


HI0752
810250
811071
diaminopimelate epimerase (dapF) (Escherichia coli)
77.0
85.8
274


HI0256
284972
285865
dihydrodipicolinate synthetase (dapA) (Escheichia coli)
58.2
79.8
292


HI1638
1693968
1694330
lysine-sensitive aspartokinase III (lysC) (Escherichia coli)
55.3
73.2
449


HI0102
109226
108096
succinyl-diaminopimelate desuccinylase (dapE) (Escherichia coli)
61.6
79.7
374


HI1640
1696728
1695820
tetrahydrodipicolinate N-succinyltransferase (dapD) (Actinobacillus
96.7
98.5
273





pleuropneumoniae)


HI0089
96280
93836
aspartokinase-homoserine dehydrogenase (thrA)
62.2
77.4
814





(Serratia marcescens)


HI0088
93820
92879
homoserine kinase (thrB) (Serratia marcescens)
61.8
80.6
306


HI0087
92833
91559
threonine synthase (thrC) (Serratia marcescens)
67.0
80.9
425


HI1044
1107725
1105876
B12-dependent homocysteine-N5-methyltetrahydrofolate
54.2
70.4
12.7





transmethylase (metH) (Escherichia coli)


HI0122
137932
136745
beta-cystathionase (metC) (Escherichia coli)
65.4
84.1
390


HI0086
90743
89601
cystathionine gamma-synthase (met8) (Escherichia coli)
41.9
62.2
374


HI1266
1339983
1341056
homoserine acetyltransferase (met2) (Saccharomyces cerevisiae)
38.1
57.1
387


HI1708
1773488
1771221
tetrahydropteroyltriglutamate methyltransferase (metE) (Escherichia coli)
52.4
68.0
747









Serine family













HI0891
942366
943628
serine hydroxymethyltransferase (serine methylase) (glyA) (Actinobacillus
85.7
93.6
419





actinomyceterncomitans)


HI0467
486594
487823
phosphoglycerate dehydrogenase (serA) (Escherichia coli)
71.1
83.9
408


HI1170
1238587
1237502
phosphoserine aminotransferase (serC) (Escherichia coli)
53.4
72.3
358


HI1035
1097573
1098514
phosphoserine phosphatase (o-phosphoserine phosphohydrolase) (serB)
52.3
69.5
303





(Escherichia coli)


HI1105
1165130
1168077
cysteine synthetase (cysK) (Escherichia coli)
70.0
83.9
309


HI0608
636187
636987
serine acetyltransferase (cysE) (Escherichia coli)
73.0
86.3
256









Aromatic amino acid family













HI0972
1026936
1027382
3-dehydroquinase (aroQ) (Actincobacillus pleuropneumoniae)
67.1
82.5
143


HI0209
222169
223254
3-dehydroquinate synthase (aroB) (Escherichia coli)
62.1
76.7
356


HI0197
211424
212494
chorismate synthase (aroC) (Escherichia coli)
77.3
88.4
350


HI0609
637000
637812
dehydroquinase shikimate dehydrogenase (Nicotiana tabacum)
30.0
51.5
242


HI1595
1656463
1657758
enolpyruvylshikimatephosphatesynthase (aroA) (Haemophilus influenzae)
97.7
98.4
432


HI0657
698939
698124
shikimate 5-dehydrogenase (aroE) (Escherichia coli)
49.1
70.1
270


HI0208
221607
222146
shikimic acid kinase I (aroK) (Escherichia coli)
75.0
87.5
104


HI1148
1213767
1214921
chorismate mutase/prephenate dehydratase pheA polypeptide (pheA)
54.3
74.7
375





(Escherichia coli)


HI1553
1618339
1617254
DAHP synthetase (phenylalanine repressible) (aroG) (Escherichia coli)
72.0
83.8
345


HI1293
1370448
1371578
chorismate mutase (tyrA) (Erwinia herbicola)
58.6
76.8
366


HI1392
1481917
1483470
anthranilate synthase component I (trpE) (Escherichia coli)
52.9
73.2
494


HI1393
1483718
1485554
anthranilate synthase component II (trpD) (Escherichia coli)
56.6
74.2
452


HI1174
1240757
1241335
anthnanilate synthese glutamine amidotransferase (trpG) (Acinetobacter
34.0
59.0
191





calcoaceticus)


HI1437
1519794
1520597
tryptophan synthase alpha chain (trpA) (Salmonella typhimurium)
57.8
72.8
267


HI1436
1518601
1519791
tryptophan synthase beta chain (trpB) (Escherichia coli)
82.4
90.3
391


HI0474
494758
495354
amidotransferase (hisH) (Escherichia coli)
55.9
70.3
195


HI0470
490033
490941
ATP phosphoribosyltransferase (hisC) (Escherchia coli)
72.2
82.0
295


HI0476
496124
496897
hisF cyclase (hisF) (Escherichia coli)
62.0
91.0
256


HI0472
492389
493489
histidinol-phosphate aminotransferase (hisC) (Escherichia coli)
60.1
77.5
351


HI1169
1237411
1236314
histidinol-phosphate aminotransferase (hisH) (Bacillus subtilis)
38.7
61.0
354


HI0473
493604
494689
imidazoleglycerol-phosphate dehydratase (hisB) (Escherichia coli)
65.0
80.5
353


HI0477
496900
497562
phosphoribosyl-AMP cyclohydrolase (hislE) (Escherichia coli)
60.7
77.0
195


HI0475
495393
496139
phosphoribosylformimino-5-aminoimidazole caroxamide ribotide isomerase
62.9
77.1
245





(hisA) (Escherichia coli)









Pyruvate family













HI1581
1642613
1643692
alamine racemase, biosynthetic (air) (Escherichia coli)
56.3
74.9
358









Branched chain family













HI0739
791174
791968
acetohydroxy acid synthase II (ilvG) (Escherichia coli)
63.6
78.5
386


HI1591
1652923
1651205
acetolactate synthase III large chain (ilvI) (Escherichia coli)
69.1
83.9
527


HI1590
1651202
1650714
acetolactate synthase III small chain (ilvH) (Escherichia coli)
65.6
85.0
160


HI1196
1259031
1258003
branched-chain-amino-acid transaminase (Salmonella typhimunium)
32.9
49.8
298


HI0740
791969
793960
dihydroxyacid dehydrase (ilvD) (Escherichia coli)
77.9
89.5
614


HI0684
723320
724795
ketol-acid reductoisomerase (ilvC) (Escherichia coli)
81.7
89.6
491


HI0991
1047074
1047673
3-isopropylmalate dehydratase (isopropylmalate isomerase) (leuD)
71.1
86.3
197





(Salmonella typhimunum)


HI0989
1044390
1045463
3-isopropylmalate dehydrogenase (beta-IPM dehydrogenaset (leuB)
68.0
80.1
353





(Salmonella typhimunium)


HI0985
1040319
1039678
leuA protein (leuA) (Haemophilus influenzae)
99.5
100.0
193







Biosynthesis of cofactors, prosthetic groups, carriers









Biotin













HI1560
1625092
1823803
7,8-diamino-pelargonic acid aminotransferase (bioA) (Escherichia coli)
58.0
74.1
420


HI1559
1623791
1622652
7-keto-8-aminopelargonic acid synthetase (bioF) (Bacillus sphaericus)
33.5
56.3
370


HI1557
1622004
1621225
biotin biosynthesis: reaction prior to pimeloyl CoA (bioC) (Escherichia coli)
28.6
46.8
151


HI0645
687346
684872
biotin sulfoxide reductase (BDS reductase) (bisC) (Escherchia coli)
54.0
71.8
734


HI1024
1085538
1086535
biotin synthetase (bioB) (Escherichia coli)
59.6
77.5
307


HI1556
1621212
1620640
dethiobiotin synthase (bioD) (Bacillus sphaericus)
42.1
59.6
175


HI1449
1532932
1532207
dethiobiotin synthetase (bioD) (Escherichia coli)
41.3
62.4
217









Folic acid













HI1448
1531237
1532112
5,10 methylenetetrahydrofolate reductase (metF) (Escherichia coli)
72.8
83.4
290


HI0611
640325
639480
5,10-methylene-tetrahydrofolate dehydrogenese (lolD) (Escherchia coli)
67.6
82.0
278


HI0064
67257
67760
7,8-dihydro-5-hydroxymethylptenn-pyrophosphokinase (folK) (Escherichia
56.3
77.8
158





coli)


HI0459
478432
477392
aminodeoxylchorismate lyase (pabC) (Escherichia coli)
40.1
66.5
243


HI1635
1691986
1691351
dedA protein (dedA) (Escherichia coli)
30.4
55.1
158


HI0901
955417
954938
dehydrofolate reductase, type I (lolA) (Escherichia coli)
53.2
68.4
158


HI1339
1412130
1412954
dihydropteroate synthase (lolP) (Escherichia coli)
54.5
70.9
275


HI1469
1547395
1548370
dihydropteroate synthase (lolP) (Escherichia coli)
54.5
70.9
275


HI1264
1337544
1338854
folylpolyglutamate synthase (lolC) (Escherichia coli)
51.7
68.4
409


HI1451
1534018
1533365
GTP cyclohydrolase I (lolE) (Escherichia coli)
63.9
79.0
219


HI1173
1240715
1239732
p-aminobenzoate synthetase (pabB) (Escherichia coli)
31.0
53.6
257









Lipoate













HI0026
28610
27651
lipoate biosynthesis protein A (lipA) (Escherichia coli)
73.8
84.1
321


HI0027
29302
28667
lipoate biosynthesis protein B (lipB) (Escherichia coli)
66.7
64.2
181









Molybdopterin













HI1681
1743523
1743044
moaC protein (moaC) (Escherichia coli)
79.1
89.2
157


HI1682
1744628
1743618
molybdenum colactor biosynthesis protein A (moaA) (Escherichia coli)
61.8
78.3
327


HI1373
1461582
1461376
molybdenum-pterin binding protein (mopl) (Clostridium pasteurianum)
51.5
74.2
66


HI1680
1743078
1742797
molybdopterin (MPT) converting factor, subunit 1 (moaD) (Escherichia coli)
59.3
79.0
81


HI1452
1534156
1535367
molybdopterin biosynthesis protein (chIE) (Escherichia coli)
56.4
72.5
403


HI0118
132351
133133
molybdopterin biosynthesis protein (chIN) (Escherichia coli)
27.9
52.9
135


HI1453
1535374
1536102
molybdopterin biosynthesis protein (chIE) (Escherichia coli)
63.9
78.4
241


HI1679
1742793
1742344
molybdopterin converting factor, subunit 2 (moaE) (Escherichia coli)
58.0
76.0
150


HI0846
892779
892204
molybdopterin-guanine dinucleotide (mob) (Escherichia coli)
39.4
61.7
187









Pantothenate













HI0633
670462
669530
aniothenate kinase (coaA) (Escherichia coli)
64.1
78.2
314









Pyridoxine













HI0865
913165
913851
pyridoxamine phosphate oxidase (pdxH) (Escherichia coli)
46.0
65.3
213









Riboflavin













HI0766
827249
827893
3,4-dihydroxy-2-butanone 4-phosphate synthase (ribB) (Escherichia coli)
69.6
82.7
213


HI0213
225991
226662
GTP cyclohydrolase II (nbA) (Escherichia coli)
68.0
81.4
193


HI0946
1002788
1003883
riboflavin biosynthesis protein RIBG (ribD) (Escherichia coli)
57.6
76.5
361


HI1619
1678899
1679510
riboflavin synthase alha chain (ribC) (Escherichia coli)
65.5
82.3
203


HI1306
1382553
1383071
riboflavin synthase beta chain (ribE) (Escherichia coli)
76.3
89.7
156









Thioredoxin, glutaredoxin, glutathione













HI0162
177496
176129
glutathinone reductase (gor) (Escherichia coli)
74.2
85.0
450


HI1118
1181697
1181197
thioredoxin (trxA) (Anabaena sp.)
36.6
58.5
82


HI1162
1228652
1228002
thioredoxin (trxA) (Anabaena sp.)
33.3
61.5
39


HI0084
86470
88150
thioredoxin m (trxM) (Anacystis nidulans)
53.3
79.4
107









Menaquinone, ubiquinone













HI0285
317765
316062
2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (menD)
46.8
64.4
551





(Escherichia coli)


HI0971
1025835
1026875
4-(2′-carboxyphenyl)-4-oxybutyric acid synthase (menC) (Escherichia coli)
57.3
74.2
312


HI1192
1256548
1255916
coenzyme PQQ synthesis protein III (pqqIII) (Acinetobacter calcoaceticus)
25.4
48.6
211


HI0970
1024963
1025817
DHNA synthase (menB) (Escherichia coli)
86.7
95.1
285


HI1442
1525823
1526707
famesyldiphosphate synthase (ispA) (Escherichia coli)
53.6
71.2
297


HI0195
206694
208049
o-succinylbenzoate-CoA synthase (menE) (Escherichia coli)
46.0
66.8
426









Heme, porphyrin













HI1163
1229908
1228940
ferrochelatase (visA) (Escherichia coli)
51.6
69.4
315


HI0113
119848
122079
heme utilization (hxuC) (Haemophilus influenzae)
26.4
46.1
695


HI0265
293930
295824
heme-hemopexin utilization (hxuB) (Haemophilus influenzae)
96.1
98.9
585


HI0604
631034
629751
hemY protein (hemY) (Escherichia coli)
38.9
64.4
355


HI0465
484621
485769
oxygen-independent coproporphyrinogen III oxidase (hemN) (Salmonella
31.5
52.3
241





typhimurium)


HI1204
1267418
1266477
protoporphrinogen oxidase (hemG) (Escherichia coli)
36.1
56.8
153


HI1565
1629849
1628974
protoporphrinogen oxidase (hemG) (Escherichia coli)
59.1
72.6
203


HI0605
631035
632562
uroporphyrinogen III methylase (hemX) (Escherichia coli)
39.9
60.3
358







Cell envelope









Membranes, lipoproteins, porins













HI1585
1647711
1647247
15 kd peptidoglycan-associated lipoprotein (lpp) (Haemophilus influenzae)
94.8
95.5
154


HI0622
653682
652864
28 kDa membrane protein (hlpA) (Haemophilus influenzae)
99.6
100.0
273


HI0304
335684
337249
apolipoprotein N-acyltransferase (eute) (Escherichia coli)
45.2
64.1
497


HI0362
384880
384035
hydrophobic membrane protein (Steptococcus gordonii)
37.2
66.5
268


HI0409
428260
427478
hydrophobic membrane protein (Steptococcus gordonii)
34.4
61.3
254


HI1573
1634553
1636106
iron-regluated outer membrane protein A (iroA) (Neisseria meningitidis)
28.9
50.9
398


HI0895
736825
737646
lipoprotein (hel) (Haemophilus influenzae)
99.6
99.6
274


HI0707
749215
750429
lipoprotein (nlpD) (Escherichia coli)
48.6
64.8
364


HI0705
748419
748994
lipoprotein B (lppB) (Haemophilus somnus)
72.3
89.5
191


HI0896
946675
947916
membrane fusion protein (mtrC) (Neisseria gonorrhoeae)
30.9
53.6
337


HI0403
421547
422923
outer membrane protein P1 (ompP1) (Haemophilus influenzae)
93.0
97.2
459


HI0140
153446
154522
outer membrane protein P2 (ompP2) (Haemophilus influenzae)
96.7
97.5
361


HI1167
1234699
1235757
outer membrane protein P5 (ompA) (Haemophilus influenzae)
94.1
95.8
353


HI0906
958098
958901
prolipoprotein diacylglyceryl transferase (lgt) (Escherichia coli)
62.8
80.1
285


HI0030
31698
30838
rare lipoprotein A (rlpA) (Escherichia coli)
34.5
57.8
288


HI0924
979182
979727
rare lipoprotein B (rlpB) (Escherichia coli)
33.5
62.1
163









Surface polysaccharides, lipopolysaccharides & antigens













HI1563
1628153
1627302
2-dehydro-3-deoxyphosphooctonate aldolase (kdsA) (Escherichia coli)
81.3
91.5
283


HI0654
696743
695463
3-deoxy-d-mann-octulosonic-acid transferase (kdtA) (Escherichia coli)
50.7
69.9
420


HI1108
1169176
1168139
ADP-heptose-lps heptosyltransferase II (rfaF) (Escherichia coli)
63.6
78.9
345


HI1117
1181141
1180218
ADP-L-glycero-D-mannoheptose-6-epimerase (rfaD) (Escherichia coli)
78.2
87.7
308


HI0058
59659
58898
CTP:CMP-3-deoxy-D-manno-octulosonate-cylidylyl-transferase (kdsB)
65.0
81.7
245





(Escherichia coli)


HI0917
970233
969211
firA protein (firA) (Pasteurella multocida)
84.9
91.1
338


HI0870
919974
920723
glycosyl transferase (lglD) (Neisseria gonorrhoeae)
30.3
55.3
200


HI1584
1646090
1647058
glycosyl transferase (lglD) (Neisseria gonorrhoeae)
47.3
64.0
328


HI0653
695463
694996
KDTB protein (kdtB) (Escherichia coli)
52.3
75.8
153


HI1684
1746281
1747291
kpsF protein (kpsF) (Escherichia coli)
49.3
70.6
294


HI1543
1607986
1608967
lic-1 operon protein (licA) (Haemophilus influenzae)
99.1
100.0
321


HI1544
1608970
1609885
lic-1 operon protein (licB) (Haemophilus influenzae)
99.0
99.3
303


HI1545
1609845
1610543
lic-1 operon protein (licC) (Haemophilus influenzae)
96.5
99.5
198


HI1546
1610546
1611340
lic-1 operon protein (licD) (Haemophilus influenzae)
88.7
94.0
268


HI1052
1125450
1124254
lipid A disaccharide synthetase (lpxB) (Escherichia coli)
63.2
77.3
382


HI0552
571001
570096
lipooligosacchardie biosynthesis protein (Haemophilus influenzae)
98.3
99.0
298


HI0767
827911
828756
lipooligosacchardie biosynthesis protein (Haemophilus influenzae)
36.4
59.5
267


HI0869
918779
919990
lsg locus hypothetical protein (GB:M94855_1) (Haemophilus influenzae)
60.5
82.5
400


HI1706
1770127
1768916
lsg locus hypothetical protein (GB:M94855_1) (Haemophilus influenzae)
99.3
100.0
401


HI1705
1768916
1768005
lsg locus hypothetical protein (GB:M94855_2) (Haemophilus influenzae)
98.4
98.7
304


HI1704
1768000
1767322
lsg locus hypothetical protein (GB:M94855_3) (Haemophilus influenzae)
96.0
97.4
226


HI1703
1766957
1766157
lsg locus hypothetical protein (GB:M94855_4) (Haemophilus influenzae)
96.1
98.4
257


HI1702
1766142
1765261
lsg locus hypothetical protein (GB:M94855_5) (Haemophilus influenzae)
96.9
98.3
294


HI1701
1765256
1764456
lsg locus hypothetical protein (GB:M94855_6) (Haemophilus influenzae)
98.9
99.3
267


HI1700
1763577
1764341
lsg locus hypothetical protein (GB:M94855_7) (Haemophilus influenzae)
98.4
98.4
255


HI1899
1763439
1762678
lsg locus hypothetical protein (GB:M94855_8) (Haemophilus influenzae)
98.6
99.0
209


HI0263
290317
291357
opsX locus protein (opsX) (Xanthomonas campestris)
35.2
56.7
261


HI1722
1768547
1787483
rfe (CGSC No 294) protein (Escherichia coli)
59.0
77.2
344


HI1147
1212723
1213637
UDP-3-0-acyl N-acetylglcosamine deacetylase (envA) (Escherichia coli)
77.3
88.2
304


HI1063
1126278
1125493
UDP-N-acetylglucosamine acetyltransferase (lpxA) (Escherichia coli)
66.0
79.4
262


HI0875
925083
926096
UDP-N-acetylglucosamine epimerase (rffE) (Escherichia coli)
65.5
79.5
336


HI0874
923609
925021
undecaprenyl-phosphate galactosephosphotransferase (rfbP) (Salmonella
57.9
75.1
465





typhimurium)









Surface structures













HI1738
1808251
1804281
adhesin (aidA-1) (Escherichia coli)
29.3
45.8
1196


HI0119
133314
134324
adhesin B precursor (fimA) (Streptococcus parasanguis)
24.5
48.3
309


HI0364
386685
385807
adhesin B precursor (fimA) (Streptococcus parasanguis)
34.6
61.6
302


HI0332
356770
358062
cell envelope protein (oapA) (Haemophilus influenaze)
99.8
100.0
431


HI0713
757120
757425
flagellar switch protein (fliM) (Salmonella typhimurium)
34.1
61.0
41


HI1464
1542848
1542296
invasin precursor (outer membrane adhesin) (yopA) (Yersinia enterocolitica)
38.5
62.1
291


HI0333
358125
358526
opacity associated protein (oapB) (Haemophilus influenzae)
99.2
99.2
132


HI0416
436627
436836
opacity protein (opa66) (Neisseria gonorrhoeae)
74.5
90.9
55


HI1177
1243585
1243947
opacity protein (opa66) (Neisseria gonorrhoeae)
37.7
59.0
181


HI1451
1540805
1540272
opacity protein (opaD) (Neisseria meningitidis)
34.5
55.8
230


HI0300
333052
331661
pilin biogenesis protein (pilB) (Pseudomonas aeruginosa)
44.1
64.8
485


HI0919
973373
970950
protective surface antigen D15 (Haemophilus influenzae)
98.6
99.5
797









Murein saccufus, peptidoglycan













HI1674
1737564
1735481
carboxy-terminal protease, penicillin-binding protein 3 (prc) (Escherichia
52.3
69.5
860





coli)


HI1143
1208355
1209272
D-alanine-D-alanine ligase (ddlB) (Escherichia coli)
59.9
75.8
303


HI1333
1408286
1406850
D-alanyl-D-alanine carboxypeptidase (dacB) (Escherichia coli)
43.9
68.2
454


HI0066
68323
69618
N-acetylmuramoyl-L-alanine amidase (amiB) (Escherichia coli)
59.5
77.0
221


HI0383
401990
401532
PC protein (15kd peptidoglycan-associated outer membrane lipoprotein)
100.0
100.0
153





(pal) (Haemophilus influenzae)


HI1731
1795566
1797908
penicillin-binding protein 1B (ponB) (Escherichia coli)
47.0
67.5
767


HI0032
34810
32858
penicillin-binding protein 2 (pbp2) (Escherichia coli)
58.8
73.8
609


HI0029
30819
29641
penicillin-binding protein 5 (dacA) (Escherichia coli)
54.8
68.4
362


HI0198
212582
213439
penicillin-insensitive murein endopeptidase (mepA) (Escherichia coli)
49.3
66.7
269


HI1138
1201927
1203006
phospho-N-acetylmuramoyl-pentapeptide-transferas E (mraY) (Escherichia
76.7
88.9
360





coli)


HI0038
40689
41741
rod shape-determining protein (mreC) (Escherichia coli)
50.3
74.5
293


HI0031
32865
31753
rod shape-determining protein (mreB) (Escherichia coli)
63.1
80.7
358


HI0037
39473
40605
rod shape-determining protein (mreB) (Escherichia coli)
79.6
89.9
347


HI0039
41744
42229
rod shape-determining protein (mreD) (Escherichia coli)
40.6
71.6
154


HI0831
878792
880570
soluble lytic murein transglycosylase (slt) (Escherichia coli)
40.4
59.3
378


HI1141
1205663
1206715
transferase, peptidoglycan synthesis (murG) (Escherichia coli)
61.7
76.0
350


HI1137
1200560
1201930
UDP-murnac-pentapeptide synthetase (murF) (Escherichia coli)
51.4
68.2
452


HI1136
1199080
1200543
UDP-MurNac-tripeptide synthetase (murE) (Escherichia coli)
55.7
72.6
463


HI0270
301246
302267
UDP-N-acetylenolpyruvoylglucosamine reductase (murB) (Escherichia coli)
57.6
75.6
340


HI1083
1148434
1147163
UDP-N-acetylglucosamine enolpyruvyl transferase (murZ) (Escherichia coli)
72.4
64.5
419


HI1142
1206856
1208280
UDP-N-acetylmuramate-alanine ligase (murC) (Escherichia coli)
68.2
81.8
470


HI1139
1203132
1204442
UDP-N-acetylmuramoylalanine-D-glutamate ligase (murD) (Escherichia coli)
61.0
73.7
437


HI1499
1569479
1569826
N-acetylmuramoyl-L-alanine amidase (Bacteriophage T3)
42.9
62.2
97







Central intermediary metabolism









Phosphorus compounds













HI0697
739608
738640
exopolyphosphatase (ppx) (Escherichia coli)
55.2
76.7
318


HI0124
139861
139334
inorganic pyrophosphatase (ppe) (Escherichia coli)
36.3
50.3
157


HI0647
689574
688637
lysophospholipase L2 (pldB) (Escherichia coli)
31.2
53.1
317









Sulfur metabolism













HI1374
1462019
1461693
desulfoviridin gamma subunit (dsvC) (Desulfovibrio vulgaris)
36.0
58.0
99


HI0807
854438
853741
putative arylsulfatase regulatory protein (aslB) (Escherichia coli)
47.4
67.0
381


HI0561
578539
577856
sulfite synthesis pathway protein (cysQ) (Escherichia coli)
35.9
56.0
205









Polyamine biosynthesis













HI0099
106307
107374
nucleotide binding protein (potG) (Escherichia coli)
42.6
66.9
340


HI0593
614187
612028
amithine decarboxylase (speF) (Escherichia coli)
66.4
80.2
717









Polysaccharides - (cytoplasmic)













HI1360
1436170
1438359
1,4-alpha-glucan branching enzyme (glgB) (Escherichia coli)
64.5
80.1
723


HI1362
1440427
1441758
ADP-glucose synthetase (glgC) (Escherichia coli)
55.0
74.3
407


HI1364
1443545
1446007
alpha-glucan phosphorylase (glgP) (Escherichia coli)
61.1
79.1
809


HI1361
1438458
1440434
glycogen operon protein (glgX) (Escherichia coli)
54.3
67.8
501


HI1363
1441869
1443296
glycogen synthase (glgA) (Escherichia coli)
56.2
71.2
475









Degradation of polysaccharides













HI1359
1434061
1436157
amylomaltase (malQ) (Escherichia coli)
40.9
62.0
615


HI1420
1507662
1507063
endochitinase (Oryza sativa)
38.9
50.9
106









Amino sugars













HI0431
452989
451160
glutamine amidotransferase (glmS) (Escherichia coli)
72.1
84.3
609


HI0141
155859
154717
N-acetylglucosamine-6-phosphate deacetylase (nagA) (Escherichia coli)
54.5
72.1
376


HI0142
156944
156135
nagB protein (nagB) (Escherichia coli)
74.2
88.1
260









Other













HI0048
49257
48403
7-alpha-hydroxysteroid dehydrogenase (hdhA) (Escherichia coli)
32.4
55.1
244


HI1207
1271536
1270334
acetate kinase (ackA) (Escherichia coli)
69.1
83.9
396


HI0951
1009728
1008367
GABA transaminase (gabT) (Escherichia coli)
34.4
55.8
420


HI0111
118858
119484
glutathione transferase (bphH) (Pseudomonas sp.)
37.6
57.4
200


HI0693
734488
735996
glycerol kinase (glpK) (Escherichia coli)
76.9
89.2
502


HI0586
606429
605161
hippuricase (hipO) (Campylobacter jejuni)
27.8
49.6
376


HI0543
564874
564575
urease (ureA) (Helicobacter heilmannii)
62.4
76.2
101


HI0539
561668
561087
urease accessory protein (UreF) (Bacillus sp.)
31.8
54.9
194


HI0541
564179
562464
urease alpha subunit (urea amidohydrolase) (ureC) (Bacilus sp.)
67.3
82.1
569


HI0540
562333
561779
urease protein (ureE) (Helicobacter pylori)
31.0
56.8
155


HI0538
560981
560307
urease protein (ureG) (Helicobacter pylori)
70.7
86.9
198


HI0537
560229
559447
urease protein (ureH) (Helicobacter pylori)
31.5
53.9
213


HI0542
564180
564574
urease subunit B (ureB) (Escherichia coli)
61.8
77.5
103







Energy metabolism









Amino acids, amines













HI0536
559266
557842
aspartase (aspA) (Escherichia coli)
78.2
89.1
468


HI0597
617739
616810
carbamate kinase (arcC) (Pseudomonas aeruginosa)
78.3
67.7
309


HI0747
802651
803697
L-asparaginase II (ansB) (Escherichia coli)
70.5
81.2
329


HI0290
323270
321907
L-serine deaminase (sdaA) (Escherichia coli)
68.6
83.3
454









Sugars













HI0820
869307
868288
aldose 1-epimerase precursor (mutarotase) (mro) (Acinetobacter
35.8
54.7
326





calcoaceticus)


HI0055
55016
56197
O-mannonate hydrolase (uxuA) (Escherichia coli)
72.8
85.8
394


HI1119
1181808
1182476
deoxyribose aldolase (deoC) (Mycoplasma hominis)
49.0
68.5
200


HI0615
644708
643299
fucokinase (fucK) (Escherichia coli)
41.4
64.5
459


HI0613
642828
642181
fuculose-1-phosphate aldolase (fucA) (Escherichia coli)
64.7
81.4
215


HI1014
1075981
1076610
fuculose-1-phosphate aldolase (fucA) (Escherichia coli)
32.9
51.8
163


HI0821
870510
869320
galactokinase (galK) (Haemophilus influenzae)
98.4
99.0
384


HI0145
159883
158984
glucose kinase (glk) (Streptomyces coelicolor)
33.6
53.2
303


HI0616
646595
644784
L-fucose isomerase (fucl) (Escherichia coli)
69.5
84.5
583


HI1027
1090247
1089519
L-ribulose-phosphate 4-epimerase (araD) (Escherichia coli)
72.3
81.8
231


HI1111
1173107
1171938
mal inducer biosynthesis blocker (malY) (Escherichia coli)
28.1
51.6
375


HI0143
158111
157233
N-acetylneuraminate lyase (nanA) (Escherichia coli)
36.2
61.4
291


HI0507
521330
522247
ribokinase (rbsK) (Escherichia coli)
56.0
74.8
302


HI1115
1177307
1178623
xylose isomerase (xytA) (Escherichia coli)
71.3
87.2
439


HI1116
1178629
1180161
xylulose kinase (xylulokinase) (Escherichia coli)
33.1
50.0
479









Glycolysis













HI0449
470280
469342
1-phosphofructokinase (fruK) (Escherichia coli)
55.4
74.1
304


HI0984
1039579
1038617
6-phosphofructokase (pfkA) (Escherichia coli
74.4
84.4
319


HI0934
990636
989329
enolase (eno) (Bacillus subtilis)
65.9
78.5
413


HI0526
547668
546592
fructose-bisphosphate aldolase (fba) (Escherichia coli)
71.3
85.8
359


HI1582
1643750
1645438
glucose-6-phosphate isomerase (pgi) (Escherichia coli)
76.9
68.7
548


HI0001
1
600
glyceraldehyde-3-phosphate dehydrogenase (gapdH) (Escherichia coli)
85.6
90.3
133


HI0527
548939
547782
phosphoglycerate kinase (pgk) (Escherichia coli)
81.1
90.7
387


HI0759
820852
821533
phosphoglyceromutase (gpmA) (Zymomonas mobilis)
58.9
74.6
222


HI1579
1639619
1641052
pyruvate kinase type II (pykA) (Escherichia coli)
77.2
87.5
480


HI0680
719664
720452
triosephosphate isomerase (tpiA) (Escherichia coli)
74.4
80.7
253









Pyruvate dehydrogenase













HI1235
1303195
1301495
dihydrolipoamide acetyltransferase (aceF) (Escherichia coli)
72.8
82.4
526


HI0194
206108
205248
dihydrolipoamide acetyltransferase (acoC) (Pseudomonas putida)
27.8
49.1
235


HI1234
1301378
1299945
lipoamide dehydrogenase (lpdA) (Escherichia coli)
81.5
91.6
474


HI1236
1305918
1303261
pyruvate dehydrogenase (aceE) (Escherichia coli)
68.6
84.0
885









TCA cycle













HI1668
1731748
1728899
2-oxoglutarate dehydrogenase (sucA) (Escherichia coli)
69.0
80.7
930


HI0025
27397
26393
acetate: SH-citrate lyase ligase (AMP) (Klebsiella pneumoniae)
48.9
68.4
321


HI0022
25179
23680
citrate lyase alpha chain (acyl lyase subunit) (citF) (Klebsiella pneumoniae)
72.1
86.1
469


HI0023
26068
25457
citrate lyase beta chain (acyl lyase subunit) (Klebsiella pneumoniae)
62.3
81.9
203


HI0024
26352
26068
citrate lyase gamma chain (acyl lyase subunit) (citD) (Klebsiella
52.1
71.9
97





pneumoniae)


HI1667
1728793
1727567
dihydrolipoamide succinyltransferase (sucB) (Escherichia coli)
73.6
84.5
403


HI1403
1493925
1495316
fumarate hydralase class II (fumarase) (fumC) (Escherichia coli)
61.8
74.2
460


HI1213
1275907
1276839
malate dehydrogenase (mdh) (Escherichia coli)
78.5
85.1
303


HI1248
1317431
1319698
malic acid enzyme (Bacillus stearothemophilus)
49.5
68.3
376


HI1200
1262687
1263565
succinyl-CoA synthetase alpha-subunit (sucD) (Escherichia coli)
83.4
91.7
289


HI1199
1261518
1262684
succinyl-CoA synthetase beta-subunit (sucC) (Escherichia coli)
64.7
80.2
388









Pentose phosphate pathway













HI0555
574159
572708
6-phosphogluconate dehydrogenase, decarboxylating (gnd) (Escherichia
54.0
71.1
464





coli)


HI0560
577777
576296
glucose-6-phosphate 1-dehydrogenase (G6PD) (Synechococcus sp.)
46.2
65.3
483


HI1025
1088660
1086666
transketolase 1 (TK 1) (tktA) (Escherichia coli)
77.1
87.5
664









Entner-Doudoroff













HI0047
48381
47746
2-keto-3-deoxy-6-phosphogluconate aldolase (eda) (Escherichia coli)
37.3
63.2
193


HI0049
50201
49260
2-keto-3-deoxy-D-gluconate kinase (kdgK) (Erwinia chrysanthemi)
44.2
64.5
300









Aerobic













HI1655
1715678
1713987
D-lactate dehydrogenase (dld) (Escherichia coli)
59.5
77.7
560


HI1166
1234330
1231250
D-lactate dehydrogenase (dld) (Saccharomyces cerevisiae)
27.6
47.7
427


HI0607
635168
636172
glycerol-3-phosphate dehydrogenase (gpsA) (Escherichia coli)
66.6
81.5
335


HI0749
805382
806713
NADH dehydrogenase (ndh) (Escherichia coli)
57.8
75.4
430









Anaerobic













HI1049
1112944
1110527
anaerobic dimethyl sulfoxide reductase A (dmsA) (Escherichia coli)
74.0
86.3
785


HI1048
1110513
1109899
anaerobic dimethyl sulfoxide reductase B (dmsB) (Escherichia coli)
72.1
84.8
204


HI1047
1109894
1109058
anaerobic dimethyl sulfoxide reductase C (dmsC) (Escherichia coli)
41.0
65.0
287


HI0646
688485
687382
cytochrome C-type protein (torC) (Escherichia coli)
37.4
54.7
365


HI0350
374535
375134
denitrification system component (nirT) (Pseudomonas stutzeri)
51.7
71.6
176


HI0009
9878
10783
fdhE protein (fdhE) (Escherichia coli)
50.8
71.6
307


HI0006
5067
8156
formate dehydrogenase, nitrate-inducible major subunit (fdnG) (Escherichia
64.4
79.2
1016





coli)


HI0005
4802
3993
formate dehydrogenase-N affector (fdhD) (Escherichia coli)
57.7
71.0
249


HI0008
9035
9805
formate dehydrogenase-O gamma subunit (fdoI) (Escherichia coli)
52.8
72.1
195


HI0007
8161
9096
formate dehydrogenase-O, beta subunit (fdoH) (Escherichia coli)
72.2
85.5
297


HI1071
1133439
1131826
formate-dependent nitrite reductase (cytochrome C552) (nrfA)
56.7
75.3
450





(Escherichia coli)


HI1070
1131779
1131102
formate-dependent nitrite reductase (nrfB) (Escherichia coli)
50.0
66.9
134


HI1069
1131102
1130428
formate-dependent nitrite reductase protein Fe—S centers (nrfC)
64.2
81.2
217





(Escherichia coli)


HI1068
1130428
1129466
formate-dependent nitrite reductase transmembrane protein (nrfD)
48.2
68.4
312





(Escherichia coli)


HI0835
882094
882529
fumarate reductase (frdC) (Escherichia coli)
49.2
72.3
129


HI0834
882093
881752
fumarate reductase 13 kDa hydrophobic protein (frdD) (Escherichia coli)
53.0
76.5
119


HI0837
885089
883293
fumarate reductase, flavoprotein subunit (frdA) (Escherichia coli)
75.4
87.2
602


HI0836
883357
882530
fumarate reductase, iron-sulfur protein (frdB) (Escherichia coli)
75.5
85.3
244


HI0681
720855
720541
glpE protein (glpE) (Escherichia coli)
43.3
63.5
103


HI0620
651184
651759
glpG protein (glpG) (Escherichia coli)
39.1
64.8
178


HI0687
729180
727492
glycerol-3-phosphate dehydrogenase, subunit A (glpA) (Escherichia coli)
69.9
82.7
531


HI0686
727529
726204
glycerol-3-phosphate dehydrogenase, subunit B (glpB) (Escherichia coli)
42.3
60.3
414


HI0685
726189
724912
glycerol-3-phosphate dehydrogenase, subunit C (glpC) (Escherichia coli)
58.8
76.0
393


HI1395
1487067
1487358
hydrogenase isoenzymes formation protein (hypC) (Escherichia coli)
63.2
81.6
76









Electron transport













HI0887
936816
938552
C-type cytochrome biogenesis protein (copper tolerance) (cycZ)
48.8
67.7
557





(Escherichia coli)


HI1078
1141318
1139756
cytochrome oxidase d subunit I (cydA) (Escherichia coli)
64.3
82.4
515


HI1077
1139738
1138605
cytochrome oxidase d subunit II (cydB) (Escherichia coli)
60.9
78.4
379


HI0529
549872
550341
ferredoxin (fdx) (Chromatium vinosum)
59.5
77.2
78


HI0374
394564
394226
ferredoxin (fdx) (Escherichia coli)
64.5
83.6
110


HI0192
205148
204627
flavodoxin (fldA) (Escherichia coli)
76.9
87.3
173


HI1365
1446272
1447807
NAD(P) transhydrogenase subunit alpha (pntA) (Escherichia coli)
73.7
84.1
509


HI1366
1447821
1449242
NAD(P) transhydrogenase subunit beta (pntB) (Escherichia coli)
80.5
87.7
462


HI1281
1355273
1354614
NAD(P)H-flavin oxidoreductase (Vibrio fischeri)
33.3
54.8
211









Fermentation













HI0501
514365
515657
aldehyde dehydrogenase (aldH) (NAD(P) transhydrogenase subunit alpha (pntA))
41.2
61.8
236





(Escherichia coli)


HI0776
836864
836114
butyrate-acetoacetate coa-transferase subunit A (ctfA) (Clostridium
53.3
75.2
214





acetobutylicum)


HI0186
200017
198884
glutathione-dependent formaldehyde dehydrogenase (gd-laldH) (Paracoccus
58.5
77.6
375





denitrificans)


HI1308
1383529
1384563
hydrogenase gene region (hypE) (Alcaligenes eutrophus)
28.1
48.2
237


HI1642
1698196
1700833
phosphoenolpyruvate carboxylase (ppc) (NAD(P) transhydrogenase subunit alpha (pntA))
64.8
80.0
683





(Escherichia coli)


HI0181
193936
191621
pyruvate formate-lyase (plf) (NAD(P) transhydrogenase subunit alpha (pntA))
86.1
92.9
760





(Escherichia coli)


HI0180
191487
190750
pyruvate formate-lyase activating enzyme (act) (NAD(P) transhydrogenase subunit
74.0
85.4
246





alpha (pntA)) (Escherichia coli)


HI1435
1517826
1518581
short chain alcohol dehydrogenase (ORFB) (Dichelobacter nodosus)
51.9
69.2
104









Gluconeogenesis













HI1651
1709919
1710917
fructose-1,6-bisphosphatase (fbp) (NAD(P) transhydrogenase subunit alpha (pntA))
70.5
84.0
331





(Escherichia coli)


HI0811
859038
857425
phosphoenolpyruvate carboxykinase (pckA) (NAD(P) transhydrogenase subunit alpha
71.7
83.0
444





(pntA)) (Escherichia coli)









ATP-protein motive force interconversion













HI0486
504824
504573
ATP synthase C chain (atpE) (Vibrio alginolyticus)
62.7
81.9
83


HI0487
505668
504883
ATP synthase F0 subunit a (alpB) (Escherichia coli)
58.2
78.1
261


HI0485
504520
504053
ATP synthase F0 subunit b (alpF) (Escherichia coli)
63.5
79.5
156


HI0483
503491
501953
ATP synthase F1 alpha subunit (atpA) (Escherichia coli)
86.5
94.7
513


HI0481
501081
499678
ATP synthase F1 beta subunit (atpD) (Escherichia coli)
89.3
96.1
460


HI0484
504037
503507
ATP synthase F1 delta subunit (atpH) (Escherichia coli)
58.0
78.4
176


HI0480
499645
499220
ATP synthase F1 epsilon subunit (atpC) (Escherichia coli)
59.6
75.7
136


HI0482
501934
501068
ATP synthase F1 gamma subunit (atpG) (Escherichia coli)
65.3
83.0
287


HI1277
1349508
1350221
ATP synthase subunit 3 region protein (atp) (Rhodopseudomonas blastica)
31.9
50.0
237







Fatty acid/phospholipid metabolism













HI0773
834230
832896
acetyl coenzyme A acetyltranferase (thiolase) (fadA) (Clostridium
63.0
80.4
391





acetobutylicum)


HI0428
448891
448169
fadR protein involved in fatty acid metabolism (fadR) (Escherichia coli)
47.4
68.4
234


HI1064
1126738
1126295
(3R)-hydroxymyristol acyl carrier protein dehydrase (fabZ) (Escherichia coli)
68.1
85.1
141


HI0156
171552
170827
3-ketoacyl-acyl carrier protein reductase (fabG) (Escherichia coli)
73.4
88.4
241


HI0408
427385
426441
acetyl-CoA carboxylase (accA) (Escherichia coli)
75.3
88.3
318


HI0155
170566
170341
acyl carrier protein (acpP) (Escherichia coli)
82.7
90.7
75


HI0076
82175
83032
acyl-CoA thioesterase II (tesB) (Escherichia coli)
52.3
73.1
283


HI1539
1605754
1604537
beta-ketoacyl-ACP synthase I (fabB) (Escherichia coli)
72.8
83.7
403


HI0158
174085
173138
beta-ketoacyl-acyl-carrier protein synthase III (fabH) (Escherichia coli)
85.9
79.8
317


HI0973
1027538
1028002
biotin carboxyl carrier protein (accB) (Escherichia coli)
71.2
82.7
156


HI0974
1028180
1029523
bioton carboxylase (accC) (Escherichia coli)
81.5
91.3
448


HI1328
1404041
1404571
D-3-hydroxydecanoyl-(acyl carrier-protein) dehydratase (fabA) (Escherichia
79.2
91.7
168





coli)


HI0337
362881
363234
diacylglycerol kinase (dgkA) (Escherichia coli)
50.9
71.8
110


HI0002
601
2421
long chain fatty acid coA ligase (Homo sapiens)
29.5
52.8
575


HI0157
172507
171572
malonyl coenzyme A-acyl carrier protein transacylase (fabD) (Escherichia
71.0
81.6
309





coli)


HI1740
1811556
1810672
short chain alcohol dehydrogenase homolog (envM) (Escherichia coli)
75.3
84.9
259


HI1438
1521691
1520741
USG-1 protein (usg) (Escherichia coli)
32.7
53.9
334


HI0736
788371
787652
1-acyl-glycerol-3-phosphate acyltransferase (plsC) (Escherichia coli)
62.2
78.2
238


HI0921
975561
974698
CDP-diglyceride synthetase (cdsA) (Escherichia coli)
48.4
66.5
246


HI0750
809228
806799
glycerol-3-phosphate acyltransferase (plsB) (Escherichia coli)
57.3
75.7
804


HI0212
225946
225224
phosphatidylglycerophosphate phosphatase B (pgpB) (Escherichia coli)
35.7
60.3
220


HI0123
138207
138761
phosphatidylglycerophosphate synthase (pgsA) (Escherichia coli)
66.5
83.0
182


HI0161
175145
176014
phosphatidylserine decarboxylase proenzyme (psd) (Escherichia coli)
57.6
75.5
280


HI0427
446754
448118
phosphatidylserine synthase (pssA) (Escherichia coli)
49.2
70.8
452


HI0691
732349
733440
protein D (hpd) (Haemophilus influenzae)
98.4
99.2
364







Purines, pyrimidines, nucleosides and nucleotides









Purine ribonucleotide biosynthesis













HI1622
1682920
1684005
5′-phosphoribosyl-5-amino-4-imidazole carboxylase II (purK) (Escherichia
56.8
71.9
351





coli)


HI1434
1517646
1516615
5′-phosphoribosyl-5-aminoimidazole synthetase (purM) (Escherichia coli)
76.5
86.7
344


HI1749
1829283
1828660
5′-guanylate kinase (gmk) (Escherichia coli)
64.7
81.6
206


HI0351
375941
375300
adenylate kinase (ATP-AMP transphosphorylase) (adk) (Haemophilus
99.5
99.5
214





influenzae)


HI0641
679574
681094
adenylosuccinate lyase (purB) (Escherichia coli)
76.5
87.9
456


HI1639
1694462
1695757
adenylosuccinate synthetase (purA) (Escherichia coli)
75.7
87.3
432


HI1210
1272783
1274297
amidophosphoribosyltransferase (purF) (Escherichia coli)
69.1
84.0
504


HI0754
812369
816328
formylglycineamide ribonucleotide synthetase (purL) (Escherichia coli)
69.7
82.0
1290


HI1594
1655627
1656460
formyltetrahydrofolate hydrolase (purU) (Escherichia coli)
72.6
85.2
277


HI0223
250532
252100
guaA protein (guaA) (Escherichia coli)
78.1
87.6
525


HI0222
248355
249818
inosine-5′-monophosphate dehydrogenase (guaB) (Acinetobacter
62.7
80.9
487





calcoaceticus)


HI0678
928811
929233
nucleoside diphosphate kinase (ndk) (Escherichia coli)
63.0
73.9
138


HI0890
940953
942239
phosphoribosylamine-glycine ligase (purD) (Escherichia coli)
75.2
84.5
427


HI1621
1682355
1682847
phosphoribosylaminoimidazole carboxylase catalytic subunit (purE)
94.4
96.9
161





(Haemophilus influenzae)


HI0889
939259
940854
phosphoribosylaminoimidazolecarboxamide formyltransferase (purH)
77.2
86.5
525





(Escherichia coli)


HI1433
1516557
1515922
phosphoribosylglycinamide formyltransferase (purN) (Escherichia coli)
51.9
71.4
210


HI1615
1674317
1675261
phosphoribosylpyrophosphate synthetase (prsA) (Salmonella typhimurium)
84.1
91.1
314


HI1732
1798036
1798953
SAICAR synthetase (purC) (Streptococcus pneumoniae)
29.8
54.6
204









Pyrimidine ribonucleotide biosyn













HI1406
1497997
1496981
dihydroorotate dehydrogenase (dihydroorotate oxidase) (pyrD) (Escherichia
60.7
77.4
334





coli)


HI0274
305799
305161
orotate phosphoribosyltransferase (pyrE) (Escherichia coli)
69.0
83.6
213


HI1228
1293965
1294282
pyrF operon encoding orotidine 5′-monophosphate (OMP) decarboxylase
77.1
87.6
105





(Escherichia coli)


HI1227
1293266
1293955
pyrF protein (pyrF) (Escherichia coli)
62.3
79.4
228


HI0461
480053
479517
uracil phosphoribosyltranferase (pyrR) (Bacillus caldolyticus)
52.2
73.9
179









2′-deoxyribonucleotide metabolism













HI0075
79934
82054
anaerobic ribonucleoside-triphosphate reductase (nrdD) (Escherichia coli)
77.4
86.2
702


HI0133
146656
147240
deoxycytidine triphosphate deaminase (dcd) (Escherichia coli)
75.6
86.5
193


HI0956
1012787
1013239
deoxyuridinetriphosphatase (dut) (Escherichia coli)
75.5
90.7
151


HI1538
1604204
1604464
glutaredoxin (grx) (Escherichia coli)
69.9
79.5
83


HI1686
1726318
1727445
nrdB protein (nrdB) (Escherichia coli)
85.4
92.6
376


HI1665
1723831
1726173
ribonucleoside-diphosphate reductase 1 alpha chain (nrdA) (Escherichia coli)
83.4
92.2
761


HI1161
1227925
1226972
thioredoxin reductase (trxB) (Escherichia coli)
75.9
85.8
316


HI0907
958914
959762
thymidylate synthetase (thyA) (Escherichia coli)
35.3
55.0
264









Salvage of nucleosides and nucleotides













HI0585
605064
603094
2′,3′-cyclic-nucleotide 2′-phosphodiesterase (cpdB) (Escherichia coli)
62.4
77.7
641


HI1233
1299794
1299255
adenine phosphoribosyltransferase (apt) (Escherichia coli)
66.1
83.1
177


HI0553
571120
571943
adenosine-tetraphosphatase (apaH) (Escherichia coli)
52.4
73.1
271


HI1353
1426390
1427265
cytidine deaminase (cytidine aminohydrolase) (cda) (Escherichia coli)
50.0
63.4
253


HI1222
1288579
1289628
cytidylate kinase (cmk) (Escherichia coli)
64.5
79.3
217


HI1652
1711636
1710842
cytidylate kinase (cmk) (Escherichia coli)
63.5
76.8
202


HI0520
540879
540166
purine-nucleoside phosphorylase (deoD) (Escherichia coli)
84.3
90.2
235


HI0531
552177
551599
thymidine kinase (tdk) (Escherichia coli)
68.6
82.4
188


HI1231
1297050
1296427
uracil phosphoribosyltransferase (upp) (Escherichia coli)
83.2
93.8
208


HI0282
312879
313655
uridine phosphorylase (udp) (Escherichia coli)
72.0
84.8
250


HI0676
716559
716095
xanthine guanine phosphorbiosyl transferase gpt (xgprt) (Escherichia coli)
72.1
87.7
152


HI0694
736541
736077
xanthine-guanine phosphoribosyltransferase (xgprt) (Salmonella
74.0
87.7
152





typhimurium)


HI1280
1353404
1354561
putative ATPase (mrp) (Escherichia coli)
66.0
79.0
353









Sugar-nucleotide biosynthesis, conversions













HI0207
219511
221319
5′-nucleotidase (ushA) (Homo sapiens)
34.5
54.8
487


HI1282
1355376
1356061
CMP-NeuNAc synthetase (siaB) (Neisseria meningitidis)
47.1
64.3
221


HI0822
871597
870551
galactose-1-phosphate uridylyltransferase (galT) (Haemophilus influenzae)
99.1
100.0
349


HI0814
862632
861748
glucosephosphate uridylyltranferase (galU) (Escherichia coli)
74.0
86.1
287


HI0353
378461
377448
udp-glucose 4-epimerase (galactowaldenase) (galE) (Haemophilus
99.1
99.1
338





influenzae)


HI0644
682446
683813
UDP-N-acetylglucosamine pyrophosphorylase (glmU) (Escherichia coli)
68.6
83.1
456









Nucleotide and nucleoside interconversions













HI1302
1376759
1378139
deoxyguanosine triphosphate triphosphohydrolase (dgt) (Escherichia coli)
38.2
57.6
469


HI1079
1141970
1143603
pyrG protein (pyrG) (Escherichia coli)
80.4
90.5
545


HI0132
146006
146644
uridine kinase (uridine monophosphokinase (udk) (Escherichia coli)
67.8
84.7
202







Regulatory functions













HI0606
632563
635091
adenylate cyclase (cyaA) (Haemophilus influenzae)
100.0
100.0
843


HI0886
936624
935917
aerobic respiration control protein ARCA (DYE resistance protein) (arcA)
77.2
87.8
237





(Escherichia coli)


HI0221
238723
248354
aerobic respiration control sensor protein (arcB) (Escherichia coli)
45.7
70.4
768


HI1054
1117872
1116979
araC-like transcription regulator (Streptomyces lividans)
25.7
47.7
303


HI1212
1275700
1275248
arginine repressor protein (argR) (Escherichia coli)
69.1
81.2
149


HI0237
265657
265310
arsC protein (arsC) (Plasmid R773)
38.3
56.5
114


HI0464
482094
484502
ATP-dependent proteinase (lon) (Escherichia coli)
74.5
87.9
769


HI0336
360636
362863
ATP:GTP 3′-pyrophosphotransferase (relA) (Escherichia coli)
62.9
80.5
741


HI1130
1193658
1195126
carbon starvation protein (cstA) (Escherichia coli)
32.1
53.5
499


HI0815
862845
862657
carbon storage regulator (csrA) (Escherichia coli)
68.4
91.2
57


HI0806
853619
853063
cyclic AMP receptor protein (crp) (Haemophilus influenzae)
27.2
46.7
174


HI0959
1014161
1014832
cyclic AMP receptor protein (crp) (Haemophilus influenzae)
100.0
100.0
224


HI1203
1265444
1266412
cys regulon transcriptional activator (cysB) (cyclic AMP receptor protein (crp)
63.3
79.3
324





(Haemophilus influenzae))


HI0191
204595
204158
ferric uptake regulation protein (fur) cyclic AMP receptor protein (crp)
61.4
75.0
139





(Escherichia coli))


HI1457
1537858
1537391
fimbrial transcription regulation repressor (pilB) (Neisseria gonorrhaeae)
32.3
53.2
124


HI1459
1539614
1538556
fimbrial transcription regulation repressor (pilB) (Neisseria gonorrhaeae)
59.0
72.6
325


HI1263
1336661
1337548
folylpolyglutamate-dihydrofolate synthetase expression regulator (accD)
69.5
82.5
290





(Escherichia coli)


HI1430
1512975
1513745
fumarate (and nitrate) reduction regulatory protein (fnr) (Escherichia coli)
78.8
88.8
240


HI0823
871805
872800
galactose operon repressor (galS) (Haemophilus influenzae)
99.1
99.4
332


HI0756
817661
818569
glucokinase regulator (Rattus norvegicus)
31.8
56.1
512


HI0621
651792
652556
glycerol-3-phosphate regulon repressor (glpR) (Escherichia coli)
61.5
77.4
252


HI1011
1073676
1073047
glycerol-3-phosphate regulon repressor (glpR) (Escherichia coli)
28.6
50.3
198


HI1197
1259493
1260395
glycine cleavage system transcriptional activator (gcvA) (glycerol-3-phosphate regulon
51.7
69.1
298





repressor (glpR)) (Escherichia coli)


HI0013
13742
12837
GTP-binding protein (era) (Escherichia coli)
77.9
87.0
299


HI0879
930478
929309
GTP-binding protein (obg) (Bacillus subtilis)
47.7
70.9
332


HI0573
592001
591099
hydrogen peroxide-inducible activator (oxyR) (Escherichia coli)
71.1
85.9
298


HI0617
647526
646780
L-fucose operon activator (fucR) (Escherichia coli)
35.1
56.1
229


HI0401
450131
420952
lacZ expression regulator (icc) (Escherichia coli)
52.9
71.3
261


HI0225
253133
253636
leucine responsive regulatory protein (lrp) (Escherichia coli)
29.6
52.6
152


HI1602
1663150
1662653
leucine responsive regulatory protein (lrp) (Escherichia coli)
77.2
86.7
158


HI0751
809477
810103
LEXA repressor (lexA) (Escherichia coli)
68.1
85.3
202


HI1465
1542848
1542810
lipoligosaccharide protein (lex2A) (Haemophilus influenzae)
44.4
66.7
9


HI1466
1542849
1543428
lipoligosaccharide protein (lex2A) (Haemophilus influenzae)
50.0
66.7
48


HI0296
328190
327876
metF aporepressor (metJ) (Escherichia coli)
81.9
93.3
105


HI1478
1558154
1557312
molybdenum transport system alternative nitrogenase regulator (modD)
31.8
51.7
259





(Rhodobacter capsulatus)


HI0200
214274
215227
msbB protein (msbB) (Escherichia coli)
45.3
67.0
301


HI0411
429238
430662
msbB protein (msbB) (Escherichia coli)
50.9
69.3
284


HI0712
756824
757117
negative regulator of translation (relB) (Escherichia coli)
28.3
48.3
60


HI0631
667822
668406
negative rpo regulator (mctA) (Escherichia coli)
40.1
62.9
199


HI0269
299532
301232
nitrate sensor protein (narQ) (Escherichia coli)
38.6
63.0
555


HI0728
778003
777380
nitrate/nitrite response regulator protein (narP) (Escherichia coli)
59.6
79.3
205


HI0339
363915
364250
nitrogen regulatory protein P-II (glnB) (Escherichia coli)
77.7
93.8
112


HI1747
1828067
1826037
penta-phosphate guanosine-3′-pyrophosphohydrolase (spoT) (Escherichia
58.8
76.6
675





coli)


HI1381
1475017
1473741
phosphate regulon sensor protein (phoR) (Escherichia coli)
41.8
66.8
335


HI1382
1475709
1475017
phosphate regulon transcriptional regulatory protein (phoB) (Escherichia
52.9
71.8
227





coli)


HI0765
827030
825768
probable nadAB transcriptional regulator (nadR) (Escherichia coli)
54.6
75.1
349


HI1641
1697003
1698115
purine nucleotide synthesis repressor protein (purR) (Escherichia coli)
55.9
74.5
328


HI0164
178405
178713
putative murein gene regulator (bolA) (Escherichia coli)
47.1
65.7
102


HI0508
522278
523273
rbs repressor (rbsR) (Escherichia coli)
48.8
71.0
329


HI0565
582225
581776
regulatory protein (asnC) (Escherichia coli)
68.0
81.0
147


HI1617
1677452
1676583
regulatory protein sfs1 involved in maltose metabolism (sfsA) (Escherichia
54.3
71.2
218





coli)


HI0895
946128
946688
repressor for cytochrome P450 (Bm3R1) (Bacillus magaterium)
23.3
50.6
182


HI0271
302396
303238
RNA polymerase sigma-32 factor (heat shock regulatory protein F334)
70.8
86.8
281





(rpoH) (Escherichia coli)


HI0535
555646
557532
RNA polymerase sigma-70 factor (rpoD) (Escherichia coli)
68.9
80.8
608


HI0630
667228
667794
RNA polymerase sigma-E factor (rpoE) (Escherichia coli)
73.0
87.8
189


HI1713
1781137
1779785
sensor protein for basR (basS) (Escherichia coli)
30.0
55.7
253


HI1444
1529117
1528668
stringent starvation protein (sspB) (Escherichia coli)
63.2
81.1
106


HI1445
1529755
1529120
stringent starvation protein A (sspA) (Haemophilus somnus)
76.9
87.3
212


HI1745
1815630
1814704
trans-activator of metE and-metH (metR) (Escherichia coli)
39.5
60.8
294


HI0360
382477
383121
transcription activator (tenA) (Bacillus subtilis)
27.8
48.3
208


HI0683
722643
721768
transcriptional activator protein (ilvY) (Escherichia coli)
47.4
70.3
293


HI1714
1781799
1781137
transcriptional regulatory protein (basR) (Escherichia coli)
43.5
59.7
216


HI0412
430780
431733
transcriptional regulatory protein (tyrR) (Escherichia coli)
48.2
66.8
306


HI0832
880611
880913
tryptophan repressor (trpR) (Enterobacter aerogenes)
39.8
67.0
88


HI0054
54188
54985
uxu operon regulator (uxuR) (Escherichia coli)
50.0
72.1
246


HI1109
1170415
1169255
xylose operon regulatory protein (xylR) (Escherichia coli)
57.3
75.3
384







Replication









DNA - replication, restr/modification, recombination













HI0761
822003
823136
A/G-specific adenine glycosylase (mutY) (Escherichia coli)
61.6
75.1
341


HI0995
1056674
1055313
chromosomal replication initiator protein (dnaA) (Escherichia coli)
61.7
79.7
464


HI1229
1294415
1294317
chromosomal replication initiator protein (dnaA) (Escherichia coli)
50.0
75.0
12


HI0316
345720
345151
crossover junction endodeoxyribonuclease (ruvC) (chromosomal replication
78.5
88.3
163





initiator protein (dnaA)) (Escherichia coli)


HI0955
1011537
1012736
dfp protein (dfp) (Escherichia coli)
61.1
76.6
402


HI0210
223259
224116
DNA adenine methylase (dam) (Escherichia coli)
55.4
71.4
266


HI1267
1343755
1341116
DNA gyrase, subunit A (gyrA) (Escherichia coli)
70.6
84.9
859


HI0569
587397
584980
DNA gyrase, subunit B (gyrB) (Escherichia coli)
74.7
85.9
803


HI1191
1255302
1253122
DNA helicase II (uvrD) (Haemophilus influenzae)
96.8
97.5
727


HI1102
1162989
1160953
DNA ligase (lig) (Escherichia coli)
63.7
79.9
666


HI0405
423539
424207
DNA mismatch protein (mutH) (Escherichia coli)
60.4
80.7
212


HI0709
750565
753147
DNA mismatch repair protein (mutS) (Escherichia coli)
71.0
84.0
853


HI0067
69622
71508
DNA mismatch repair protein MUTL (mutL) (Escherichia coli)
50.2
67.3
612


HI0858
904919
902130
DNA polymerase I (potA) (Escherichia coli)
63.1
77.0
928


HI0994
1055297
1054200
DNA polymerase III beta-subunit (dnaN) (Escherichia coli)
62.6
80.3
366


HI0457
476761
475763
DNA polymerase III delta prime subunit (holB) (Escherichia coli)
35.3
57.4
316


HI0925
979730
980761
DNA polymerase III delta subunit (holA) (Escherichia coli)
45.2
62.0
332


HI0138
152669
151902
DNA polymerase III epsilon subunit (dnaQ) (Escherichia coli)
61.3
76.5
236


HI0741
799019
795544
DNA polymerase III, alpha chain (dnaE) (Escherichia coli)
71.9
85.7
1159


HI1402
1493690
1493259
DNA polymerase III, chi subunit (holC) (Haemophilus influenzae)
98.9
98.9
88


HI0011
11672
11271
DNA polymerase III, psi subunit (holD) (Escherichia coli)
34.4
59.2
123


HI0534
553659
555645
DNA primase (dnaG) (Escherichia coli)
56.5
73.8
571


HI1746
1826037
1823959
DNA recombinase (recG) (Escherichia coli)
56.5
80.1
693


HI0070
77166
75493
DNA repair protein (recN) (Escherichia coli)
48.6
67.3
533


HI0659
699507
700058
DNA topoisomerase I (topA) (Bacillus subtilis)
34.2
55.0
110


HI0656
698124
697570
DNA-3-methyladenine glycosidase I (tagI) (Escherichia coli)
62.6
76.0
179


HI0730
779457
781969
DNA-dependent ATPase, DNA helicase (recQ) (Escherichia coli)
62.9
77.6
589


HI0568
584860
584159
dod protein (dod) (Serratia marceucens)
81.4
93.3
210


HI0062
65230
65664
dosage-dependent dnaK suppressor protein (dksA) (Escherichia coli)
73.9
83.8
142


HI0948
1005798
1004986
formamidopyrimidine-DNA glycosylase (fpg) (Escherichia coli)
57.6
74.7
269


HI0584
602405
600519
glucose inhibited division protein (gidA) (Escherichia coli)
76.1
87.3
627


HI0488
506816
506208
glucose inhibited division protein (gidB) (Escherichia coli)
64.0
78.0
200


HI0982
1037496
1037792
Hin recombinational enhancer binding protein (fis) (Escherichia coli)
81.6
92.9
97


HI0514
528338
527565
HincII endonuclease (HincII) (Haemophilus influenzae)
98.4
98.4
258


HI1397
1491189
1490263
HindIII modification methyltransferase (hindIIIM) (Haemophilus influenzae)
99.4
99.4
309


HI1398
1492072
1491173
HindIII restriction endonuclease (hindIIIR) (Haemophilus influenzae)
99.7
99.7
300


HI0315
345085
344474
holiday junction DNA helicase (ruvA) (Escherichia coli)
58.8
79.9
203


HI0314
344463
343459
holiday junction DNA helicase (ruvB) (Escherichia coli)
80.9
90.0
330


HI0678
719064
718180
integrase/recombinase protein (xerC) (Escherichia coli)
58.0
74.4
293


HI1316
1391102
1391389
integration host factor alpha-subunit (himA) (Escherichia coli)
63.6
83.0
94


HI1224
1291400
1291681
integration host factor beta-subunit (IHF-beta) (himD) (Escherichia coli)
56.5
77.2
92


HI0404
422970
423539
methylated-DNA-protein-cystein methyltransferase (dat1) (Bacillus
40.1
61.7
163





subtilis)


HI0671
713369
713806
mioC protein (mioC) (Escherichia coli)
53.5
71.5
144


HI1043
1104813
1105724
modification methylase HgiDl (MHgiDl) (Herpelosiphon aurantiacus)
56.4
70.5
297


HI0515
529891
528338
modification methylase HincII (hincIIM) (Haemophilus influenzae)
98.2
98.6
502


HI0912
963611
964312
mutator mutT (AT-GC transversion) (Escherichia coli)
48.8
72.0
125


HI0193
206098
206688
negative modulator of initiation of replication (seqA) (Escherichia coli)
53.1
71.8
177


HI0548
568202
567879
primosomal protein n precursor (priB) (Escherichia coli)
57.4
75.2
101


HI0341
367532
365343
primosomal protein replication factor (priA) (Escherichia coli)
52.3
70.2
729


HI0389
406402
408321
probable ATP-dependent helicase (dinG) (Escherichia coli)
32.2
51.1
680


HI0993
1054243
1053119
recF protein (recF) (Escherichia coli)
57.0
75.8
356


HI0334
358532
359239
recO protein (recO) (Escherichia coli)
64.6
76.5
226


HI0602
621957
620896
recombinase (recA) (Haemophilus influenzae)
100.0
100.0
354


HI0061
64971
62573
recombination protein (rec2) (Haemophilus influenzae)
99.9
99.9
800


HI0445
464118
464717
recR protein (recR) (Escherichia coli)
74.9
88.4
199


HI0601
620735
620358
regulatory protein (recX) (Pseudomonas fluorescans)
28.6
50.4
117


HI0651
694852
692768
rep helicase (rep) (Escherichia coli)
66.9
82.7
669


HI1232
1299240
1297177
replication protein (dnaX) (Escherichia coli)
52.9
69.8
843


HI1580
1641089
1642600
replicative DNA helicase (dnaB) (Escherichia coli)
68.6
82.8
462


HI1042
1103812
1104813
restriction enzyme (hgiDIR) (Herpetosiphon giganteus)
44.2
63.9
350


HI1175
1241423
1242574
S-adenosylmethionine synthetase 2 (metX) (Escherichia coli)
82.3
91.7
383


HI1429
1512463
1511552
shufflon-specific DNA recombinase (rci) (Escherichia coli)
31.1
55.5
259


HI0251
281830
282333
single-stranded DNA binding protein (ssb) (Haemophilus influenzae)
95.8
98.2
168


HI1578
1839113
1638016
site-specific recombinase (rcb) (Escherichia coli)
36.3
57.0
265


HI1368
1450325
1452928
topoisomerase I (topA) (Escherichia coli)
72.0
84.3
865


HI0446
464736
466688
topoisomerase III (topB) (Escherichia coli)
65.9
79.4
645


HI1535
1599641
1601881
topoisomerase IV subunit A (parC) (Escherichia coli)
71.4
85.4
727


HI1534
1597676
1599571
topoisomerase IV subunit B (parE) (Escherichia coli)
76.5
88.6
630


HI1261
1331575
1335011
transcription-repair coupling factor (trcF) (mld) (Escherichia coli)
64.3
82.7
1134


HI0217
232884
234038
type I restriction enzyme ecokl specificity protein (hsdS) (Escherichia coli)
36.1
58.6
394


HI0216
231281
232797
type I restriction enzyme ECOR124/3 I M protein (hsdM) (Escherichia coli)
81.2
89.3
512


HI1290
1368549
1367223
type I restriction enzyme ECOR124/3 I M protein (hsdM) (Escherichia coli)
30.4
53.7
332


HI1288
1365756
1362592
type I restriction enzyme ECOR124/3 I R protein (hsdR) (Escherichia coli)
30.4
52.7
991


HI1059
1123091
1121205
type III restriction-modification ECOP15 enzyme (mod) (Escherichia coli)
36.5
55.5
384


HI0018
18087
18743
uracil DNA glycosylase (ung) (Escherichia coli)
70.2
79.5
215


HI0311
342051
342941
xprB protein (xerD) (Escherichia coli)
68.9
84.8
296









Degradation of DNA













HI1695
1758680
1759312
endonuclease III (nth) (Escherichia coli)
83.4
91.9
211


HI0250
278528
281829
excinuclease ABC subunit A (uvrA) (Escherichia coli)
81.2
91.0
940


HI1250
1323924
1321888
excinuclease ABC subunit B (uvrB) (Escherichia coli)
78.0
87.7
869


HI0057
58893
57067
excinuclease ABC subunit C (uvrC) (Escherichia coli)
65.9
80.0
588


HI1380
1471626
1473044
exodeoxyribonucleasae I (sbcB) (Escherichia coli)
57.5
74.9
462


HI1324
1395898
1399530
exodeoxyribonucleasae V (recB) (Escherichia coli)
37.1
58.2
1165


HI0944
998895
1002257
exodeoxyribonucleasae V (recC) (Escherichia coli)
40.1
61.2
1114


HI1325
1399533
140142
exodeoxyribonucleasae V (recD) (Escherichia coli)
40.0
59.3
570


HI0041
43872
43072
exonuclease III (xthA) (Escherichia coli)
71.9
83.9
267


HI0399
417972
419288
exonuclease VII, large subunit (xseA) (Escherichia coli)
57.8
74.4
437


HI1217
1280795
1282519
single-stranded-DNA-specific exonuclease (recJ) (Escherichia coli)
59.2
77.3
554







Transcription









RNA synthesis, modification and DNA transcription













HI0618
647724
650492
ATP-dependent helicase HEPA (hepA) (Escherichia coli)
53.6
73.6
968


HI0424
444751
443435
ATP-dependent RNA helicase (srmB) (Escherichia coli)
39.8
60.9
448


HI0232
260978
262816
ATP-dependent RNA helicase DEAD (deaD) (Escherichia coli)
64.0
78.6
613


HI0804
851485
852468
DNA-directed RNA polymerase alpha chain (rpoA) (Escherichia coli)
91.8
97.0
329


HI0517
534212
538870
DNA-directed RNA polymerase beta chain (rpoB) (Salmonella typhimurium)
83.3
91.9
1342


HI0516
534211
529967
DNA-directed RNA polymerase beta chain (rpoC) (Escherichia coli)
83.0
90.7
1399


HI1307
1383078
1383509
N utilization substance protein B (nusB) (Escherichia coli)
54.9
71.4
133


HI0063
65915
67269
plasmid copy number control protein (pcnB) (Escherichia coli)
55.7
73.4
404


HI0230
257702
259828
polynucleotide phosphorylase (pnp) (Escherichia coli)
74.2
86.7
708


HI0894
944630
945883
putative ATP-dependent RNA helicase (rhlB) (Escherichia coli)
73.9
64.1
410


HI1748
1828594
1828331
RNA polymerase omega subunit (rpoZ) (Escherichia coli)
64.6
76.1
88


HI1463
1542205
1541624
sigma factor (algU) (Pseudomonas aeruginosa)
27.6
48.8
168


HI0719
764847
765401
transcription antitermination protein (nusG) (Escherichia coli)
73.7
84.4
179


HI0571
589932
590405
transcription elongation factor (greB) (Escherichia coli)
61.5
79.5
156


HI1286
1358486
1360006
transcription factor (nusA) (Salmonella typhimurium)
70.8
84.1
499


HI0297
328437
329696
transcription termination factor rho (rho) (Escherichia coli)
87.4
95.2
419









Degradation of RNA













HI0219
234848
237923
anticodon nuclease masking-agent (prrD) (Escherichia coli)
72.9
85.6
291


HI1739
1810586
1808610
exoribonuclease II (RNaseII) (Escherichia coli)
50.8
68.0
588


HI0392
411354
412550
ribonuclease D (md) (Escherichia coli)
41.3
65.5
365


HI0415
433540
436392
ribonuclease E (me) (Escherichia coli)
60.3
72.3
1058


HI0139
152730
153191
ribonuclease H (mh) (Escherichia coli)
64.9
76.0
154


HI1061
1124258
1123668
ribonuclease HII (EC31264) (RNASE HII) (Escherichia coli)
73.7
82.8
185


HI0014
14422
13742
ribonuclease III (mc) (Escherichia coli)
65.3
80.2
221


HI0275
306539
305826
ribonuclease PH (rph) (Escherichia coli)
78.9
87.8
237


HI1001
1063336
1063743
RNase P (mpA) (Escherichia coli)
69.7
80.7
119


HI0326
351726
352412
RNase T (mt) (Escherichia coli)
65.7
80.9
204







Translation









Ribosomal proteins - synthesis, modification













HI0518
539557
538871
ribosomal protein L1 (rpL1) (Escherichia coli)
85.6
93.4
229


HI0642
681369
681857
ribosomal protein L10 (rpL10) (Salmonella typhimurium)
80.5
89.0
165


HI0519
539990
539565
ribosomal protein L11 (rpL11) (Escherichia coli)
86.6
94.4
142


HI0980
1035484
1036371
ribosomal protein L11 methyltransferase (prmA) (Escherichia coli)
69.2
83.2
291


HI1447
1530773
1530348
ribosomal protein L13 (rpL13) (Haemophilus somnus)
94.4
95.8
142


HI0790
844379
844747
ribosomal protein L14 (rpL14) (Escherichia coli)
94.3
98.4
123


HI0799
847996
848427
ribosomal protein L15 (rpL15) (Escherichia coli)
82.6
91.0
144


HI0780
842244
842651
ribosomal protein L16 (rpL16) (Escherichia coli)
89.7
95.6
136


HI0805
852512
852895
ribosomal protein L17 (rplQ) (Escherichia coli)
89.8
92.1
127


HI0796
846938
847288
ribosomal protein L18 (rpL18) (Escherichia coli)
84.6
91.5
117


HI0202
216787
216440
ribosomal protein L19 (rpL19) (Escherichia coli)
89.5
93.2
114


HI0782
840039
840857
ribosomal protein L2 (rpL2) (Escherichia coli)
85.7
93.4
273


HI1323
1395432
1395782
ribosomal protein L20 (rpL20) (Escherichia coli)
94.0
96.6
117


HI0882
932097
931789
ribosomal protein L21 (rpL21) (Escherichia coli)
79.6
86.4
103


HI0784
841173
841502
ribosomal protein L22 (rpL22) (Escherichia coli)
91.8
97.3
110


HI0781
839722
840018
ribosomal protein L23 (rpL23) (Escherichia coli)
71.7
82.8
99


HI0791
844761
845069
ribosomal protein L24 (rpL24) (Escherichia coli)
76.7
86.4
103


HI1636
1692153
1692437
ribosomal protein L25 (rpL25) (Escherichia coli)
61.9
77.4
84


HI0881
931428
931788
ribosomal protein L27 (rpL27) (Escherichia coli)
87.1
90.6
85


HI0953
1010494
1010261
ribosomal protein L28 (rpL28) (Escherichia coli)
85.7
94.8
77


HI0787
842654
842842
ribosomal protein L29 (rpL29) (Escherichia coli)
75.8
87.1
62


HI0779
838481
839104
ribosomal protein L3 (rpL3) (Escherichia coli)
85.2
92.3
209


HI0798
847813
847989
ribosomal protein L30 (rpL30) (Escherichia coli)
79.7
85.4
59


HI0760
821826
821617
ribosomal protein L31 (rpL31) (Escherichia coli)
71.4
85.7
70


HI0159
174441
174274
ribosomal protein L32 (rpL32) (Escherichia coli)
77.2
86.0
57


HI0952
1010246
1010079
ribosomal protein L33 (rpL33) (Escherichia coli)
81.5
90.7
54


HI1000
1063233
1063364
ribosomal protein L34 (rpL34) (Escherichia coli)
86.4
93.2
44


HI1322
1395096
1395269
ribosomal protein L35 (rpL35) (Escherichia coli)
75.0
90.6
32


HI0780
839123
839722
ribosomal protein L4 (rpL4) (Escherichia coli)
83.6
93.0
201


HI0792
845090
845626
ribosomal protein L5 (rpL5) (Escherichia coli)
90.5
96.1
179


HI0795
846391
846921
ribosomal protein L6 (rpL6) (Escherichia coli)
75.1
90.4
177


HI0643
681915
682283
ribosomal protein L7/L12 (rpL7/L12) (Escherichia coli)
82.0
91.8
121


HI0546
567619
587173
ribosomal protein L9 (rpL9) (Escherichia coli)
72.5
85.9
149


HI1223
1289629
1291274
ribosomal protein S1 (rpS1) (Escherichia coli)
79.3
88.7
657


HI0778
838108
838461
ribosomal protein S10 (rpS10) (Escherichia coli)
98.1
99.0
103


HI0802
850416
850802
ribosomal protein S11 (rpS11) (Escherichia coli)
92.2
96.1
129


HI0801
850045
850397
ribosomal protein S13 (rpS13) (Escherichia coli)
86.4
93.2
118


HI0793
845641
845943
ribosomal protein S14 (rpS14) (Escherichia coli)
89.9
94.9
99


HI1331
1405806
1406072
ribosomal protein S15 (rpS15) (Escherichia coli)
80.9
86.5
89


HI1473
1554091
1553825
ribosomal protein S15 (rpS15) (Escherichia coli)
80.9
86.5
89


HI0205
218422
218177
ribosomal protein S16 (rpS16) (Escherichia coli)
70.7
85.4
82


HI0788
842845
843099
ribosomal protein S17 (rpS17) (Escherichia coli)
85.7
94.0
84


HI0547
567863
567639
ribosomal protein S18 (rpS18) (Escherichia coli)
92.0
94.7
75


HI0783
840885
841158
ribosomal protein S19 (rpS19) (Escherichia coli)
90.1
97.8
91


HI0915
967289
968041
ribosomal protein S2 (rpS2) (Escherichia coli)
82.2
89.2
241


HI0533
553446
553658
ribosomal protein S21 (rpS21) (Escherichia coli)
83.1
87.3
71


HI0785
841523
842227
ribosomal protein S3 (rpS3) (Escherichia coli)
87.2
93.2
233


HI0803
850833
851450
ribosomal protein S4 (rpS4) (Escherichia coli)
89.3
94.7
206


HI0797
847306
847803
ribosomal protein S5 (rpS5) (Escherichia coli)
92.8
95.8
166


HI0549
568566
568192
ribosomal protein S6 (rpS6) (Escherichia coli)
76.8
87.2
125


HI1537
1604087
1603182
ribosomal protein S6 modification protein (rimK (Escherichia coli)
45.3
69.0
272


HI0582
599803
599336
ribosomal protein S7 (rpS7) (Escherichia coli)
89.7
94.2
155


HI0794
845983
846372
ribosomal protein S8 (rpS8) (Escherichia coli)
86.2
90.8
130


HI1446
1530328
1529939
ribosomal protein S9 (rpS9) (Haemophilus somnus)
94.6
98.5
130


HI0010
11292
10828
ribosomal-protein-alanine acetyltransferase (rml) (Escherichia coli)
55.9
73.1
144


HI0583
600334
599963
streptomycin resistance protein (strA) (Haemophilus influenzae)
100.0
100.0
124









Amino acyl tRNA Synthetases, tRNA modification













HI0816
865547
862926
alanyl-tRNA synthetase (alaS) (Escherichia coli)
68.2
82.6
873


HI1589
1646685
1850415
arginyl-tRNA synthetase (argS) (Escherichia coli)
71.2
83.5
577


HI1305
1382405
1380975
asparaginyl-tRNA synthetase (asrS) (Escherichia coli)
80.6
90.8
465


HI0319
348931
347168
aspartyl-tRNA synthetase (aspS) (Escherichia coli)
76.2
85.5
585


HI0078
85367
83991
cys-tRNA synthetase (cysS) (Escherichia coli)
75.7
87.0
461


HI0710
753356
754738
cysteinyl-tRNA (ser) selenium transferase (selA) (Escherichia coli)
58.8
75.8
454


HI1357
1431798
1433466
glutaminyl-tRNA synthetase (glnS) (Escherichia coli)
75.7
86.9
547


HI0276
308282
306843
glutamyl-tRNA synthetase (gltX) (Escherichia coli)
72.4
84.3
464


HI0929
985024
984119
glycyl-tRNA synthetase alpha chain (glyQ) (Escherichia coli)
90.6
94.6
299


HI0926
983065
981002
glycyl-tRNA synthetase beta chain (glyS) (Escherichia coli)
69.7
81.9
689


HI0371
392076
393344
histidine-tRNA synthetase (hisS) (Escherichia coli)
66.8
79.1
421


HI0964
1021072
1018250
isoleucyl-tRNA ligase (ileS) (Escherichia coli)
66.0
78.5
934


HI0923
976547
979129
leucyl-tRNA synthetase (leuS) (Escherichia coli)
72.3
82.2
859


HI1214
1278435
1276930
lysyl-tRNA synthetase (lysU) (Escherichia coli)
70.2
84.3
505


HI0838
885271
886269
lysyl-tRNA synthetase analog (genX) (Escherichia coli)
62.7
78.5
331


HI0625
662613
663586
methionyl-tRNA formyltransferase (fml) (Escherichia coli)
65.0
77.4
313


HI1279
1353301
1351256
methionyl-tRNA synthetase (melG) (Escherichia coli)
69.0
83.3
677


HI0396
416278
415697
peptidyl-tRNA hydrolase (pth) (Escherichia coli)
64.2
80.5
190


HI1314
1387690
1388676
phenylalenyl-tRNA synthetase beta-subunit (pheS) (Escherichia coli)
75.0
82.0
327


HI1315
1388713
1391097
phenylalenyl-tRNA synthetase beta-subunit (pheT) (Escherichia coli)
65.3
80.1
795


HI0731
781970
783684
prolyl-tRNA synthetase (proS) (Escherichia coli)
74.9
86.8
570


HI1650
1709685
1708879
pseudouridylate synthase I (hisT) (Escherichia coli)
69.2
82.7
260


HI0246
273589
272501
queuosine biosynthesis protein (queA) (Escherichia coli)
72.5
85.7
346


HI0201
215333
216439
selenium metabolism protein (selD) (Escherichia coli)
66.1
80.6
330


HI0110
117234
118520
seryl-tRNA synthetase (serS) (Escherichia coli)
77.6
86.5
430


HI1370
1453876
1455804
threonyl-tRNA synthetase (thrS) (Escherichia coli)
77.9
86.1
642


HI0245
272154
271009
transfer RNA-guanine transglycosylase (tgt) (Escherichia coli)
81.3
91.5
374


HI0203
217564
216827
tRNA (guanine-N1)-methyltransferase (M1G-methyltransferase) (trmD)
83.2
93.0
244





(Escherichia coli)


HI0850
894301
895389
tRNA (uracil-5-)-methyltransferase (trmA) (Escherichia coli)
64.6
80.4
362


HI0068
71519
72451
tRNA delta(2)-isopentenylpyrophosphate transferase (trpX) (Escherichia
69.8
87.4
300





coli)


HI1612
1671420
1672667
tRNA nucleotidyltransferase (cca) (Escherichia coli)
58.4
73.4
404


HI0242
270097
269807
tRNA-guanine-transglycosylase (tgt) (Escherichia coli)
62.4
81.7
92


HI0639
678958
677957
tryptophenyl-tRNA synthetase (trpS) (Escherichia coli)
78.1
86.2
334


HI1616
1676533
1675331
tyrosyl tRNA synthetase (tyrS) (Thiobacillus ferroxidans)
53.6
72.6
398


HI1396
1490259
1487398
valyl-tRNA synthetase (valS) (Escherichia coli)
70.8
83.3
951









Nucleoproteins













HI0187
200140
200544
DNA binding protein (probable) (Bacillus subtilis)
43.4
64.2
106


HI1496
1568461
1568885
DNA-binding protein (rdgB) (Erwinia carolovora)
42.4
60.5
57


HI1593
1655153
1655554
DNA-binding protein H—NS (hns) (Escherichia coli)
47.4
65.2
135


HI0432
453511
453104
DNA-binding protein HU-ALPHA (NS2) (HU-2) (Escherichia coli)
78.9
86.7
90









Proteins - translation and modification













HI0848
893035
893757
disulfide oxidoreductase (por) (Haemophilus influenzae)
100.0
100.0
205


HI0987
1042200
1041082
DNA processing chain A (dprA) (Escherichia coli)
44.8
60.2
358


HI0916
968177
969025
elongation factor EF-Ts (tsf) (Escherichia coli)
71.4
85.0
280


HI0580
597082
595901
elongation factor EF-Tu (duplicate) (tufB) (Escherichia coli)
92.6
95.9
394


HI0634
671167
672348
elongation factor EF-Tu (duplicate) (tufB) (Escherichia coli)
92.6
95.9
394


HI0581
599249
597150
elongation factor G (fusA) (Escherichia coli)
84.6
92.0
704


HI0330
355617
355054
elongation factor P (elp) (Escherichia coli)
75.0
85.6
188


HI0069
72460
75402
glutamate-ammonia-ligase adenylyltransferase (glnE) (Escherichia coli)
52.5
69.7
914


HI1321
1394551
1394954
initiation factor 3 (infC) (Escherichia coli)
82.8
94.6
134


HI0550
569019
568768
initiation factor IF-1 (infA) (Escherichia coli)
94.4
98.6
72


HI1287
1360021
1362507
initiation factor IF-2 (infB) (Escherichia coli)
70.9
84.5
842


HI1155
128859
1220211
maturation of antibiotic MccB17 (pmbA) (Escherichia coli)
60.8
78.7
450


HI1728
1794724
1793921
methionine aminopeptidase (map) (Escherichia coli)
64.3
79.8
262


HI0430
450570
451100
oxido-reductase (dsbB) (Escherichia coli)
43.8
68.8
174


HI1215
1279684
1278589
peptide chain release factor 2 (prfB) (Salmonella typhimurium)
81.7
93.7
365


HI1741
1811638
1813216
peptide-chain-release factor 3 (prfC) (Escherichia coli)
86.0
93.4
527


HI0079
85470
85976
peptidyl-prolyl cis-trans isomerase B (ppiB) (Escherichia coli)
71.3
80.5
163


HI1567
1631427
1630345
polypeptide chain release factor 1 (prfA) (Salmonella typhimurium)
72.5
88.3
360


HI0624
662011
662517
polypeptide deformylase (formalymethionine deformylase) (def) (Escherichia
65.1
79.9
169





coli)


HI0810
857270
856716
ribosome releasing factor (rrf) (Escherichia coli)
68.1
84.9
185


HI0575
593158
592940
rotamase, peptidyl prolyl cis-trans isomerase (slyD) (Escherichia coli)
50.7
73.1
67


HI0701
745982
745413
rotamase, peptidyl prolyl cis-trans isomerase (slyD) (Escherichia coli)
68.3
79.4
187


HI1334
1408450
1408923
transcription elongation factor (greA) (Escherichia coli)
79.7
89.9
158


HI0711
754738
756593
translation factor (selB) (Escherichia coli)
44.0
64.7
606


HI1216
1279817
1280503
xprA protein (xprA) (Escherichia coli)
45.4
67.4
227









Degradation of proteins, peptides, glycopeptides













HI0877
927500
928801
aminopeptidase A (pepA) (Rickettsia prowazekii)
39.6
57.9
313


HI1711
1775967
1777439
aminopeptidase a/i (pepA) (Escherichia coli)
57.3
77.5
497


HI1620
1682194
1879588
aminopeptidase N (pepN) (Escherichia coli)
60.9
75.6
864


HI0818
867554
866265
aminopeptidase P (pepP) (Escherichia coli)
54.6
73.6
435


HI0716
762461
763039
ATP-dependent clp protease proteolytic component (clpP) (Escherichia coli)
71.0
88.1
193


HI0717
763052
764284
ATP-dependent protease ATPase subunit (clpX) (Escherichia coli)
70.2
83.2
413


HI0861
906379
908946
ATP-dependent protease binding subunit (clpB) (Escherichia coli)
77.4
88.6
857


HI0421
440910
442289
collagenase activity collagenase (prtC) (Porphyromonas gingivalis)
31.1
53.4
206


HI0151
166695
165811
HFLC protein (hflC) (Escherichia coli)
58.5
78.2
329


HI0248
274175
276400
IgA1 protease (iga1) (Haemophilus influenzae)
28.6
51.5
759


HI0992
1047674
1053118
IgA1 protease (iga1) (Haemophilus influenzae)
99.8
99.9
1702


HI0249
278527
276401
IgA1 protease (iga1) (Haemophilus influenzae)
45.2
62.5
791


HI1327
1402067
1403869
Ion protease (ion) (Bacillus brevis)
24.2
46.6
714


HI0215
229004
231046
oligopeptidase A (prtC) (Escherichia coli)
72.0
84.8
676


HI0677
716670
718121
peptidase D (pepD) (Escherichia coli)
56.8
72.2
485


HI0589
608542
607865
peptidase E (pepE) (Escherichia coli)
41.4
60.0
214


HI1351
1423832
1425067
peptidase T (pepT) (Salmonella typhimurium)
53.3
71.4
398


HI1262
1336467
1335070
periplasmic senne protease Do and heal shock protein (htrA) (Escherichia
55.8
73.9
489





coli)


HI1603
1664636
1663212
probable ATP-dependent protease (sms) (Escherichia coli)
80.0
92.2
480


HI0724
768169
768786
proline dipeptidase (pepQ) (Escherichia coli)
53.7
70.2
204


HI0137
151209
151901
protease (prtH) (Porphyromonas gingivalis)
52.6
64.9
57


HI1547
1613228
1611384
protease IV (sppA) (Escherichia coli)
43.7
64.0
607


HI0152
167927
166698
protease specific for phage lambda cll repressor (hflK) (Escherichia coli)
55.8
72.6
396


HI1688
1751031
1752089
putative protease (sohB) (Escherichia coli)
53.3
74.5
348


HI0532
553214
552189
sialoglycoprotease (gcp) (Pasteurella haemolytica)
81.8
91.5
319







Transport/binding proteins









Amino acids, peptides, amines













HI1183
1247387
1246659
arginine transport ATP-binding protein anP (anP) (Escherichia coli)
65.8
83.1
242


HI1180
1245250
1244570
arginine transport system permease protein (artM) (Escherichia coli)
55.7
79.9
218


HI1181
1245915
1245253
arginine transport system permease protein (artQ) (Escherichia coli)
59.0
77.8
229


HI0254
284235
283786
biopolymer transport protein (exbB) (Haemophilus influenzae)
96.0
98.7
150


HI0253
283779
283339
biopolymer transport protein (exbD) (Escherichia coli)
28.8
55.1
118


HI1734
1801710
1800520
branched chain as transport system II carrier protein (braB) (Pseudomonas
28.4
49.8
279





aeruginosa)


HI0885
935516
934149
O-alanine permease (dagA) (Alteromonas haloplanktis)
43.2
65.5
527


HI1188
1251117
1250128
dipeptide transport ATP-binding protein (dppD) (Escherichia coli)
74.2
84.0
326


HI1187
1250122
1249142
dipeptide transport ATP-binding protein (dppF) (Escherichia coli)
76.4
87.1
325


HI1126
1189626
1188709
dipeptide transport system permease protein (dppB) (Escherichia coli)
34.1
50.7
337


HI1190
1253029
1252031
dipeptide transport system permease protein (dppB) (Escherichia coli)
61.1
79.2
337


HI1189
1252013
1251130
dipeptide transport system permease protein (dppC) (Escherichia coli)
63.8
83.3
287


HI1536
1601926
1603137
glutamate permease (gltS) (Escherichia coli)
53.9
73.0
391


HI1081
1146102
1145389
glutamine transport system permease protein (glnP) (Escherichia coli)
37.6
59.0
212


HI1082
1146859
1146089
glutamine-binding periplasmic protein (glnH) (Escherichia coli)
28.4
48.2
222


HI0410
429066
428263
leucine-specific transport protein (livG) (Escherichia coli)
28.1
55.2
250


HI0227
255068
256375
membrane-associated component, LIV-II transport system (bmO)
32.9
60.4
425





(Salmonella typhimurium)


HI0214
228528
226987
oligopeptide binding protein (oppA) (Escherichia coli)
31.7
53.5
473


HI1127
1191333
1189710
oligopeptide binding protein (oppA) (Escherichia coli)
52.6
69.0
527


HI1124
1187751
1186783
oligopeptide transport ATP-binding protein (oppD) (Salmonella
77.2
85.0
320





typhimurium)


HI1123
1186783
1185788
oligopeptide transport ATP-binding protein (oppF) (Salmonella typhimurium)
71.5
83.9
329


HI1125
1188696
1187764
oligopeptide transport system permease protein (oppC) C (Salmonella
71.1
87.4
300





typhiumurium)


HI1644
1702355
1704049
peptide transport periplasmic protein (sapA) (Salmonella typhimurium)
39.3
63.8
504


HI1647
1705898
1706944
peptide transport system ATP-binding protein (sapD) (Salmonella
62.4
80.0
330





typhimurium)


HI1646
1705007
1705891
dipeptide transport system permease protein (dppC) (Escherichia coli)
36.2
59.9
279


HI1645
1704052
1705014
peptide transport system permease protein (sapB) (Salmonella
34.4
63.8
319





typhimurium)


HI1182
1246638
1245922
periplasmic arginine-binding protein (artI) (Pastuerella haemolytica)
58.6
73.4
234


HI1157
1221270
1222589
proton glutamate symport protein (gltP) (Bacillus caldotenax)
26.6
53.6
395


HI0592
611920
610616
putrescine transport protein (potE) (Escherichia coli)
77.2
88.0
434


HI0291
324543
323308
serine transporter (sdaC) (Escherichia coli)
61.0
77.8
411


HI1350
1423563
1422421
spermidine/putrescine transport ATP-binding protein (potA) (Escherichia
68.1
83.1
378





coli)


HI1349
1422434
1421577
spermidine/putrescine transport system permease protein (potB)
61.5
83.6
275





(Escherichia coli)


HI1348
1421548
1420808
spermidine/putrescine transport system permease protein (potC)
72.4
88.9
243





(Escherichia coli)


HI0500
514110
513175
spermidine/putrescine-binding periplasmic protein precursor (potD)
59.2
75.2
309





(Escherichia coli)


HI1347
1420732
1419596
spermidine/putrescine-binding periplasmic protein precursor (potD)
54.1
71.6
330





(Escherichia coli)


HI0289
320539
321792
typtophan-specific permease (mtr) (Escherichia coli)
55.8
72.5
396


HI0479
497829
499028
tyrosine-specific transport protein (tyrP) (Escherichia coli)
46.1
68.2
401


HI0530
551559
550342
tyrosine-specific transport protein (tyrP) (Escherichia coli)
45.4
65.4
404









Cations













HI0255
284871
284407
bacterioferritin comigratory protein (bcp) (Escherichia coli)
62.3
79.9
154


HI1275
1347862
1348650
ferric enterobactin transport ATP-binding protein (fepC) (Escherichia coli)
29.4
51.3
238


HI1475
1555193
1554435
ferric enterobactin transport ATP-binding protein (fepC) (Escherichia coli)
33.2
54.8
220


HI1471
1549654
1551853
ferrichrome-iron receptor (fhuA) (Escherichia coli)
26.4
48.9
710


HI1388
1479930
1480475
ferritin like protein (rsgA) (Escherichia coli)
57.4
79.0
162


HI1389
1480494
1480988
ferritin like protein (rsgA) (Escherichia coli)
57.3
73.8
164


HI0363
385804
384887
iron(III) dicitrate transport ATP-binding protein FECE (Escherichia coli)
35.9
56.4
220


HI1274
1347324
1347861
iron(III) dicitrate transport system permease protein (fecD) (Escherichia
36.0
64.0
255





coli)


HI1037
1099321
1100265
magnesium and cobalt transport protein (corA) (Escherichia coli)
70.3
84.8
316


HI0097
103798
104679
major ferric iron binding protein precursor (fbp) (Neisseria gonorrhoeae)
69.7
82.3
293


HI1051
1114308
1114635
mercuric transport protein (merT) (Pseudomonas aeruginosa)
25.0
55.2
99


HI1052
1114651
1114926
mercury scavenger protein (merP) (Pseudomonas fluorescens)
29.3
45.7
91


HI0294
327396
327193
mercury scavenger protein (merP) (Psudomonas fluorescens)
32.8
67.2
67


HI1531
1594953
1594219
molybdate-binding periplasmic protein precursor (modB) (Azotobacter
21.7
43.0
245





vinefandii)


HI0226
254880
253681
NA(+)/H(+) antiporter 1 (nhaA) (Escherichia coli)
52.6
74.6
380


HI0429
448992
450557
Na+/H+ antiporter (nhaB) (Escherichia coli)
70.6
87.5
501


HI1110
1171933
1170530
Na+/H+ antiporter (nhaC) (Bacillus firmus)
37.5
62.0
382


HI0098
104899
106317
periplasmic-binding-protein-dependent iron transport protein (sfuB)
38.1
59.5
457





(Serratia marcescens)


HI1479
1558763
1558167
periplasmic-binding-protein-dependent iron transport protein (sfuC)
39.9
58.0
197





(Serratia mercescens)


HI0913
964424
966276
potassium efflux system (kelC) (Escherichia coli)
40.9
65.7
594


HI0292
326934
324769
potassium/copper-transportING ATPase A (copA) (Enterococcus faecalis)
42.9
64.4
723


HI1355
1429787
1428276
sodium/proline symporter (proline permease (putP) (Escherichia coli)
62.8
79.1
489


HI0252
283326
282517
tonB protein (tonB) (Haemophilus influenzae)
96.2
98.5
261


HI0627
664922
666362
TRK system potassium uptake protein (trkA) (Escherichia coli)
85.8
83.4
458









Carbohydrates, organic alcohols & acids













HI0020
22097
20661
2-oxoglutarate/malate translocator (SOD/T1) (Spinacia cleraces)
35.8
59.6
452


HI0824
872894
873940
D-galactose-binding periplasmic protein (mglB) (Escherichia coli)
67.6
81.2
329


HI1113
1176024
1174516
D-xylose transport ATP-binding protein (xylG) (Escherichia coli)
71.5
85.8
501


HI1114
1177073
1176076
D-xylose-binding periplasmic protein (rbsB) (Escherichia coli)
76.0
88.4
328


HI1718
1785024
1783300
enzyme 1 (ptsl) (Salmonella typhimurium)
70.2
84.3
574


HI0182
194818
193967
formate transporter (formate channel) (Escherichia coli)
53.2
73.4
263


HI0450
471781
470285
fructose-permease IIA/FPR component (fruB) (Escherichia coli)
51.5
68.3
374


HI0448
469337
467670
fructose-permease IIBC component (fruA) (Escherichia coli)
57.2
72.2
552


HI0614
643282
642851
fucose operon protein (fucU) (Escherichia coli)
66.3
80.0
94


HI0692
733673
734464
glpF protein (glpF) (Escherichia coli)
73.6
87.2
258


HI1019
1080518
1081194
glpF protein (glpF) (Escherichia coli)
30.6
54.6
208


HI1017
1078404
1079867
gluconate permease (gntP) (Bacillus subtilis)
29.1
56.4
442


HI1717
1783237
1782740
glucose phosphotransferase enzyme III-glc (crr) (Escherichia coli)
73.2
83.3
169


HI0688
729474
730914
glycerol-3-phosphatase transporter (glpT) (Escherichia coli)
64.5
78.9
445


HI0504
517869
519347
high affinity ribose transport protein (rbsA) (Escherichia coli)
71.1
85.4
494


HI0505
519363
520331
high affinity ribose transport protein (rbsC) (Escherichia coli)
68.0
86.5
303


HI0503
517436
517852
high affinity ribose transport protein (rbsD) (Escherichia coli)
59.0
78.4
139


HI0612
642139
640856
L-fucose permease (fucP) (Escherichia coli)
35.6
57.9
413


HI1221
1288578
1286983
L-lactate permease (lctP) (Escherichia coli)
30.2
53.9
532


HI1735
1802527
1801757
lactam utilization protein (lamB) (Emericella ridulans)
41.3
60.3
130


HI0825
874009
875526
mglA protein (mglA) (Escherichia coli)
73.9
84.6
506


HI0826
875546
876553
mglC protein (mglC) (Escherichia coli)
79.2
90.2
336


HI0506
520354
521229
periplasmic ribose-binding protein (rbsB) (Escherichia coli)
73.9
86.6
291


HI1719
1785361
1785107
phosphohistidinoprotein-hexose phosphotransferase (ptsH) (Escherichia
77.6
88.2
85





coli)


HI0830
878480
876773
potassium channel homolog (kch) (Escherichia coli)
67.7
80.2
96


HI0154
170140
168807
putative aspartate transport protein (dcuA) (Escherichia coli)
46.4
59.9
436


HI0748
803856
805175
putative aspartate transport protein (dcuA) (Escherichia coli)
42.6
70.1
435


HI1112
1174509
1173385
ribose transport permease protein (xylH) (Escherichia coli)
69.8
84.1
371


HI1696
1759373
1760743
sodium- and chloride-dependent GABA transporter (Homo sapiens)
29.3
52.6
471


HI0738
790926
789403
sodium-dependent noradrenaline transporter (Homo sapiens)
31.1
54.2
523









Nucleosides, purines & pyrimidines













HI1089
1151815
1151024
ribonucleotide transport ATP-binding protein (mkl) (Mycrobacterium leprae)
42.2
61.5
244


HI1230
1296319
1295078
uracil permease (uraA) (Escherichia coli)
37.2
61.6
400









Anions













HI1104
1164213
1165028
cysteine synthetase (cysZ) (Escherichia coli)
53.7
76.3
190


HI1697
1761825
1760773
hydrophilic membrane-bound protein (modC) (Escherichia coli)
55.9
74.5
263


HI1698
1762501
1761815
hydrophobic membrane-bound protein (modB) (Escherichia coli)
65.9
84.8
223


HI1384
1477430
1476585
integral membrane protein (pstA) (Escherichia coli)
59.6
77.6
272


HI0358
380045
380764
nitrate transporter ATPase component (nasD) (Klebsiella pneumoniae)
34.9
57.8
254


HI1383
1475710
1476584
peripheral membrane protein B (pstB) (Escherichia coli)
77.0
86.8
256


HI1385
1478379
1477435
peripheral membrane protein C (pstC) (Escherichia coli)
57.3
78.7
300


HI1386
1479246
1478473
periplasmic phosphate-binding protein (pstS) (Escherichia coli)
49.8
67.7
256


HI1387
1479247
1479929
periplasmic phosphate-binding protein (pstS) (Escherichia coli)
63.8
75.4
89


HI1610
1669474
1670733
phosphate permease (YBR296C) (Saccharomyces cerevisiae)
35.6
60.0
551









Other













HI0060
62564
60804
ATP dependent translocator homolog (msbA) (Haemophilus influenzae)
100.0
100.0
458


HI0623
653683
662010
ATP-binding protein (abc) (Escherichia coli)
74.0
86.5
200


HI1625
1686470
1686186
cystic fibrosis transmembrane conductance regulator (Bos taurus)
35.3
60.8
233


HI0855
899042
900688
heme-binding lipoprotein (dppA) (Haemophilus influenzae)
98.9
99.3
547


HI0266
295639
298353
heme-hemopexin-binding protein (hxuA) (Haemophilus influenaze)
82.1
89.5
928


HI1476
1556199
1555189
hemin permease (hemU) (Yersinia enterocolitica)
36.1
62.7
325


HI0264
291684
293852
hemin receptor precursor (hemR) (Yersinia enterocolitica)
28.5
45.9
578


HI1712
1779487
1777481
high-affinity choline transport protein (betT) (Escherichia coli)
34.7
61.6
653


HI0663
705327
703054
lactoferrin binding protein (lbpA) (Neisseria meningitidis)
30.2
47.9
763


HI0610
637954
639336
Na+/sulfate cotransporter (Rattus norvegicus)
34.4
57.8
562


HI0977
1032420
1033871
pantothenate permease (panF) (Escherichia coli)
60.2
77.9
478


HI0714
760739
757488
transferrin binding protein 1 precursor (tbp1) (Neisseria meningitidis)
29.9
48.6
894


HI0996
1059604
1056869
transferrin binding protein 1 precursor (tbp1) (Neisseria meningitidis)
51.2
69.5
885


HI1220
1286725
1283987
transferrin binding protein 1 precursor (tbp1) (Neisseria meningtidis)
28.4
46.8
902


HI0997
1061509
1059635
transferrin binding protein 1 precursor (tbp1) (Neisseria meningtidis)
39.9
54.7
692


HI0975
1029676
1030542
transferrin-binding protein (ttbA) (Actinobacillus pleuropneumoniae)
28.9
48.0
578


HI1571
1633105
1633993
transferrin-binding protein 1 (tbp1) (Neisseria meningitidis)
41.3
59.5
727


HI0837
676956
674098
transferrin-binding protein 1 (tbp2) (Neisseria gonorrhoeae)
31.6
51.7
828


HI0665
706622
708309
transport ATP-binding protein (cydD) (Escherichia coli)
26.4
54.0
561


HI1160
1228897
1225140
transport ATP-binding protein (cydD) (Escherichia coli)
50.7
73.5
588







Cellular processes









Chaperones













HI0544
565037
565324
chaperonin (groES) (mopB) (Escherichia coli)
87.5
94.8
96


HI0545
565350
566993
heat shock protein (groEL) (mopA) (Haemophilus ducreyi)
89.8
94.9
547


HI1241
1310497
1311678
heat shock protein (dnaJ) (Escherichia coli)
68.0
82.5
376


HI0104
111572
109680
heat shock protein C62.5 (htpG) (Escherichia coli)
75.4
88.3
621


HI0375
396463
394607
hsc66 protein (hsc66) (Escherichia coli)
69.2
82.0
616


HI1240
1308539
1310443
hsp70 protein (dnaK) (Escherichia coli)
78.5
88.2
638









Cell division













HI0771
831200
831853
cell division ATP-binding protein (tts) (Escherichia coli)
64.1
78.3
216


HI1211
1275245
1274358
cell division inhibitor (sulA) (Vibrio cholerae)
33.9
55.7
116


HI1145
1210058
1211332
cell division protein (itsA) (Escherichia coli)
52.8
74.2
420


HI1338
1410017
1412129
cell division protein (itsH) (Escherichia coli)
75.2
87.8
624


HI1470
1549516
1548374
cell division protein (itsH) (Escherichia coli)
77.8
88.3
369


HI1337
1409390
1410016
cell division protein (itsJ) (Escherichia coli)
81.7
90.4
208


HI1134
1196901
1197221
cell division protein (itsL) (Escherichia coli)
36.6
60.4
101


HI1144
1209275
1210036
cell division protein (itsQ) (Escherichia coli)
40.6
58.5
231


HI1140
1204467
1205648
cell division protein (itsW) (Escherichia coli)
52.3
74.9
374


HI0770
829937
831178
cell division protein (itsY) (Escherichia coli)
66.0
61.1
497


HI1146
1211419
1212681
cell division protein (itsZ) (Escherichia coli)
67.2
83.1
306


HI1377
1465224
1469760
cell division protein (mukB) (Escherichia coli)
61.4
77.3
1455


HI1356
1429903
1431375
cytoplasmic axial filament protein (cefA) (Escherichia coli)
71.0
66.3
488


HI0772
831866
832795
ItsX protein (ltsX) (Escherichia coli)
43.5
69.9
292


HI1067
1128511
1129221
mukB suppressor protein (smba) (Escherichia coli)
77.4
90.2
235


HI1135
1197237
1199067
penicillin-binding protein 3 (ftsI) (Escherichia coli)
52.8
70.7
564









Protein, peptide secretion













HI0016
17278
15485
GTP-binding membrane protein (lepA) (Escherichia coli)
85.6
91.0
597


HI1472
1551915
1553681
colicin V secretion ATP-binding protein (cvaB) (Escherichia coli)
29.9
56.0
373


HI1008
1070885
1071397
lipoprotein signal peptidase (lspA) (Escherichia coli)
51.3
71.5
158


HI1648
1706947
1707753
peptide transport system ATP-binding protein SAPF (sapF) (Escherichia coli)
49.6
70.8
264


HI0718
764525
764842
preprotein translocase (secE) (Escherichia coli)
40.6
62.3
106


HI0600
848438
849760
preprotein translocase SECY subunit (secY) (Escherichia coli)
74.7
86.9
443


HI0241
269734
267887
protein-export membrane protein (secD) (Escherichia coli)
59.6
77.3
615


HI0240
267876
266902
protein-export membrane protein (secF) (Escherichia coli)
48.0
73.0
302


HI0447
466800
467135
protein-export membrane protein (secG) (Escherichia coli)
58.9
81.3
110


HI0745
801965
801459
protein-export protein (secB) (Escherichia coli)
56.2
80.8
145


HI0911
961135
963837
secA protein (secA) (Escherichia coli)
88.0
81.7
896


HI0015
15473
14427
signal peptidase I (lepB) (Escherichia coli)
46.3
65.1
319


HI0106
114073
112688
signal recognition particle protein (54 homolog) (lfh) (Escherichia coli)
79.9
90.9
452


HI0715
761040
762335
trigger factor (tig) (Escherichia coli)
64.4
80.3
432


HI0298
330445
329758
type 4 prepilin-like protein specific leader peptidase (hopD) (Escherichia
27.2
49.0
208





coli)


HI0299
331661
330445
xcpS protein (xcpS) (Pseudomonas putida)
29.2
56.7
398









Detoxification













HI0930
985290
966813
KW20 catalase (hktE) (Haemophilus influenzae)
99.2
99.4
508


HI1090
1152892
1152248
superoxide dismutase (sodA) (Haemophilus influenzae)
99.0
99.5
209


HI1004
1065726
1067108
thiophene and furan oxidation protein (thdF) (Escherichia coli)
73.8
85.4
451









Cell killing













HI0303
334801
335697
hemolysin (tlyC) (Serpulina hyodysenteriae)
36.9
57.5
252


HI1664
1723070
1723648
hemolysin, 21 kDa (hly) (Actinobacillus pleuropneumoniae)
54.5
72.4
156


HI1376
1464493
1465221
killing protein (kicA) (Escherichia coli)
69.0
83.6
222


HI1375
1463019
1464443
killing protein suppressor (kicB) (Escherichia coli)
66.9
83.0
440


HI1053
1116898
1115057
leukotoxin secretion ATP-binding protein (lktB) (Actinobacillus
34.2
55.1
512





actinomycetemcomitans)









Transformation













HI0436
456360
455674
com101A protein (comF) (Haemophilus influenzae)
100.0
100.0
229


HI1010
1072519
1072854
competence locus E (comE1) (Bacillus subtilis)
46.7
70.0
59


HI0603
622277
622927
tfoX protein (tfoX) (Haemophilus influenzae)
99.5
99.5
217


HI0443
462729
463571
transformation gene cluster hypothetical protein (GB:M62809_1) (com)
100.0
100.0
281





(Haemophilus influenzae)


HI0435
455595
455002
transformation gene cluster hypothetical protein (GB:M62809_10) (com)
99.5
99.5
198





(Haemophilus influenzae)


HI0442
460047
462638
transformation gene cluster hypothetical protein (GB:M62809_2) (com)
100.0
100.0
864





(Haemophilus influenzae)


HI0441
459948
459154
transformation gene cluster hypothetical protein (GB:M62809_3) (com)
100.0
100.0
265





(Haemophilus influenzae)


HI0440
459150
458647
transformation gene cluster hypothetical protein (GB:M62809_4) (com)
100.0
100.0
168





(Haemophilus influenzae)


HI0439
458647
458129
transformation gene cluster hypothetical protein (GB:M62809_5) (com)
100.0
100.0
173





(Haemophilus influenzae)


HI0438
458129
457719
transformation gene cluster hypothetical protein (GB:M62809_6) (com)
100.0
100.0
137





(Haemophilus influenzae)


HI0437
457706
456385
transformation gene cluster hypothetical protein (GB:M62809_7) (com)
99.8
99.8
441





(Haemophilus influenzae)







Other categories









Colicin-related functions













HI0384
403297
402017
colicin tolerance protein (tolB) (Escherichia coli)
63.9
78.1
409


HI1209
1272281
1272769
colicin V production protein (pur regulon) (cvpA) (Escherichia coli)
64.7
79.5
156


HI0387
405650
404967
inner membrane protein (tolQ) (Escherichia coli)
68.8
83.3
221


HI0386
404892
404476
inner membrane protein (tolR) (Escherichia coli)
61.8
78.7
136


HI0385
404457
403342
outer membrane integrity protein (tolA) (Escherichia coli)
42.6
57.1
406


HI1691
1753623
1756079
outer membrane integrity protein (tolA) (Escherichia coli)
28.9
47.7
345









Phage-related functions and prophages













HI1493
1566955
1567509
E16 protein (muE16) (Bacteriophage mu)
28.5
52.8
143


HI1508
1576485
1576922
G protein (muG) (Bacteriophage mu)
38.3
52.5
147


HI1574
1636594
1636181
G protein (muG) (Bacteriophase mu)
33.3
54.0
138


HI1488
1564685
1565191
gam protein (Bacteriophage mu)
57.1
73.8
168


HI0071
78159
78860
heat shock protein B253 (grpE) (Escherichia coli)
45.9
66.5
193


HI0413
432108
431836
host factor-1 (HF-1) (hfq) (Escherichia coli)
90.5
97.3
74


HI1509
1577156
1578220
I protein (mul) (Bacteriophase mu)
50.0
55.4
58


HI1485
1563429
1564289
MuB protein (muB) (Bacteriophase mu)
46.4
70.4
277


HI1521
1584995
1586365
N protein (muN) (Bacteriophage mu)
31.5
52.1
452


HI1522
1586368
1587105
P protein (Bacteriophage mu)
39.5
67.3
220


HI1416
1505940
1505428
terminase subunit 1 (Bacteriophase SF6)
32.3
52.3
128


HI1483
1560600
1562660
transposase A (muA) (Bacteriophage mu)
40.6
60.1
596









Transposon-related functions













HI1106
1166078
1166803
insertion sequence IS1016 (V-4) hypothetical protein (GB:X58176_2)
43.6
66.7
139





(Haemophilus influenzae)


HI1020
1081916
1081346
IS1016-V6 protein (IS1016-V6) (Haemophilus influenzae)
91.7
93.8
191


HI1332
1406795
1406150
IS1016-V6 protein (IS1016-V6) (Haemophilus influenzae)
54.7
74.7
170


HI1583
1645515
1645991
IS1016-V6 protein (IS1016-V6) (Haemophilus influenzae)
45.4
61.2
153









Drug/analog sensitivity













HI0897
947919
951014
acriflavine resistance protein (acrB) (Escherichia coli)
32.7
55.0
1027


HI0302
333614
334165
ampD signalling protein (ampD) (Escherichia coli)
56.1
75.1
172


HI1245
1315822
1314629
bicyclomycin resistance protein (bcr) (Escherichia coli)
42.6
68.7
383


HI1629
1688581
1689111
mercury resistance regulatory protein (merR2) (Thiobacillus ferroxidans)
37.7
57.5
105


HI0650
692523
691900
modulator of drug activity (mda66) (Escherichia coli)
58.1
75.4
191


HI0899
953570
951041
multidrug resistance protein (emrB) (Escherichia coli)
67.7
84.8
499


HI0900
954752
953583
multidrug resistance protein (ermA) (Escherichia coli)
46.5
66.3
389


HI0036
37441
39472
multidrug resistance protein (mdl) (Escherichia coli)
29.0
51.2
1094


HI1467
1543471
1544832
modulation protein T (nodT) (Rhizobium leguminosarum)
20.1
46.3
390


HI0551
569189
570049
rRNA (adenosine-N6,N6-)-dimethyltransferase (ksgA) (Escherichia coli)
69.3
81.5
269


HI0513
527345
526362
tellurite resistance protein (tehA) (Escherichia coli)
36.9
62.0
317


HI1278
1351140
1350283
tellurite resistance protein (tehB) (Escherichia coli)
55.2
70.6
194









Radiation sensitivity













HI0954
1011412
1010711
radC protein (radC) (Escherichia coli)
49.8
71.7
219









Adaptations, atypical conditions













HI1532
1596570
1595143
autotrophic growth protein (aut) (Atcaligenea sutrophus)
45.0
60.9
154


HI0722
766921
767769
heat shock protein (htpX) (Escherichia coli)
66.3
82.1
288


HI1533
1596655
1597599
heat shock protein B (ibpB) (Escherichia coli)
55.9
71.2
304


HI0947
1003887
1004906
htrA-like protein (htrH) (Escherichia coli)
55.2
72.6
282


HI0903
956705
957292
invasion protein (invA) (Bartonella bacilliformis)
39.5
60.5
187


HI1550
1615090
1814485
NAD(P)H:menadione oxidoreductase (Mus musculus)
35.9
54.9
200


HI0460
479443
478505
survival protein (surA) (Escherichia coli)
33.0
58.5
424


HI0817
866160
865738
uspA protein (uspA) (Escherichia coli)
68.6
87.1
140


HI0323
350541
350774
virulence plasmid protein (vagC) (Salmonella dublin)
35.9
57.8
62


HI1254
1326770
1327090
virulence associated protein A (vapA) (Dichelobacter nodosus)
40.8
57.7
71


HI0324
350774
351175
virulence associated protein C (vapC) (Dichelobacter nodosus)
35.4
56.9
128


HI0949
1007984
1007589
virulence associated protein C (vapC) (Dichelobacter nodosus)
40.9
60.6
131


HI0452
472751
472479
virulence associated protein D (vapD) (Dichelobacter nodosus)
40.7
87.0
91


HI1310
1385051
1385680
virulence plasmid protein (mfgA) (Shewanella colwelliana)
23.8
56.3
124









Undetermined













HI1164
1230321
1229908
f5 kDa protein (P15) (Escherichia coli)
49.3
68.4
136


HI0085
89585
88593
2-hydroxyaciddehydrogenase homolog (ddh) (Zymomonas mobilis)
51.5
72.8
324


HI0462
480185
480973
beta-lactamase regulatory homolog (mazG) (Escherichia coli)
48.3
72.6
257


HI1676
1738223
1737753
conjugative transfer co-repressor (finO) (Escherichia coli)
32.5
51.9
76


HI0309
340039
340851
delta-1-pyrroline-5-carboxylate reductase (proC) (Pseudomonas aeruginosa)
44.0
60.1
267


HI1555
1620490
1619810
devA protein (devA) (Anabaena sp.)
42.7
66.4
219


HI0558
576002
575514
devB protein (devB) (Anabaena sp.)
32.7
51.5
166


HI1342
1415087
1415473
embryonic abundant protein, group 3 (Triticum aestivum)
33.3
50.0
102


HI0939
996457
995658
extragenic suppressor (suhB) (Escherichia coli)
64.7
80.2
258


HI0370
390980
392083
GCPE protein (protein E) (gpcE) (Escherichia coli)
88.2
93.9
362


HI0095
102616
101864
GerC2 protein (gerC2) (Bacillus subtilis)
32.9
55.2
191


HI0669
712892
711894
glpX protein (glpX) (Escherichia coli)
69.2
83.4
325


HI1015
1076616
1077389
glyoxylate-induced protein (Escherichia coli)
39.1
57.8
258


HI0499
511702
513099
hslU protein (hslU) (Escherichia coli)
80.4
90.1
443


HI0498
511230
511754
hslV protein (hslV) (Escherichia coli)
79.8
89.0
172


HI1120
1184041
1182516
ilv-related protein (Escherichia coli)
59.7
77.0
504


HI0287
319073
317784
isochorismate synthase (entC) (Bacillus subtilis)
31.5
48.9
311


HI1624
1686217
1685567
membrane associated ATPase (cbiO) (Propionibacterium freudenreichii)
33.7
52.7
184


HI0463
481901
481029
membrane protein (lapB) (Pasteurella haemolytica)
34.2
56.0
221


HI1122
1184867
1185742
membrane protein (lapB) (Pasteurella haemolytica)
63.1
80.2
216


HI0590
608642
609874
N-carbamyl-L-amino acid amidohydrolase (Bacillus stearothermphilus)
35.9
59.2
406


HI0380
399796
398579
nitrogen fixation protein (nifS) (Anabaena sp.)
48.2
67.0
379


HI1298
1375045
1373735
nitrogen fixation protein (nifS) (Mycrobacterium leprae)
33.4
56.2
402


HI1346
1418238
1417523
nitrogen fixation protein (nifS) (Mycrobacterium leprae)
38.6
58.5
186


HI0379
398591
398139
nitrogen fixation protein (nifU) (Klebsiella pneumoniae)
50.8
74.2
122


HI0167
180354
181586
nitrogen fixation protein (mfE) (Rhodobacter capsulatus)
30.1
47.9
292


HI1692
1756087
1757180
nitrogen fixation protein (mfE) (Rhodobacter capsulatus)
32.7
59.5
290


HI0129
143015
144800
nitrogenase C (nifC) (Clostridium pasteurianum)
27.1
52.6
248


HI1480
1559124
1558768
nitrogenase C (nifC) (Clostridium pasteurianum)
40.9
60.2
92


HI0359
381523
382464
nmt1 protein (nmt1) (Aspergillus parasiticus)
25.6
54.7
289


HI1299
1375415
1374682
partitioning system protein (parB) (Plasmid RP4)
43.6
67.7
141


HI0224
252941
252168
rarD protein (rarD) (Escherichia coli)
26.5
53.0
230


HI0682
721733
720840
rarD protein (rarD) (Escherichia coli)
27.1
55.0
289


HI0918
970839
970249
skp protein (skp) (Pasteurella multocide)
55.5
76.4
191


HI0983
1038375
1037893
small protein (smpB) (Escherichia coli)
78.8
91.3
160


HI1598
1661468
1659882
spoIIIE protein (spoIIIE) (Coxiella burnettii)
56.1
74.5
504


HI0898
951407
952018
suppressor protein (msgA) (Escherichia coli)
30.2
56.1
254


HI1080
1145382
1144612
surfactin (slpo) (Bacillus subtilis)
58.2
77.9
246


HI0753
811790
811296
toxR regulon (tagD) (Vibrio cholerae)
45.7
64.0
164


HI1412
1502860
1501311
traN protein (traN) (Plasmid RP4)
40.2
61.5
233


HI0666
708305
709960
transport ATP-binding protein (cydC) (Escherichia coli)
26.3
51.7
536


HI1159
1225137
1223410
transport ATP-binding protein (cydC) (Escherichia coli)
48.5
70.1
568


HI1562
1627239
1626295
vanH protein (vanH) (Transposon Tn1546)
39.7
57.1
251


HI0632
658489
669433
mucoid status locus protein (mucB) (Pseudomonas aeruginosa)
25.4
51.8
309


HI0172
183553
184785
phenolhydroxylase (ORF6) (Acinetobacter calcoaceticus)
33.0
58.9
313


HI1390
1481177
1481266
plasma protease C1 inhibitor (Homo sapiens)
75.0
79.2
23

















TABLE 1(b)







HI0060
ATP dependent translocator homolog (msbA)


HI0140
outer membrane protein P2 (ompP2)


HI0251
single-stranded DNA binding protein (ssb)


HI0252
tonB protein (tonB)


HI0266
heme-hemopexin-binding protein (hxuA)


HI0351
adenylate kinase (ATP-AMP transphosphorylase) (adk)


HI0352
hypothetical protein (SP:P24326)


HI0353
udp-glucose 4-epimerase (galactowaldenase) (galE)


HI0354
hypothetical protein (SP:P24324)


HI0383
PC protein (15kd peptidoglycan-associated outer membrane



lipoprotein) (pal)


HI0403
outer membrane protein P1 (ompP1)


HI0435
transformation gene cluster hypothetical protein



(GB:M62809_10) (com)


HI0436
com101A protein (comF)


HI0437
transformation gene cluster hypothetical protein



(GB:M62809_7) (com)


HI0438
transformation gene cluster hypothetical protein



(GB:M62809_6) (com)


HI0439
transformation gene cluster hypothetical protein



(GB:M62809_5) (com)


HI0440
transformation gene cluster hypothetical protein



(GB:M62809_4) (com)


HI0441
transformation gene cluster hypothetical protein



(GB:M62809_3) (com)


HI0442
transformation gene cluster hypothetical protein



(GB:M62809_2) (com)


HI0443
transformation gene cluster hypothetical protein



(GB:M62809_1) (com)


HI0514
HincII endonuclease (HincII)


HI0515
modification methylase HincII (hincIIM)


HI0552
lipooligosaccharide biosynthesis protein


HI0583
streptomycin resistance protein (strA)


HI0602
recombinase (recA)


HI0603
tfoX protein (tfoX)


HI0606
adenylate cyclase (cyaA)


HI0622
28 kDa membrane protein (hlpA)


HI0691
protein D (hpd)


HI0695
lipoprotein (hel)


HI0820
aldose 1-epimerase precursor (mutarotase) (mro)


HI0821
galactokinase (galK)


HI0822
galactose-1-phosphate uridylyitransferase (galT)


HI0823
galactose operon repressor (galS)


HI0847
hypothetical protein (GB:M94205_1)


HI0848
disulfide oxidoreductase (por)


HI0855
heme-binding lipoprotein (dppA)


HI0919
protective surface antigen D15


HI0930
KW20 catalase (hktE)


HI0959
cyclic AMP receptor protein (crp)


HI1090
superoxide dismutase (sodA)


HI1167
outer membrane protein P5 (ompA)


HI1191
DNA helicase II (uvrD)


HI1397
HindIII modification methyltransferase (hindIIIM)


HI1398
HindIII restriction endonuclease (hindIIIR)


HI1402
DNA polymerase III, chi subunit (holC)


HI1545
lic-1 operon protein (licC)


HI1546
lic-1 operon protein (licD)


HI1585
15 kd peptidoglycan-associated lipoprotein (lpp)


HI1594
formyltetrahydrofolate hydrolase (purU)


HI1595
enolpyruvylshikimatephosphatesynthase (aroA)


HI1699
Isg locus hypothetical protein (GB:M94855_8)


HI1700
Isg locus hypothetical protein (GB:M94855_7)


HI1701
Isg locus hypothetical protein (GB:M94855_6)


HI1702
Isg locus hypothetical protein (GB:M94855_5)


HI1703
Isg locus hypothetical protein (GB:M94855_4)


HI1704
Isg locus hypothetical protein (GB:M94855_3)


HI1705
Isg locus hypothetical protein (GB:M94855_2)


HI1706
Isg locus hypothetical protein (G8:M94855_1)
















TABLE 2





UNKNOWNS



















HI0003
3249
2464



HI0004
3729
3268



HI0012
11778
12767



HI0017
17829
17449



HI0019
20239
18819



HI0021
23349
22102



HI0028
29582
29307



HI0033
35298
34834



HI0034
35660
35355



HI0035
37440
35788



HI0040
43059
42286



HI0042
44594
43923



HI0043
45658
44597



HI0044
46380
45721



HI0045
47261
46710



HI0046
47328
47687



HI0050
51426
50224



HI0051
51998
51504



HI0052
53023
52040



HI0053
54078
53053



HI0056
56966
56256



HI0059
60728
59733



HI0065
67839
68312



HI0072
78167
77313



HI0073
79220
78879



HI0074
79653
79216



HI0077
83046
83909



HI0080
85983
86411



HI0081
86556
87341



HI0082
87601
87864



HI0083
87882
88094



HI0090
96604
97314



HI0091
98493
97360



HI0092
99761
98505



HI0093
100989
99886



HI0094
101511
101194



HI0096
102950
103522



HI0100
107807
107415



HI0101
108091
107654



HI0103
109598
109257



HI0105
111789
112625



HI0107
114405
115612



HI0108
115744
116634



HI0109
117067
116729



HI0112
119485
119847



HI0114
122424
122311



HI0115
128606
130242



HI0116
130860
130246



HI0117
131552
131800



HI0120
134883
134380



HI0121
136357
134999



HI0125
140096
141409



HI0126
142556
141573



HI0127
142955
143011



HI0128
142718
142584



Hl0130
145160
144804



HI0131
145840
145136



HI0134
147247
148419



HI0135
148422
149609



HI0136
151208
149695



HI0144
159021
158125



HI0146
160156
159932



HI0147
160966
161952



HI0148
161966
163864



HI0149
164031
165167



HI0150
165574
165762



HI0153
168744
168040



HI0160
174988
174467



HI0163
178311
177715



HI0165
179007
180080



HI0166
180130
180348



HI0168
181582
182313



HI0169
182316
182567



HI0170
182570
182938



HI0171
182945
183537



HI0173
184932
185969



HI0174
185975
186232



HI0175
186247
187500



HI0176
188281
187550



HI0177
189257
188286



HI0178
189365
190150



HI0179
190715
190236



HI0183
195295
196233



HI0184
196413
197855



HI0185
198872
198048



HI0188
200705
201555



HI0189
201568
202335



HI0196
208646
208611



HI0199
213460
214224



HI0204
218138
217605



HI0206
218715
219485



HI0211
225095
225199



HI0218
234170
234697



HI0220
238722
238084



HI0228
256953
256489



HI0229
257403
257032



HI0231
259913
260854



HI0233
262997
264382



HI0234
264390
264539



HI0235
264822
264679



HI0236
265239
265033



HI0238
265736
266389



HI0239
266350
266781



HI0243
270426
270208



HI0244
270941
270426



HI0247
274159
273716



HI0257
285979
286623



HI0258
286796
286879



HI0259
286880
288054



HI0260
288240
288058



HI0261
288839
288180



HI0262
289503
288919



HI0267
298808
298450



HI0268
298891
299487



HI0272
304213
303284



HI0273
305079
304216



HI0277
309032
310684



HI0278
311516
310710



HI0279
311998
311516



HI0280
312417
312004



HI0281
312664
312371



HI0283
315199
313886



HI0284
315200
316061



HI0286
318836
319252



HI0293
327115
326912



HI0295
327473
327858



HI0301
333498
333052



HI0305
337302
338036



HI0306
338036
338593



HI0307
338596
339012



HI0308
339973
339068



HI0310
340854
342017



HI0312
343117
343401



HI0313
343271
343092



HI0317
346507
345770



HI0318
347143
346670



HI0320
349150
349665



HI0321
349721
350002



HI0322
349998
350444



HI0325
351245
351649



HI0327
352729
354078



HI0328
354114
354374



HI0329
354653
354697



HI0331
355855
356668



HI0335
359242
360555



HI0338
363320
363910



HI0340
364253
365296



HI0342
367615
368352



HI0343
368440
368781



HI0344
368990
369516



HI0345
389512
369790



HI0346
369815
372311



HI0347
372369
373205



HI0348
373208
374068



HI0349
374068
374517



HI0352
377303
376029



HI0354
379329
378637



HI0355
379330
380044



HI0357
380765
381167



HI0358
381227
381171



HI0361
384039
383227



HI0365
386932
387009



HI0366
387928
387053



HI0367
388154
389323



HI0368
389428
389964



HI0369
390039
390947



HI0372
393364
393975



HI0373
394223
394032



HI0376
397168
396485



HI0377
397743
397222



HI0378
398079
397759



HI0381
400309
399860



HI0382
401087
400365



HI0388
406077
405670



HI0390
408337
409044



HI0391
409072
409620



HI0393
413144
412599



HI0394
414371
413637



HI0395
415645
414557



HI0397
416445
416750



HI0398
416756
417967



HI0400
419458
420118



HI0402
421340
421056



HI0406
425499
424210



HI0407
426365
425502



HI0414
433167
432202



HI0417
437163
437957



HI0418
437953
438759



HI0419
438773
439450



HI0420
439398
440738



HI0422
442434
442730



HI0423
443077
442916



HI0425
444797
445516



HI0426
446607
445555



HI0433
454103
453516



HI0434
454932
454142



HI0444
463691
464053



HI0451
472389
471856



HI0453
472951
472763



HI0454
474321
473026



HI0455
474896
474375



HI0456
475705
474926



HI0458
477453
476743



HI0466
485905
486561



HI0468
486712
487873



HI0469
489585
488725



HI0471
491037
492317



HI0478
497647
497796



HI0489
507333
506959



HI0490
507449
508048



HI0491
508051
508521



HI0492
508274
508038



HI0493
508854
509354



HI0494
509815
509856



HI0495
509856
510253



HI0496
510797
510306



HI0497
511011
510814



HI0502
516228
517265



HI0509
523382
523930



HI0510
524561
524076



HI0511
525540
524616



HI0512
525587
526303



HI0521
542216
540966



HI0522
543103
542318



HI0523
544656
543115



HI0524
544869
545522



HI0525
546551
545484



HI0528
549859
549044



HI0554
571956
572576



HI0556
575147
574608



HI0557
575547
575211



HI0559
576210
576091



HI0562
578540
580381



HI0563
581038
580382



HI0564
581352
581744



HI0567
584110
583439



HI0570
587757
587551



HI0572
591096
590482



HI0574
592124
592846



HI0576
593256
593978



HI0577
594070
594732



HI0578
594735
595112



HI0579
595480
595764



HI0587
607340
606504



HI0588
607795
607361



HI0591
610092
610508



HI0594
614632
614441



HI0595
616566
616775



HI0596
616702
615176



HI0599
619155
619970



HI0600
620322
619999



HI0619
650498
651154



HI0626
663569
664921



HI0628
666387
666770



HI0629
666863
667117



HI0635
672600
672893



HI0636
672899
673879



HI0638
677932
677645



HI0640
679087
679701



HI0649
691619
690908



HI0652
694996
694787



HI0655
696806
697567



HI0658
699494
698946



HI0660
701972
700059



HI0661
702429
702136



HI0662
702781
702425



HI0664
706058
705867



HI0667
711078
710050



HI0668
711395
711078



HI0670
713054
713269



HI0672
713806
714236



HI0673
715017
714544



HI0674
715691
714544



HI0675
715969
715694



HI0679
719498
719061



HI0689
731017
731928



HI0690
732026
732334



HI0696
737789
738508



HI0698
743511
739619



HI0699
744964
743524



HI0700
745259
744239



HI0702
746523
746065



HI0703
746632
747648



HI0704
747649
748418



HI0706
749006
749188



HI0708
749180
749148



HI0720
765555
766304



HI0721
766361
766750



HI0723
768095
767817



HI0725
768792
770060



HI0726
776311
776858



HI0727
776875
777312



HI0732
786122
783778



HI0733
788625
786245



HI0734
788731
786582



HI0735
787647
788715



HI0737
788457
789167



HI0742
799454
800908



HI0743
801060
801388



HI0744
801027
800965



HI0746
802425
801982



HI0755
816503
817648



HI0757
819456
818531



HI0758
820676
819447



HI0762
823117
823386



HI0763
823404
824474



HI0764
825768
825091



HI0768
829290
828811



HI0769
829882
829304



HI0774
835432
834092



HI0775
836100
835432



HI0777
836970
837914



HI0789
843493
844095



HI0808
854572
855375



HI0809
858603
855413



HI0812
860092
859214



HI0819
868114
867569



HI0827
876702
877433



HI0828
877442
877996



HI0829
877999
878480



HI0833
881059
881640



HI0839
887221
886541



HI0840
887844
887276



HI0841
888779
887757



HI0842
888896
889111



HI0843
869116
890870



HI0844
891071
891898



HI0845
891925
892059



HI0847
892866
893129



HI0849
893822
894164



HI0851
895374
896144



Hl0852
896141
896572



HI0853
896977
897510



HI0854
897510
898898



HI0856
900867
901625



HI0857
902112
901768



HI0859
905068
905357



HI0860
905688
906248



HI0862
909726
908989



HI0863
912130
909785



HI0864
913029
912325



HI0866
915792
913945



HI0868
918419
918538



HI0871
920692
921246



HI0872
921338
921439



HI0873
922696
923613



HI0878
927351
926155



HI0880
931427
930509



HI0883
932310
933296



HI0884
933350
934084



HI0888
938667
939068



HI0892
943690
944319



HI0893
944315
944518



HI0904
957295
958086



HI0905
957488
957174



HI0908
959765
960283



HI0909
960628
960317



HI0910
960708
961007



HI0914
966380
967141



HI0920
974685
973357



HI0922
976298
975582



HI0927
983767
983405



HI0928
984057
983800



HI0931
988229
987051



HI0932
988850
988233



HI0933
989308
988826



HI0935
991961
990760



HI0936
993112
991961



HI0937
993639
993112



HI0938
995546
993642



HI0940
996553
997110



HI0941
997170
997883



HI0942
997886
998566



HI0943
998544
998846



HI0945
1002315
1002762



HI0950
1008217
1007987



HI0957
1013246
1013899



HI0958
1013924
1014091



HI0960
1016378
1015203



HI0961
1017426
1016374



HI0962
1017780
1017433



HI0963
1018172
1017783



HI0965
1022039
1021104



HI0966
1023606
1022077



HI0967
1023993
1024175



HI0968
1024843
1024944



HI0969
1024817
1024254



HI0976
1030609
1031712



HI0978
1033994
1034863



HI0979
1034868
1035440



HI0981
1036523
1037512



HI0986
1041067
1040252



HI0988
1042709
1044301



HI0990
1045642
1047047



HI0998
1061607
1062044



HI0999
1062363
1063049



HI1002
1063710
1063967



HI1003
1063970
1065592



HI1005
1067299
1067478



HI1006
1067384
1069165



HI1007
1069256
1070812



HI1009
1071385
1072338



HI1012
1073835
1074737



HI1013
1074743
1075981



HI1016
1077448
1078392



HI1018
1079890
1080315



HI1021
1082175
1083170



HI1022
1083178
1084791



HI1023
1084736
1085422



HI1026
1089466
1088792



HI1028
1091065
1090208



HI1029
1091066
1092597



HI1030
1093581
1092598



HI1031
1094889
1093615



HI1032
1095371
1094889



HI1033
1096441
1095446



HI1034
1096617
1097420



HI1036
1098535
1099023



HI1038
1100259
1100810



HI1039
1101878
1100997



HI1040
1102257
1103456



HI1041
1103535
1103386



HI1045
1108332
1107835



HI1046
1108943
1108335



HI1050
1113198
1114304



HI1055
1117984
1118322



HI1056
1119807
1118428



HI1057
1121239
1119698



HI1058
1123210
1123287



HI1060
1123449
1122868



HI1065
1127036
1126827



HI1066
1128454
1127000



HI1072
1135049
1133604



HI1073
1135234
1134995



HI1074
1137513
1135267



HI1075
1137884
1137513



HI1076
1138337
1137888



HI1084
1148702
1148448



HI1085
1149040
1148726



HI1086
1149695
1149054



HI1087
1150228
1149728



HI1088
1151024
1150242



HI1091
1153141
1153776



HI1092
1153784
1154446



HI1093
1154507
1155244



HI1094
1155289
1155489



HI1095
1155489
1156007



HI1096
1156007
1157950



HI1097
1158092
1158634



HI1098
1158637
1160013



HI1099
1160451
1160492



HI1100
1160501
1160632



HI1101
1160637
1160942



HI1103
1164060
1163077



HI1107
1166804
1168024



HI1121
1184774
1184115



HI1128
1191829
1192577



HI1129
1193461
1193234



HI1131
1195069
1195242



HI1132
1195447
1195899



HI1133
1195933
1196895



HI1149
1215838
1214972



HI1150
1216338
1215847



HI1151
1217066
1218344



HI1152
1217588
1217073



HI1153
1218198
1217572



HI1154
1218770
1218237



HI1156
1220425
1220961



HI1158
1223159
1222695



HI1165
1231243
1230773



HI1168
1235872
1236231



HI1171
1238778
1239119



HI1172
1239729
1239166



HI1176
1242916
1243383



HI1178
1244125
1244051



HI1179
1244360
1244142



HI1184
1248098
1247517



HI1185
1248305
1248859



HI1186
1248934
1249107



HI1193
1256974
1256552



HI1194
1257654
1257067



HI1195
1257810
1257950



HI1198
1260250
1261479



HI1201
1263689
1264309



HI1202
1264360
1255430



HI1205
1267550
1268050



HI1206
1270263
1268131



HI1208
1271751
1272191



HI1218
1282515
1283219



HI1219
1283219
1283904



HI1225
1291759
1292049



HI1226
1292052
1293239



HI1237
1306218
1306673



HI1238
1307299
1306835



HI1239
1308273
1307173



HI1243
1313696
1313037



HI1244
1313794
1314591



HI1246
1316522
1315827



HI1247
1317233
1316616



HI1249
1319911
1321851



HI1251
1325506
1324541



HI1252
1326129
1325512



HI1253
1326454
1326756



HI1255
1327256
1328923



HI1256
1328946
1329326



HI1257
1329334
1330392



HI1258
1330618
1330839



HI1259
1330839
1331300



HI1260
1331300
1331470



HI1265
1339879
1339148



HI1268
1346269
1345733



HI1269
1346756
1346836



HI1270
1346624
1348241



HI1271
1346849
1347025



HI1272
1347022
1347135



HI1273
1347135
1347323



HI1276
1348650
1349453



HI1283
1356439
1356654



HI1284
1356655
1357185



HI1285
1358080
1358502



HI1289
1367227
1365851



HI1291
1369064
1369447



HI1292
1369450
1370385



HI1294
1372453
1371817



HI1295
1373365
1372583



HI1296
1373601
1373359



HI1297
1373735
1373532



HI1300
1375530
1375949



HI1301
1375971
1376663



HI1303
1378236
1380176



HI1304
1380896
1380210



HI1309
1384563
1385051



HI1312
1386755
1386510



HI1313
1386780
1387538



HI1317
1391445
1391927



HI1318
1392096
1392410



HI1319
1392802
1393383



HI1320
1393468
1394280



HI1326
1401970
1401527



HI1329
1404808
1405533



HI1330
1405533
1405667



HI1335
1409063
1408968



HI1338
1409263
1408968



HI1340
1412995
1414329



HI1341
1414391
1414882



HI1343
1416879
1415557



HI1344
1417617
1417009



HI1345
1418133
1419509



HI1352
1426116
1425637



HI1154
1428276
1427314



HI1358
1433535
1433996



HI1367
1450229
1449366



HI1369
1453591
1453010



HI1371
1458706
1455929



HI1372
1461329
1458813



HI1378
1469827
1470732



HI1379
1470738
1471610



HI1391
1481365
1481808



HI1394
1484556
1485554



HI1399
1492391
1492023



HI1400
1493035
1492616



HI1401
1493171
1493004



HI1404
1495447
1496052



HI1405
1496978
1496157



HI1407
1498433
1498230



HI1408
1499014
1498469



HI1409
1499166
1499050



HI1410
1500612
1499515



HI1411
1501029
1500676



HI1413
1503610
1504028



HI1414
1504094
1502787



HI1415
1505280
1504099



HI1417
1506471
1505953



HI1418
1506880
1506602



HI1419
1507067
1506795



HI1421
1507987
1507634



HI1422
1508392
1508327



HI1423
1509030
1508428



HI1424
1509352
1509648



HI1425
1509648
1509938



HI1426
1510250
1509975



HI1427
1510403
1510975



HI1428
1511264
1511545



HI1431
1513776
1514795



HI1432
1514998
1515831



HI1439
1521750
1522223



HI1440
1522224
1525568



HI1441
1525569
1525820



HI1443
1526752
1528626



HI1450
1533358
1533038



HI1454
1536172
1536492



HI1455
1536633
1536668



HI1456
1537150
1536566



HI1458
1538541
1537903



HI1460
1540315
1539812



HI1462
1541101
1541340



HI1468
1547394
1546060



HI1474
1554422
1554078



HI1477
1557241
1556189



HI1481
1560071
1559355



HI1482
1560378
1560563



HI1484
1562720
1562989



HI1486
1563395
1562928



HI1487
1564353
1564667



HI1489
1565191
1565349



HI1490
1565824
1566042



HI1491
1566045
1566215



HI1492
1566221
1566778



HI1494
1567509
1568060



HI1495
1566255
1568467



HI1497
1568697
1569200



HI1498
1569285
1569566



HI1500
1569836
1570093



HI1501
1570093
1570344



HI1502
1570465
1570689



HI1503
1570599
1571015



HI1504
1571343
1571909



HI1505
1571912
1573435



HI1506
1573450
1575009



HI1507
1575103
1578344



HI1510
1578223
1579146



HI1511
1579232
1579486



HI1512
1579501
1579614



HI1513
1579620
1580042



HI1514
1580012
1580593



HI1515
1580609
1580797



HI1516
1580800
1582260



HI1517
1582273
1582626



HI1518
1582642
1583022



HI1519
1583106
1584998



HI1520
1584526
1584371



HI1523
1587316
1587624



HI1524
1587664
1588209



HI1525
1588221
1588625



HI1526
1588626
1589692



HI1527
1589781
1590284



HI1528
1590287
1592155



HI1529
1592772
1593659



HI1530
1593826
1593975



HI1540
1605903
1606442



HI1541
1606426
1607595



HI1542
1607566
1607912



HI1548
1613326
1613877



HI1549
1614482
1613931



HI1551
1616455
1615214



HIl552
1616740
1617159



HI1554
1619807
1618560



HI1558
1622639
1621995



HI1561
1626292
1625114



HI1564
1628971
1628171



HI1566
1630319
1629852



HI1568
1631692
1631537



HI1569
1632481
1631948



HI1570
1632603
1632517



HI1572
1633105
1633257



HI1575
1636870
1636721



HI1576
1637376
1636870



HI1577
1637498
1637439



HI1586
1647922
1647857



HI1587
1648198
1648028



HI1588
1648605
1648189



HI1592
1654749
1653193



HI1596
1659183
1657846



HI1597
1659861
1659247



HI1599
1661605
1661453



HI1600
1662311
1661643



HI1601
1662648
1662328



HI1604
1665779
1664724



HI1G05
1666807
1666094



HI1606
1667750
1666800



HI1607
1668067
1667783



HI1608
1668561
1668109



HI1609
1668769
1669446



HI1611
1670802
1671410



HIl613
1672733
1673359



HI1614
1673350
1674312



HI1618
1678855
1677464



HI1626
1686816
1686316



HI1627
1687436
1686819



HI1628
1687921
1687439



HI1630
1688617
1687937



HI1631
1689671
1689177



HI1632
1690500
1680847



HI1633
1690388
1689675



HI1634
1690881
1691282



HI1637
1693111
1692542



HI1643
1702265
1700876



HI1649
1707768
1708761



HI1653
1711982
1712854



HI1654
1712909
1713433



HI1655
1715939
1716046



HI1657
1716442
1716167



HI1658
1717744
1717198



HI1659
1718225
1717860



HI1660
1720257
1719409



HI1661
1720329
1722053



HI1662
1722056
1722412



HI1663
1722428
1723010



HI1669
1732543
1731909



HI1670
1733332
1732556



HI1671
1733482
1733363



HI1672
1733919
1733539



HI1673
1735404
1733938



HI1675
1737711
1737589



HI1677
1738407
1739654



HI1678
1739641
1742283



HI1683
1745073
1745741



HI1685
1747304
1747843



HI1686
1750100
1747947



HI1687
1750833
1750171



HI1689
1752090
1753040



HI1690
1753041
1753619



HI1693
1757163
1757783



HI1694
1757788
1758492



HI1707
1770253
1770993



HI1709
1774757
1773684



HI1710
1775859
1774744



HI1715
1782227
1781865



HI1716
1782482
1782345



HI1720
1786560
1785523



HI1721
1786631
1787176



HI1723
1788842
1788747



HI1724
1769761
1788979



HI1726
1792471
1793034



HI1727
1793205
1793652



HI1729
1794860
1795201



HI1730
1795161
1795556



HI1736
1803407
1802481



HI1737
1804045
1803407



HI1742
1813528
1813298



HI1743
1813960
1813834



HI1744
1814891
1813960

















TABLE 3







Whole Genome Sequencing Strategy








Stage
Description





Random small insert
Randomly sheared genomic DNA on the order of


and large insert library
2 kb and 15-20 kb respectively


construction


Library Plating
Verify random nature of library and maximize



random selection of small insert and large



insert clones for template production


High-throughput DNA
Sequence sufficient number of sequence


sequencing
fragments from both ends for 6X coverage


Assembly
Assemble random sequence fragments and



identify repeat regions


Gap closure


a. Physical gaps
Order all contigs (fingerprints, peptide links,



lambda clones, PCR) and provide



templates for closure


b. Sequence gaps
Complete the genome sequence by primer



walking


Editing
Visual inspection and resolution of sequence



ambiguities, including frameshifts


Annotation
Identification and description of all predicted



coding regions (putative identifications,



starts and stops, role



assignments, operons, regulatory regions)
















TABLE 4







The theory of shotgun sequencing follows from the application of


the equation for the Poisson distribution px = mxem/xt where x is


the number of occurrences of an event and m is the mean number


of occurrences. The numbers below predict the assembly


of a 1.9 Mb genome with an average sequence


fragment size of 460 bp.












%
bp

Avg. Gap


N
unsequenced
unsequenced
DS Gaps
Length














250
94.44
1794304
236
7600


500
89.18
1694487
446
3800


1,000
79.54
1511204
795
1900


2,000
63.26
1201967
1265
950


3,000
50.32
956009
1509
633


5,000
31.83
604785
1592
380


10,000
10.13
192508
1013
190


15,000
3.23
61277
484
127


20,000
1.03
19505
205
95


25,000
0.33
6209
82
76


30,000
0.10
1976
31
63


50,000
0.00
20
1
38
















TABLE 5







Summary of features of whole genome sequencing


of H. influenzae Rd








Description
Number





Double stranded templates
19,687


Forward sequencing reactions (M13-21 primer)
19,346


# Successful (%)
16,240 (84%)


Average edited read length
485 bp


Reverse sequencing reactions (M13RP1 primer)
9297


# Successful (%)
7,744 (83%)


Average edited read length
444 bp


Sequence fragments in random assembly
24,304


Total # of base pairs
11,631,485


# of contigs
140


Physical gap closure
 42


PCR
 37


Southern analysis
 15


Lambda clones
 23


Peptide links
 2


Terminator sequencing reactions*
3,102


# Succesful (%)
2,024 (65%)


Average edited read length
375 bp


Genome Size
1,830,121 bp


# of N's in sequence (%)
188 (0.01%)


Coordinate of proposed origin of replication
602,483-602,764


G/C content
38%


# of rRNA
6


rmA, rmC, rmD (spacer region)
723 bp


rmB, rmE, rmF (spacer region)
478 bp


# of tRNA genes identified
54


Number of Predicted Coding Regions
1,749


# Unassigned role (%)
 724 (41%)


No database match
384


Match hypothetical proteins
340


# Assigned role (%)
1025 (59%)


Amino acid metabolism
 71 (6.9%)


Fatty acid/phospholipid metabolism
 24 (2.3%)


Biosynthesis of cofactors, prosthetic groups,
 54 (5.3%)


and carriers


Purines, pyrimidines, nucleosides, nucleotides
 54 (5.3%)


Central intermediary metabolism
 31 (3.0%)


Energy metabolism
 99 (9.7%)


Cell envelope
 82 (8.0%)


Regulatory functions
 63 (6.1%)


Replication
 88 (8.6%)


Transcription
 27 (2.5%)


Translation
 146 (14.2%)


Transport/binding proteins
 145 (14.1%)


Cellular processes
 42 (4.1%)


Other
 99 (9.7%)





Includes gap closure, walks on rRNA repeats, and random end-sequencing of lambda clones for assembly confirmation 













TABLE 6







Two component systems in H. influenzae Rd

















Length


ID
Location
Best Match
% ID
% Sim
(bp)















Sensors:







HI0221
239,378
arcB (E. coli)
39.5
63.9
200


HI0269
299,541
narQ (E. coli)
38.1
68.0
562


HI1713
1,781,143
basS (E. coli)
27.7
51.5
250


HI1381
1,475,017
phoR (E. coli)
38.1
61.6
280


Regulators:


HI0728
777,934
narP (E. coli)
59.3
77.0
209


HI0839
887,011
cpxR (E. coli)
51.9
73.0
229


HI0886
936,624
arcA (E. coli)
77.2
87.8
236


HI1382
1,475,502
phoB (E. coli)
52.9
71.4
228


HI1714
1,781,799
basR (E. coli)
43.5
59.3
219

## Claims

1. An isolated polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence encoded by ORF HI0270, represented by nucleotides 301-267 of SEQ ID NO:1.

2. The isolated polynucleotide of claim 1, wherein said polynucleotide comprises a heterologous polynucleotide sequence.

3. The isolated polynucleotide of claim 2, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.

4. A nucleic acid sequence complementary to the polynucleotide of claim 1.

5. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 1 into a vector.

6. A recombinant vector comprising the isolated polynucleotide of claim 1.

7. The recombinant vector of claim 6, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

8. A recombinant host cell comprising the isolated polynucleotide of claim 1.

9. The recombinant host cell of claim 8, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

10. A method for producing a polypeptide, comprising:
(a) culturing a cell under conditions suitable to produce a polypeptide encoded by the polynucleotide of claim 1; and 
(b) recovering the polypeptide.

11. An isolated polynucleotide comprising a nucleic acid sequence encoding a fragment of the amino acid sequence encoded by ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1, wherein said fragment specifically binds an antibody which specifically binds a polypeptide consisting of the amino acid sequence of HI0270.

12. The isolated polynucleotide of claim 11, wherein said polynucleotide comprises a heterologous polynucleotide sequence.

13. The isolated polynucleotide of claim 12, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.

14. An isolated polynucleotide complementary to the polynucleotide of claim 11.

15. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 11, into a vector.

16. A recombinant vector comprising the isolated polynucleotide of claim 11.

17. The recombinant vector of claim 16, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

18. A recombinant host cell comprising the isolated polynucleotide of claim 11.

19. The recombinant host cell of claim 18, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

20. A method for producing a polypeptide, comprising:
(a) culturing a host cell under conditions suitable to produce a polypeptide encoded by the polynucleotide of claim 11; and 
(b) recovering the polypeptide from the cell culture.

21. An isolated polynucleotide fragment comprising a nucleic acid sequence which hybridizes under hybridization conditions, comprising hybridization in 5×SSC and 50% formamide at 50-65° C. and washing in a wash buffer consisting of 0.5×SSC at 50-65° C., to the complementary strand of ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1.

22. The isolated polynucleotide of claim 21, wherein said polynucleotide comprises a heterologous polynucleotide sequence.

23. The isolated polynucleotide of claim 22, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.

24. An isolated polynucleotide complementary to the polynucleotide of claim 21.

25. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 21 into a vector.

26. A recombinant vector comprising the isolated polynucleotide of claim 21.

27. The recombinant vector of claim 26, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

28. A recombinant host cell comprising the isolated polynucleotide of claim 21.

29. The recombinant host cell of claim 28, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

30. A method for producing a polypeptide, comprising:
(a) culturing a host cell under conditions suitable to produce a polypeptide encoded by the polynucleotide of claim 21; and 
(b) recovering the polypeptide from the cell culture.

31. An isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide fragment consisting of at least 10 contiguous amino acid residues and no more than 100 amino acid residues of the amino acid sequence encoded by ORF HI0326, represented by nucleotides 301245-302267 of SEQ ID NO:1.

32. The isolated polynucleotide of claim 31, wherein said polynucleotide comprises a heterologous polynucleotide sequence.

33. The isolated polynucleotide of claim 32, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.

34. An isolated polynucleotide complementary to the polynucleotide of claim 31.

35. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 31 into a vector.

36. A recombinant vector comprising the isolated polynucleotide of claim 31.

37. The recombinant vector of claim 36, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

38. A recombinant host cell comprising the isolated polynucleotide of claim 31.

39. The recombinant host cell of claim 38, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

40. A method for producing a polypeptide, comprising:
(a) culturing a host cell under conditions suitable to produce a polypeptide encoded by the polynucleotide of claim 31; and 
(b) recovering the polypeptide from the cell culture.

41. An isolated polynucleotide fragment comprising a nucleic acid sequence consisting of at least 30 contiguous nucleotide residues and no more than 300 contiguous nucleotide residues of an ORF HI0270, represented by nucleotides 301245-302267 of SEQ ID NO:1.

42. The isolated polynucleotide of claim 41, wherein said polynucleotide comprises a heterologous polynucleotide sequence.

43. The isolated polynucleotide of claim 41, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.

44. An isolated polynucleotide complementary to the polynucleotide of claim 41.

45. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 41 into a vector.

46. A recombinant vector comprising the isolated polynucleotide of claim 41.

47. The recombinant vector of claim 46, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

48. A recombinant host cell comprising the isolated polynucleotide of claim 41.

49. The recombinant host cell of claim 48, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.

50. A method for producing a polypeptide, comprising:
(a) culturing a host cell under conditions suitable to produce a polypeptide encoded by the polynucleotide of claim 41; and 
(b) recovering the polypeptide from the cell culture.