Patent Grant
7090973
The invention relates to isolated nucleic acids and polypeptides derived from Bacteroides fragilis that are useful as molecular targets for diagnostics, prophylaxis and treatment of pathological conditions, as well as materials and methods for the diagnosis, prevention, and amelioration of pathological conditions resulting from bacterial infection.
The genus Bacteroides is a member of the family Bacteroidaceae. They are Gram-negative, obligately anaerobic, nonsporeforming rods. The genus contains at least 39 species, and are often isolated from sewage as well as the digestive tract of man, animals, and insects. Bacteroides fragilis was first described in 1898 by Veillon and Zuber, but was called Bacillus fragilis. In 1919, Castellani and Chalmers transferred it to the Bacteroides genus. The “B. fragilis group” refers to the saccharoelastic bacteroids that grow well in bile. Members of this group were previously subspecies of B. fragilis and include B. fragilis, B. distasonis, B. ovatus, B. thetaiotaomicron, and B. vulgatus (Castellani and Chalmers. 1984. Genus I. Bacteroides 1919, 959. Krieg and Holt (editors) In Bergey's Manual of Systematic Bacteriology, 1:604–631).
Bacteroides fragilis accounts for only 1% of the normal flora of the human colon, but is the most common anaerobe isolated from clinical specimens. It is associated with soft tissue infections, abscesses and bacteremia (Moncrief J., et al, 1998. Infect. Immun. 66:1735–1739). B. fragilis has also been associated with infection of the skeletal muscle (Katagiri, K., et al, 1996. J. Dermatology. 23:129–132), and meningitis (Aucher, P., et al, 1996. Eur. J. Clin. Microbiol. Infect. Dis. 15:820–823). The B. fragilis group is responsible for 65% of all anaerobic bacteremia cases, with mortality rates in excess of 19% (Redondo, M., et al, 1995. Clinical Infectious Disease. 20:1492–1496).
In 1984, strains of B. fragilis were found to cause diarrhea in newborn lambs (Myers, L., et al, 1984. Infect. Immun. 44:241–244). Subsequently, it has been shown that B. fragilis is associated with diarrhea in other livestock and young children. These strains are called enterotoxigenic strains, because they produced a 20 KD metalloprotease enterotoxin with intestinal secretory activity (Moncrief J., et al, 1995. Infect. Immun. 63:175–181).
There has been an increase in antibiotic resistance within the Bacteroides fragilis group. While there is still excellent activity of many antibiotics, even some of the most potent agents, the carbapenems and the β-lactamase-inhibitor combinations, are losing activity (Snydman, D., et al, 1996. Clinical Infectious Diseases. 23:S54–65). The cefoxitin resistance rate has increased from 0% in 1987 to 22% in 1995 (Bianchini, H., et al, 1997. Clinical Infectious Diseases. 25:S268–269). Resistance to metronidazole, co-amoxiclav, and imipenem is rare, but strains have been found that are resistant to one or all of these antibiotics. (Turner, P., et al, 1995. The Lancet. 345:1275–1277). Clindaycin resistance has been shown to be transferred between strains by either plasmid or transposon mechanisms. (Dalmau, D., et al, 1997. Clinical Infectious Diseases. 24:874–877). The increasing resistance to antibiotics commonly used against Bacteroides species may eventually lead to failures of these treatments.
Sequencing and analysis of this genome is crucial for the identification of essential genes for development of drug targets and to reduce the emerging health threat this organism poses.
The present invention fulfills the need for diagnostic tools and therapeutics by providing bacterial-specific compositions and methods for detecting Bacteroides species including B. fragilis, as well as compositions and methods useful for treating and preventing Bacteroides infection, in particular, B. fragilis infection, in vertebrates including mammals.
The present invention encompasses isolated nucleic acids and polypeptides derived from B. fragilis that are useful as reagents for diagnosis of bacterial disease, components of effective antibacterial vaccines, and/or as targets for antibacterial drugs including anti-BR fragilis drugs. They can also be used to detect the presence of B. fragilis and other Bacteroides species in a sample; and in screening compounds for the ability to interfere with the B. fragilis life cycle or to inhibit B. fragilis infection. They also have use as biocontrol agents for plants.
In one aspect, the invention features compositions of nucleic acids corresponding to entire coding sequences of B. fragilis proteins, including surface or secreted proteins or pasts thereof, nucleic acids capable of binding mRNA from B. fragilis proteins to block protein translation, and methods for producing B. fragilis proteins or parts thereof using peptide synthesis and recombinant DNA techniques. This invention also features antibodies and nucleic acids useful as probes to detect B. fragilis infection. In addition, vaccine compositions and methods for the protection or treatment of infection by B. fragilis are within the scope of this invention.
The nucleotide sequences provided in SEQ ID NO: 1–SEQ ID NO: 5222, a fragment thereof, or a nucleotide sequence at least about 99.5% identical to a sequence contained within SEQ ID NO: 1–SEQ ID NO: 5222 may be “provided” in a variety of medias 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–SEQ ID NO: 5222, a fragment thereof, or a nucleotide sequence at least about 99.5% identical to a sequence contained within SEQ ID NO: 1–SEQ ID NO: 5222. Uses for and methods for providing nucleotide sequences in a variety of media is well known in the art (see e.g., EPO Publication No. EP 0 756 006).
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 media 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 media, 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 person skilled in the art can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable media having recorded thereon a nucleotide sequence of the present invention.
As used herein, “recorded” refers to a process for storing information on computer readable media. A person skilled in the art can readily adopt any of the presently known methods for recording information on computer readable media to generate manufactures comprising the nucleotide sequence information of the present invention.
A variety of data storage structures are available to a person skilled in the art for creating a computer readable media 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 media. 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 person skilled in the art can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable media having recorded thereon the nucleotide sequence information of the present invention.
By providing the nucleotide sequence of SEQ ID NO: 1–SEQ ID NO: 5222, a fragment thereof, or a nucleotide sequence at least about 99.5% identical to SEQ ID NO: 1–SEQ ID NO: 5222 in computer readable form, a person skilled in the art can routinely access the coding sequence information for a variety of purposes. Computer software is publicly available which allows a person skilled in the art to access sequence information provided in a computer readable media. Examples of such computer software include programs of the “Staden Package”, “DNA Star”, “MacVector”, GCG “Wisconsin Package” (Genetics Computer Group, Madison, Wis.) and “NCBI Toolbox” (National Center For Biotechnology Information). Suitable programs are described, for example, in Martin J. Bishop, ed., Guide to Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif. (1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, The Internet and the New Biology. Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D.C. (1997).
Computer algorithms enable the identification of B. fragilis open reading frames (ORFs) within SEQ ID NO: 1–SEQ ID NO: 5222 which contain homology to ORFs or proteins from other organisms. Examples of such similarity-search algorithms include the BLAST [Altschul et al., J. Mol. Biol. 215:403–410 (1990)] and Smith-Waterman [Smith and Waterman (1981) Advances in Applied Mathematics, 2:482–489] search algorithms. Suitable search algorithms are described, for example, in Martin J. Bishop, ed., Guide to Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif. (1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, The Internet and the New Biology. Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D.C. (1997). Such algorithms are utilized on computer systems as exemplified below. The ORFs so identified represent protein encoding fragments within the B. fragilis 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 B. fragilis 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 person skilled in the art can readily appreciate that any one of the currently available computer-based systems is suitable for use in the present invention. 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 B. fragilis genome which are similar to, or “match”, a particular target sequence or target motif. A variety of known algorithms are known in the art and have been disclosed publicly, and a variety of commercially available software for conducting homology-based similarity searches are available and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, FASTA (GCG Wisconsin Package), Bic_SW (Compugen Bioccelerator), BLASTN2, BLASTP2, BLASTX2 (NCBI) and Motifs (GCG). Suitable software programs are described, for example, in Martin J. Bishop, ed., Guide to Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif. (1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D.C. (1997). A person skilled in the art 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 person skilled in the art 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 many genes are longer than 500 amino acids, or 1.5 kb in length, and that commercially important fragments of the B. fragilis genome, such as sequence fragments involved in gene expression and protein processing, will often be shorter than 30 nucleotides.
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 specific functional domain or three-dimensional configuration which is formed upon the folding of the target polypeptide. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzymatic active sites, membrane-spanning regions, 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 B. fragilis genome possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a person skilled in the art 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 B. fragilis genome. In the present examples, implementing software which implement the BLASTP2 and bic_SW algorithms (Altschul et al., J. Mol. Biol. 215:403–410 (1990); Compugen Biocellerator) was used to identify open reading frames within the B. fragilis genome. A person skilled in the art 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. Suitable programs are described, for example, in Martin J. Bishop, ed., Guide to Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif. (1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, The Internet and the New Biology. Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D.C. (1997).
The invention features B. fragilis polypeptides, preferably a substantially pure preparation of an B. fragilis polypeptide, or a recombinant B. fragilis polypeptide. In preferred embodiments: the polypeptide has biological activity; the polypeptide has an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least about 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least about 5, preferably at least about 10, more preferably at least about 20, still more preferably at least about 50, 100, or 150 contiguous amino acid residues of the invention contained in the Sequence Listing. In yet another preferred embodiment, the amino acid sequence which differs in sequence identity by about 7% to about 8% from the B. fragilis amino acid sequences of the invention contained in the Sequence Listing is also encompassed by the invention.
In preferred embodiments: the B. fragilis polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.
In a preferred embodiment, the subject B. fragilis polypeptide differs in amino acid sequence at about 1, 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the B. fragilis polypeptide exhibits an B. fragilis biological activity, e.g., the B. fragilis polypeptide retains a biological activity of a naturally occurring B. fragilis enzyme.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5′ or 3′ to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
In yet other preferred embodiments, the B. fragilis polypeptide is a recombinant fusion protein having a first B. fragilis polypeptide portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to B. fragilis. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
Polypeptides of the invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
In a preferred embodiment, the encoded B. fragilis polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at about 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that: the B. fragilis encoded polypeptide exhibits an B. fragilis biological activity, e.g., the encoded B. fragilis enzyme retains a biological activity of a naturally occurring B. fragilis.
In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5′ or 3′ to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
The B. fragilis strain, 14062, from which genomic sequences have been sequenced, has been deposited on Jul. 20, 1998, in the American Type Culture Collection and assigned the ATCC designation # 202158.
Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridize under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1–6.3.6, hereby incorporated by reference); and, polypeptides specifically bound by antisera to B. fragilis polypeptides, especially by antisera to an active site or binding domain of B. fragilis polypeptide. The invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as B. fragilis polypeptide analogs or variants.
The invention further provides nucleic acids, e.g., RNA or DNA and their respective complements, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In preferred embodiments, the subject B. fragilis nucleic acid will include a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the B. fragilis gene sequence, e.g., to render the B. fragilis gene sequence suitable for expression in a recombinant host cell.
In yet a further preferred embodiment, the nucleic acid which encodes an B. fragilis polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least about 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least about 12 consecutive nucleotides of the invention contained in the Sequence Listing; still more preferably to at least about 20 consecutive nucleotides of the invention contained in the Sequence Listing; most preferably to at least about 40 consecutive nucleotides of the invention contained in the Sequence Listing.
In another aspect, the invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an B. fragilis polypeptide. In preferred embodiments: the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% homologous to an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide is at least about 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least about 5, preferably at least about 10, more preferably at least about 20, still more preferably at least about 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.
In another aspect, the invention encompasses: a vector including a nucleic acid which encodes an B. fragilis polypeptide or an B. fragilis polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant B. fragilis polypeptide or B. fragilis polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating an B. fragilis or B. fragilis polypeptide variant, e.g., from the cell or from the cell culture medium.
One embodiment of the invention is directed to substantially isolated nucleic acids. Nucleic acids of the invention include sequences comprising at least about 8 nucleotides in length, more preferably at least about 12 nucleotides in length, even more preferably at least about 15–20 nucleotides in length, that correspond to a subsequence of any one of SEQ ID NO: 1–SEQ ID NO: 5222 or complements thereof. Alternatively, the nucleic acids comprise sequences contained within any ORF (open reading frame), including a complete protein-coding sequence, of which any of SEQ ID NO: 1–SEQ ID NO: 5222 forms a part. The invention encompasses sequence-conservative variants and function-conservative variants of these sequences. The nucleic acids may be DNA, RNA, DNA/RNA duplexes, protein-nucleic acid (PNA), or derivatives thereof.
In another aspect, the invention features a purified recombinant nucleic acid having at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity or % homology with a sequence of the invention contained in the Sequence Listing
The invention also encompasses recombinant DNA (including DNA cloning and expression vectors) comprising these B. fragilis-derived sequences; host cells comprising such DNA, including fungal, bacterial, yeast, plant, insect, and mammalian host cells; and methods for producing expression products comprising RNA and polypeptides encoded by the B. fragilis sequences. These methods are carried out by incubating a host cell comprising an B. fragilis-derived nucleic acid sequence under conditions in which the sequence is expressed. The host cell may be native or recombinant. The polypeptides can be obtained by (a) harvesting the incubated cells to produce a cell fraction and a medium fraction; and (b) recovering the B. fragilis polypeptide from the cell fraction, the medium fraction, or both. The polypeptides can also be made by in vitro translation.
In another aspect, the invention features nucleic acids capable of binding mRNA of B. fragilis. Such nucleic acid is capable of acting as antisense nucleic acid to control the translation of mRNA of B. fragilis. A further aspect features a nucleic acid which is capable of binding specifically to an B. fragilis nucleic acid. These nucleic acids are also referred to herein as complements and have utility as probes and as capture reagents.
In another aspect, the invention features an expression system comprising an open reading frame corresponding to B. fragilis nucleic acid. The nucleic acid further comprises a control sequence compatible with an intended host. The expression system is useful for making polypeptides corresponding to B. fragilis nucleic acid.
In another aspect, the invention encompasses: a vector including a nucleic acid which encodes an B. fragilis polypeptide or an B. fragilis polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant B. fragilis polypeptide or B. fragilis polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating the B. fragilis or B. fragilis polypeptide variant, e.g., from the cell or from the cell culture medium.
In yet another embodiment of the invention encompasses reagents for detecting bacterial infection, including B. fragilis infection, which comprise at least one B. fragilis-derived nucleic acid defined by any one of SEQ ID NO: 1–SEQ ID NO: 5222, or sequence-conservative or function-conservative variants thereof. Alternatively, the diagnostic reagents comprise nucleotide sequences that are contained within any open reading frames (ORFs), including preferably complete protein-coding sequences, contained within any of SEQ ID NO: 1–SEQ ID NO: 5222, or polypeptide sequences contained within any of SEQ ID NO: 5223–SEQ ID NO: 10444, or polypeptides of which any of the above sequences forms a part, or antibodies directed against any of the above peptide sequences or function-conservative variants and/or fragments thereof.
The invention further provides antibodies, preferably monoclonal antibodies, which specifically bind to the polypeptides of the invention. Methods are also provided for producing antibodies in a host animal. The methods of the invention comprise immunizing an animal with at least one B. fragilis-derived immunogenic component, wherein the immunogenic component comprises one or more of the polypeptides encoded by any one of SEQ ID NO: 1–SEQ ID NO: 5222 or sequence-conservative or function-conservative variants thereof; or polypeptides that are contained within any ORFs, including complete protein-coding sequences, of which any of SEQ ID NO: 1–SEQ ID NO: 5222 forms a part; or polypeptide sequences contained within any of SEQ ID NO: 5223–SEQ ID NO: 10444; or polypeptides of which any of SEQ ID NO: 5223–SEQ ID NO: 10444 forms a part. Host animals include any warm blooded animal, including without limitation mammals and birds. Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of B. fragilis —specific antigens.
In yet another aspect, the invention provides diagnostic methods for detecting B. fragilis antigenic components or anti-B. fragilis antibodies in a sample. B. fragilis antigenic components may be detected by known processes, including but not limited to detection by a process comprising: (i) contacting a sample suspected to contain a bacterial antigenic component with a bacterial-specific antibody, under conditions in which a stable antigen-antibody complex can form between the antibody and bacterial antigenic components in the sample; and (ii) detecting any antigen-antibody complex formed in step (i), wherein detection of an antigen-antibody complex indicates the presence of at least one bacterial antigenic component in the sample. In different embodiments of this method, the antibodies used are directed against a sequence encoded by any of SEQ ID NO: 1–SEQ ID NO: 5222 or sequence-conservative or function-conservative variants thereof, or against a polypeptide sequence contained in any of SEQ ID NO: 5223–SEQ ID NO: 10444 or function-conservative variants thereof.
In yet another aspect, the invention provides a method for detecting antibacterial-specific antibodies in a sample, which comprises: (i) contacting a sample suspected to contain antibacterial-specific antibodies with an B. fragilis antigenic component, under conditions in which a stable antigen-antibody complex can form between the B. fragilis antigenic component and antibacterial antibodies in the sample; and (ii) detecting any antigen-antibody complex formed in step (i), wherein detection of an antigen-antibody complex indicates the presence of antibacterial antibodies in the sample. In different embodiments of this method, the antigenic component is encoded by a sequence contained in any of SEQ ID NO: 1–SEQ ID NO: 5222 or sequence-conservative and function-conservative variants thereof, or is a polypeptide sequence contained in any of SEQ ID NO: 5223–SEQ ID NO: 10444 or function-conservative variants thereof.
In another aspect, the invention features a method of generating vaccines for immunizing an individual against B. fragilis. The method includes: immunizing a subject with an B. fragilis polypeptide, e.g., a surface or secreted polypeptide, or a combination of such peptides or active portion(s) thereof, and a pharmaceutically acceptable carrier. Such vaccines have therapeutic and prophylactic utilities.
In another aspect, the invention features a method of evaluating a compound, e.g., a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an B. fragilis polypeptide. The method includes contacting the compound to be evaluated with an B. fragilis polypeptide and determining if the compound binds or otherwise interacts with the B. fragilis polypeptide. Compounds which bind or otherwise interact with B. fragilis polypeptides are candidates as modulators, including activators and inhibitors, of the bacterial life cycle. These assays can be performed in vitro or in vivo.
In another aspect, the invention features a method of evaluating a compound, e.g., a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an B. fragilis nucleic acid, e.g., DNA or RNA. The method includes contacting the compound to be evaluated with an B. fragilis nucleic acid and determining if the compound binds or otherwise interacts with the B. fragilis nucleic acid. Compounds which bind B. fragilis are candidates as modultors, including activators and inhibitors, of the bacterial life cycle. These assays can be performed in vitro or in vivo.
A particularly preferred embodiment of the invention is directed to a method of screening test compounds for anti-bacterial activity, which method comprises: selecting as a target a bacterial specific sequence, which sequence is essential to the viability of a bacterial species; contacting a test compound with said target sequence; and selecting those test compounds which bind to said target sequence as potential anti-bacterial candidates. In one embodiment, the target sequence selected is specific to a single species, or even a single strain, such as, for example, the strain B. fragilis 14062. In a second embodiment, the target sequence is common to at least two species of bacteria. In a third embodiment, the target sequence is common to a family of bacteria. The target sequence may be a nucleic acid sequence or a polypeptide sequence. Methods employing sequences common to more than one species of microorganism may be used to screen candidates for broad spectrum anti-bacterial activity.
The invention also provides methods for preventing or treating disease caused by certain bacteria, including B. fragilis, which are carried out by administering to an animal in need of such treatment, in particular a warm-blooded vertebrate, including but not limited to birds and mammals, a compound that specifically inhibits or interferes with the function of a bacterial polypeptide or nucleic acid. In a particularly preferred embodiment, the mammal to be treated is human.
The sequences of the present invention include the specific nucleic acid and amino acid sequences set forth in the Sequence Listing that forms a part of the present specification, and which are designated SEQ ID NO: 1–SEQ ID NO: 10444. Use of the terms “SEQ ID NO: 1–SEQ ID NO: 5222”, “SEQ ID NO: 5223–SEQ ID NO: 10444, “the sequences depicted in Table 2”, etc., is intended, for convenience, to refer to each individual SEQ ID NO individually, and is not intended to refer to the genus of these sequences unless such reference would be indicated. In other words, it is a shorthand for listing all of these sequences individually. The invention encompasses each sequence individually, as well as any combination thereof.
Definitions
“Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, i.e., DNA—DNA, DNA-RNA and RNA—RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
A nucleic acid or polypeptide sequence that is “derived from” a designated sequence refers to a sequence that corresponds to a region of the designated sequence. For nucleic acid sequences, this encompasses sequences that are homologous or complementary to the sequence, as well as “sequence-conservative variants” and “function-conservative variants.” For polypeptide sequences, this encompasses “function-conservative variants.” Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Function-conservative variants are those in which a given amino acid residue in a polypeptide has been changed without altering the overall conformation and function of the native polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). “Function-conservative” variants also include any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide.
An “B. fragilis-derived” nucleic acid or polypeptide sequence may or may not be present in other bacterial species, and may or may not be present in all B. fragilis strains. This term is intended to refer to the source from which the sequence was originally isolated. Thus, an B. fragilis-derived polypeptide, as used herein, may be used, e.g., as a target to screen for a broad spectrum antibacterial agent, to search for homologous proteins in other species of bacteria or in eukaryotic organisms such as bacteria humans, etc.
A purified or isolated polypeptide or a substantially pure preparation of a polypeptide are used interchangeably herein and, as used herein, mean a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least about 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains sufficient polypeptide to allow protein sequencing; at least about 1, 10, or preferably 100 mg of polypeptide.
A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least about 10%, more preferably at least about 50%, of the subject cells.
A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3′ end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional B. fragilis DNA sequence.
A “contig” as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.
An “open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.
As used herein, a “coding sequence” is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.
A “complement” of a nucleic acid as used herein refers to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
A “gene product” is a protein or structural RNA which is specifically encoded by a gene.
As used herein, the term “probe” refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like. Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a “capture ligand”. Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions. Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions. Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stringency (such as, for example, in a solution of 0.5×SSC, at 65° C.) requires that the sequences be essentially completely homologous. Conditions of intermediate stringency (such as, for example, 2×SSC at 65° C.) and low stringency (such as, for example 2×SSC at 55° C.) require correspondingly less overall complementarity between the hybridizing sequences. (1×SSC is 0.15 M NaCl, 0.015 M Na citrate).
The terms peptides, proteins, and polypeptides are used interchangeably herein.
As used herein, the term “surface protein” refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
A polypeptide has B. fragilis biological activity if it has one, two or preferably more of the following properties: (1) if when expressed in the course of an B. fragilis infection, it can promote, or mediate the attachment of B. fragilis to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an B. fragilis protein; (3) the gene which encodes it can rescue a lethal mutation in an B. fragilis gene.
A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.
A biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the B. fragilis polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring B. fragilis polypeptides, e.g., one or more of the biological activities described herein. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in CHO (Chinese Hamster Ovary) cells. Because peptides such as B. fragilis polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful B. fragilis fragment or B. fragilis analog is one which exhibits a biological activity in any biological assay for B. fragilis activity. The fragment or analog possesses about 10%, preferably about 40%, more preferably about 60%, 70%, 80% or 90% or greater of the activity of B. fragilis, in any in vivo or in vitro assay.
Analogs can differ from naturally occurring B. fragilis polypeptides in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include B. fragilis polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the B. fragilis polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be made in view of the table below.
Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., α or γ amino acids; and cyclic analogs.
As used herein, the term “fragment”, as applied to an B. fragilis analog, will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of B. fragilis polypeptides can be generated by methods known to those skilled in the art. The ability of an Bacteroides fragment to exhibit a biological activity of B. fragilis polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are B. fragilis polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
An “immunogenic component” as used herein is a moiety, such as an B. fragilis polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal.
An “antigenic component” as used herein is a moiety, such as an B. fragilis polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
The term “antibody” as used herein is intended to include fragments thereof which are specifically reactive with B. fragilis polypeptides.
As used herein, the term “cell-specific promoter” means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
Misexpression, as used herein, refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of increased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-translational modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
As used herein, “host cells” and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
As used herein, the term “control sequence” refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.
As used herein, the term “operably linked” refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.
The “metabolism” of a substance, as used herein, means any aspect of the expression, function, action, or regulation of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modification, the substance induces in other substances. The metabolism of a substance also includes changes in the distribution of the substance. The metabolism of a substance includes changes the substance induces in the distribution of other substances.
A “sample” as used herein refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies known to those of skill in the art. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning, Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); the series, Methods in Enzymology (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.); PCR-A Practical Approach (McPherson, Quirke, and Taylor, eds., 1991); Immunology, 2d Edition, 1989, Roitt et al., C. V. Mosby Company, and New York; Advanced Immunology, 2d Edition, 1991, Male et al., Grower Medical Publishing, New York.; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning; Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); Martin J. Bishop, ed., Guide to Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif. (1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, The Internet and the New Biology: Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D.C. (1997).
Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention; however, preferred materials and/or methods are described. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted.
B. Fragilis Genomic Sequence
This invention provides nucleotide sequences of the genome of B. fragilis which thus comprises a DNA sequence library of B. fragilis genomic DNA. The detailed description that follows provides nucleotide sequences of B. fragilis, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are compositions and methods of using the disclosed B. fragilis sequences in methods including diagnostic and therapeutic applications. Furthermore, the library can be used as a database for identification and comparison of medically important sequences in this and other strains of B. fragilis.
To determine the genomic sequence of B. fragilis, DNA from strain 14062 of B. fragilis was isolated after Zymolyase digestion, sodium dodecyl sulfate lysis, potassium acetate precipitation, phenol:chloroform extraction and ethanol precipitation (Soll, D. R., T. Srikantha and S. R. Lockhart: Characterizing Developmentally Regulated Genes in B. fragilis. In Microbial Genome Methods. K. W. Adolph, editor. CRC Press. New York. p 17–37.). DNA was sheared hydrodynamically using an HPLC (Oefner, et. al., 1996) to an insert size of 2000–3000 bp. After size fractionation by gel electrophoresis the fragments were blunt-ended, ligated to adapter oligonucleotides and cloned into the pGTC (Thomann) vector to construct a “shotgun” subclone library.
DNA sequencing was achieved using established ABI sequencing methods on ABI377 automated DNA sequencers. The cloning and sequencing procedures are described in more detail in the Exemplification.
Individual sequence reads were assembled using PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, January 1996, p. 157). The average contig length was about 3–4 kb.
All subsequent steps were based on sequencing by ABI377 automated DNA sequencing methods. The cloning and sequencing procedures are described in more detail in the Exemplification.
A variety of approaches may be used to order the contigs so as to obtain a continuous sequence representing the entire B. fragilis genome. Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of B. fragilis genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined.
The B. fragilis sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides. As a result of the analysis of ORFs based on stop-to-stop codon reads, it should be understood that these ORFs may not correspond to the ORF of a naturally-occurring B. fragilis polypeptide. These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally-occurring B. fragilis polypeptide. Such start codons within the ORFs provided herein were identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded B. fragilis polypeptide is within the scope of this invention. For example, within the ORFs a codon such as AUG or GUG (encoding methionine or valine) which is part of the initiation signal for protein synthesis were identified and the portion of an ORF to corresponding to a naturally-occurring B. fragilis polypeptide was recognized. The predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARK™ (Borodovsky and McIninch, 1993, Comp. 17:123).
Each predicted ORF amino acid sequence was compared with all sequences found in current GENBANK, SWISS-PROT, and PIR databases using the BLAST algorithm. BLAST identifies local alignments occurring by chance between the ORF sequence and the sequence in the databank (Altschal et al., 1990, L Mol. Biol. 215:403–410). Homologous ORFs (probabilities less than 10−5 by chance) and ORF's that are probably non-homologous (probabilities greater than 10−5 by chance) but have good codon usage were identified. Both homologous, sequences and non-homologous sequences with good codon usage, are likely to encode proteins and are encompassed by the invention.
B. Fragilis Nucleic Acids
The present invention provides a library of B. fragilis-derived nucleic acid sequences. The libraries provide probes, primers, and markers which are used as markers in epidemiological studies. The present invention also provides a library of B. fragilis-derived nucleic acid sequences which comprise or encode targets for therapeutic drugs.
The nucleic acids of this invention may be obtained directly from the DNA of the above referenced B. fragilis strain by using the polymerase chain reaction (PCR). See “PCR, A Practical Approach” (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR is used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products is verified by conventional sequencing methods. Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
It is also possible to obtain nucleic acids encoding B. fragilis polypeptides from a cDNA library in accordance with protocols herein described. A cDNA encoding an B. fragilis polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques. Genes encoding B. fragilis polypeptides can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. Preferred nucleic acids of the invention are contained in the Sequence Listing.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
In another example, DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem. 764:17078, or other well known methods. This can be done by sequentially linking a series of oligonucleotide cassettes comprising pairs of synthetic oligonucleotides, as described below.
Nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands, antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences. As probes, primers, capture ligands and antisense agents, the nucleic acid normally consists of all or part (approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below.
Probes
A nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect B. fragilis. With the sequence information set forth in the present application, sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to B. fragilis, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least about twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules.
Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques. Individuals skilled in the art will readily recognize that the nucleic acids, for use as probes, can be provided with a label to facilitate detection of a hybridization product.
Nucleic acid isolated and synthesized in accordance with the sequence of the invention contained in the Sequence Listing can also be useful as probes to detect homologous regions (especially homologous genes) of other Bacteroides species using appropriate stringency hybridization conditions as described herein.
Capture Ligand
For use as a capture ligand, the nucleic acid selected in the manner described above with respect to probes, can be readily associated with a support. The manner in which nucleic acid is associated with supports is well known. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate B. fragilis nucleic acid from one strain from the nucleic acid of other another strain as well as from other organisms. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Bacteroides species from each other and from other organisms. Preferably, the sequence will comprise at least about twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques.
Primers
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of B. fragilis nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Bacteroides species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of ≧10–15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of B. fragilis nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods.
The copies can be used in diagnostic assays to detect specific sequences, including genes from B. fragilis and/or other Bacteroides species. The copies can also be incorporated into cloning and expression vectors to generate polypeptides corresponding to the nucleic acid synthesized by PCR, as is described in greater detail herein.
The nucleic acids of the present invention find use as templates for the recombinant production of B. fragilis-derived peptides or polypeptides
Antisense
Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of B. fragilis genes. These sequences also have utility as antisense agents to prevent expression of genes of other Bacteroides species.
In one embodiment, nucleic acid or derivatives corresponding to B. fragilis nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells. For example, a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA. Preferably, the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA. Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques. Methods for loading antisense nucleic acid in liposomes is known in the art as exemplified by U.S. Pat. No. 4,241,046 issued Dec. 23, 1980 to Papahadjopoulos et al.
The present invention encompasses isolated polypeptides and nucleic acids derived from B. fragilis that are useful as reagents for diagnosis of bacterial infection, components of effective anti-bacterial vaccines, and/or as targets for anti-bacterial drugs, including anti-B. fragilis drugs.
Expression of B. Fragilis Nucleic Acids
Table 2, which is appended herewith and which forms part of the present specification, provides a list of open reading frames (ORFs) in both strands and a putative identification of the particular function of a polypeptide which is encoded by each ORF, based on the homology match (determined by the BLASTP2 algorithm) of the predicted polypeptide with known proteins encoded by ORFs in other organisms. An ORF is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and was determined from stop to stop codons. The first column contains a designation for the ORF (“ORF Name”). The second and third columns list the SEQ ID numbers for the nucleic acid (“NT ID”) and amino acid (“AA ID”) sequences corresponding to each ORF, respectively. The fourth and fifth columns list the length of the nucleic acid ORF (“NT Length”) and the length of the amino acid ORF (“AA Length”), respectively. The nucleotide sequence corresponding to each ORF begins at the first nucleotide immediately following a stop codon and ends at the nucleotide immediately preceding the next downstream stop codon in the same reading frame. It will be recognized by one skilled in the art that the natural translation initiation sites will correspond to ATG, GTG, or TTG codons located within the ORFs. The natural initiation sites depend not only on the sequence of a start codon but also on the context of the DNA sequence adjacent to the start codon. Usually, a recognizable ribosome binding site is found within 20 nucleotides upstream from the initiation codon. In some cases where genes are translationally coupled and coordinately expressed together in “operons”, ribosome binding sites are not present, but the initiation codon of a downstream gene may occur very close to, or overlap, the stop codon of the an upstream gene in the same operon. The correct start codons can be generally identified without undue experimentation because only a few codons need be tested. It is recognized that the translational machinery in bacteria initiates all polypeptide chains with the amino acid methionine, regardless of the sequence of the start codon. In some cases, polypeptides are post-translationally modified, resulting in an N-terminal amino acid other than methionine in vivo. The sixth and seventh columns provide metrics for assessing the likelihood of the homology match (determined by the BLASTP2 algorithm), as is known in the art, to the genes indicated in the description frame (“Description”) defined further below. These genes in the Description were identified when the designated ORF was compared against a comprehensive non-redundant protein database. Specifically, the sixth column represents the Blast Score (“Score”) for the match (a higher score is a better match), and the seventh column represents the probability (“Probability”) for the match (the probability that such a match can have occurred by chance; the lower the value, the more likely the match is valid). If a BLASTP2 score of less than 100 was obtained, no value is reported in the table. The remaining fields below the columns contain additional information relating to the potential function of the sequence based on the BLASTP2 analysis. Where a match was discovered, the field “Protein name” list the protein's name identified from the match. In addition, one skilled in the art would be able to identify the match and elucidate its function using the “Locus name” and where available the accession number, “Acc#” from the database. Lastly, one skilled in the art would appreciate the “Description” field to further describe the potential function of the protein based on this analysis. This information allows one of ordinary skill in the art to determine a potential use for each identified coding sequence and, as a result, allows to use the polypeptides of the present invention for commercial and industrial purposes.
Using the information provided in SEQ ID NO: 1–SEQ ID NO: 5222, SEQ ID NO: 5223–SEQ ID NO: 10444 and in Table 2 together with routine cloning and sequencing methods, one of ordinary skill in the art will be able to clone and sequence all the nucleic acid fragments of interest including open reading frames (ORFs) encoding a large variety of proteins of B. fragilis.
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides. The nucleic acid of the invention exemplified in SEQ ID NO: 1–SEQ ID NO: 5222 and in Table 2 or fragments of said nucleic acid encoding active portions of B. fragilis polypeptides can be cloned into suitable vectors or used to isolate nucleic acid. The isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.
The function of a specific gene or operon can be ascertained by expression in a bacterial strain under conditions where the activity of the gene product(s) specified by the gene or operon in question can be specifically measured. Alternatively, a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to, Eucaryotic species such as the yeast Saccharomyces cerevisiae, Methanobacterium strains or other Archaea, and Eubacteria such as E. coli, B. Subtilis, S. Aureus, S. Pneumonia or Pseudomonas putida. In some cases the expression host will utilize the natural B. fragilis promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta-galactosidase promoter for expression in E. coli).
To express a gene product using the natural B. fragilis promoter, a procedure such as the following can be used. A restriction fragment containing the gene of interest, together with its associated natural promoter element and regulatory sequences (identified using the DNA sequence data) is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together. The recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
In the case of a gene that requires a different promoter, the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid. This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning.
A suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)), and other laboratory textbooks.
For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an B. fragilis polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. Suitable media for cell culture are well known in the art. Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides. Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
In the case of membrane bound proteins, these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be chromatographically isolated from the membrane fraction. Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
One strategy to maximize recombinant B. fragilis peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119–128). Another strategy would be to alter the nucleic acid encoding an B. fragilis peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:2111–2118). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
The present invention provides a library of B. fragilis-derived nucleic acid sequences. The libraries provide probes, primers, and markers which can be used as markers in epidemiological studies. The present invention also provides a library of B. fragilis-derived nucleic acid sequences which comprise or encode targets for therapeutic drugs.
Nucleic acids comprising any of the sequences disclosed herein or sub-sequences thereof can be prepared by standard methods using the nucleic acid sequence information provided in SEQ ID NO: 1–SEQ ID NO: 5222. For example, DNA can be chemically synthesized using, e.g., the phosphoramidite solid support method of Matteucci et al., 1981, J. Am. Chem. Soc. 103:3185, the method of Yoo et al., 1989, J. Biol. Chem. 764:17078, or other well known methods. This can be done by sequentially linking a series of oligonucleotide cassettes comprising pairs of synthetic oligonucleotides, as described below.
Of course, due to the degeneracy of the genetic code, many different nucleotide sequences can encode polypeptides having the amino acid sequences defined by SEQ ID NO: 5223–SEQ ID NO: 10444 or sub-sequences thereof. The codons can be selected for optimal expression in prokaryotic or eukaryotic systems. Such degenerate variants are also encompassed by this invention.
Insertion of nucleic acids (typically DNAs) encoding the polypeptides of the invention into a vector is easily accomplished when the termini of both the DNAs and the vector comprise compatible restriction sites. If this cannot be done, it may be necessary to modify the termini of the DNAs and/or vector by digesting back single-stranded DNA overhangs generated by restriction endonuclease cleavage to produce blunt ends, or to achieve the same result by filling in the single-stranded termini with an appropriate DNA polymerase.
Alternatively, any site desired may be produced, e.g., by ligating nucleotide sequences (linkers) onto the termini. Such linkers may comprise specific oligonucleotide sequences that define desired restriction sites. Restriction sites can also be generated by the use of the polymerase chain reaction (PCR). See, e.g., Saiki et al., 1988, Science 239:48. The cleaved vector and the DNA fragments may also be modified if required by homopolymeric tailing.
The nucleic acids of the invention may be isolated directly from cells. Alternatively, the polymerase chain reaction (PCR) method can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incorporation into a given vector for recombinant expression.
The nucleic acids of the present invention may be flanked by natural B. fragilis regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3′-noncoding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. PNAs are also included. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
The invention also provides nucleic acid vectors comprising the disclosed B. fragilis-derived sequences or derivatives or fragments thereof. A large number of vectors, including plasmid and bacterial vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts, and may be used for cloning or protein expression.
The encoded B. fragilis polypeptides may be expressed by using many known vectors, such as pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), or pRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The particular choice of vector/host is not critical to the practice of the invention.
Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. The inserted B. fragilis coding sequences may be synthesized by standard methods, isolated from natural sources, or prepared as hybrids, etc. Ligation of the B. fragilis coding sequences to transcriptional regulatory elements and/or to other amino acid coding sequences may be achieved by known methods. Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl2 mediated DNA uptake, bacterial infection, microinjection, microprojectile, or other established methods.
Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are B. fragilis, E. coli, B. Subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, and the like. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced B. fragilis-derived peptides and polypeptides.
Advantageously, vectors may also include a transcription regulatory element (i.e., a promoter) operably linked to the B. fragilis portion. The promoter may optionally contain operator portions and/or ribosome binding sites. Non-limiting examples of bacterial promoters compatible with E. coli include: b-lactamase (penicillinase) promoter; lactose promoter; tryptophan (trp) promoter; araBAD (arabinose) operon promoter; lambda-derived P1 promoter and N gene ribosome binding site; and the hybrid tac promoter derived from sequences of the trp and lac UV5 promoters. Non-limiting examples of yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Suitable promoters for mammalian cells include without limitation viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences, polyA addition sequences and enhancer sequences to increase expression. Sequences which cause amplification of the gene may also be desirable. Furthermore, sequences that facilitate secretion of the recombinant product from cells, including, but not limited to, bacteria, yeast, and animal cells, such as secretory signal sequences and/or prohormone pro region sequences, may also be included. These sequences are well described in the art.
Nucleic acids encoding wild-type or variant B. fragilis-derived polypeptides may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell, and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods such as nonhomologous recombinations or deletion of endogenous genes by homologous recombination may also be used.
The nucleic acids of the present invention find use as templates for the recombinant production of B. fragilis-derived peptides or polypeptides.
Identification and use of B. Fragilis Nucleic Acid Sequences
The disclosed B. fragilis polypeptide and nucleic acid sequences, or other sequences that are contained within ORFs, including complete protein-coding sequences, of which any of the disclosed B. fragilis-specific sequences forms a part, are useful as target components for diagnosis and/or treatment of B. fragilis-caused infection
It will be understood that the sequence of an entire protein-coding sequence of which each disclosed nucleic acid sequence forms a part can be isolated and identified based on each disclosed sequence. This can be achieved, for example, by using an isolated nucleic acid encoding the disclosed sequence, or fragments thereof, to prime a sequencing reaction with genomic B. fragilis DNA as template; this is followed by sequencing the amplified product. The isolated nucleic acid encoding the disclosed sequence, or fragments thereof, can also be hybridized to B. fragilis genomic libraries to identify clones containing additional complete segments of the protein-coding sequence of which the shorter sequence forms a part. Then, the entire protein-coding sequence, or fragments thereof, or nucleic acids encoding all or part of the sequence, or sequence-conservative or function-conservative variants thereof, may be employed in practicing the present invention.
Preferred sequences are those that are useful in diagnostic and/or therapeutic applications. Diagnostic applications include without limitation nucleic-acid-based and antibody-based methods for detecting bacterial infection. Therapeutic applications include without limitation vaccines, passive immunotherapy, and drug treatments directed against gene products that are both unique to bacteria and essential for growth and/or replication of bacteria.
Identification of Nucleic Acids Encoding Vaccine Components and Targets for Agents Effective Against B. Fragilis
The disclosed B. fragilis genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids. The invention encompasses nucleic acids encoding immunogenic components of vaccines and targets for agents effective against B. fragilis. Identification of said immunogenic components involved in the determination of the function of the disclosed sequences, which can be achieved using a variety of approaches. Non-limiting examples of these approaches are described briefly below.
Homology to Known Sequences:
Computer-assisted comparison of the disclosed B. fragilis sequences with previously reported sequences present in publicly available databases is useful for identifying functional B. fragilis nucleic acid and polypeptide sequences. It will be understood that protein-coding sequences, for example, may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80–90%) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc. In addition, many structural features of particular protein classes have been identified and correlate with specific consensus sequences, such as, for example, binding domains for nucleotides, DNA, metal ions, and other small molecules; sites for covalent modifications such as phosphorylation, acylation, and the like; sites of protein:protein interactions, etc. These consensus sequences may be quite short and thus may represent only a fraction of the entire protein-coding sequence. Identification of such a feature in an B. fragilis sequence is therefore useful in determining the function of the encoded protein and identifying useful targets of antibacterial drugs.
Of particular relevance to the present invention are structural features that are common to secretory, transmembrane, and surface proteins, including secretion signal peptides and hydrophobic transmembrane domains. B. fragilis proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
Targets for therapeutic drugs according to the invention include, but are not limited to, polypeptides of the invention, whether unique to B. fragilis or not, that are essential for growth and/or viability of B. fragilis under at least one growth condition. Polypeptides essential for growth and/or viability can be determined by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene “knockout”. Alternatively, genetic footprinting can be used (Smith et al., 1995, Proc. Natl. Acad. Sci. USA 92:5479–6433; Published International Application WO 94/26933; U.S. Pat. No. 5,612,180). Still other methods for assessing essentiality includes the ability to isolate conditional lethal mutations in the specific gene (e.g., temperature sensitive mutations). Other useful targets for therapeutic drugs, which include polypeptides that are not essential for growth or viability per se but lead to loss of viability of the cell, can be used to target therapeutic agents to cells.
Strain-Specific Sequences:
Because of the evolutionary relationship between different B. fragilis strains, it is believed that the presently disclosed B. fragilis sequences are useful for identifying, and/or discriminating between, previously known and new B. fragilis strains. It is believed that other B. fragilis strains will exhibit at least about 70% sequence homology with the presently disclosed sequence. Systematic and routine analyses of DNA sequences derived from samples containing B. fragilis strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all B. fragilis strains. In one embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of B. fragilis. Strain-specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more B. fragilis strains.
In another embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all B. fragilis strains but are not found in other bacterial species.
B. Fragilis Polypeptides
This invention encompasses isolated B. fragilis polypeptides encoded by the disclosed B. fragilis genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least about 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an B. fragilis polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region. This can be achieved, for example, by using the isolated nucleic acid encoding the ORF, or fragments thereof, to prime a polymerase chain reaction with genomic B. fragilis DNA as template; this is followed by sequencing the amplified product.
The polypeptides of the present invention, including function-conservative variants of the disclosed ORFs, may be isolated from wild-type or mutant B. fragilis cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) including B. fragilis into which an B. fragilis derived protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins.
B. fragilis polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis as described by Merrifield, 1963, J. Am. Chem. Soc. 85:2149. The synthesis is carried out with amino acids that are protected at the alpha-amino terminus. Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides. The alpha-amino protecting group is selectively removed to allow subsequent reaction to take place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove the side-chain protecting groups.
Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the B. fragilis protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against an B. fragilis protein or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.
The present invention also encompasses derivatives and homologues of B. fragilis-encoded polypeptides. For some purposes, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i.e., function-conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine); negatively charged amino acids (aspartate and glutamate); polar neutral amino acids; and non-polar amino acids.
The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.
To identify B. fragilis-derived polypeptides for use in the present invention, essentially the complete genomic sequence of a virulent, methicillin-resistant isolate of Bacteroides fragilis isolate was analyzed. While, in very rare instances, a nucleic acid sequencing error may be revealed, resolving a rare sequencing error is well within the art, and such an occurrence will not prevent one skilled in the art from practicing the invention.
Also encompassed are any B. fragilis polypeptide sequences that are contained within the open reading frames (ORFs), including complete protein-coding sequences, of which any of SEQ ID NO: 1–SEQ ID NO: 5222 forms a part. Table 2, which is appended herewith and which forms part of the present specification, provides a putative identification of the particular function of a polypeptide which is encoded by each ORF, based on the homology match (determined by the BLAST algorithm) of the predicted polypeptide with known proteins encoded by ORFs in other organisms. As a result, one skilled in the art can use the polypeptides of the present invention for commercial and industrial purposes consistent with the type of putative identification of the polypeptide.
The present invention provides a library of B. fragilis-derived polypeptide sequences, and a corresponding library of nucleic acid sequences encoding the polypeptides, wherein the polypeptides themselves, or polypeptides contained within ORFs of which they form a part, comprise sequences that are contemplated for use as components of vaccines. Non-limiting examples of such sequences are listed by SEQ ID NO in Table 2, which is appended herewith and which forms part of the present specification.
The present invention also provides a library of B. fragilis-derived polypeptide sequences, and a corresponding library of nucleic acid sequences encoding the polypeptides, wherein the polypeptides themselves, or polypeptides contained within ORFs of which they form a part, comprise sequences lacking homology to any known prokaryotic or eukaryotic sequences. Such libraries provide probes, primers, and markers which can be used to diagnose B. fragilis infection, including use as markers in epidemiological studies. Non-limiting examples of such sequences are listed by SEQ ID NO in Table 2, which is appended hereto and part hereof.
The present invention also provides a library of B. fragilis-derived polypeptide sequences, and a corresponding library of nucleic acid sequences encoding the polypeptides, wherein the polypeptides themselves, or polypeptides contained within ORFs of which they form a part, comprise targets for therapeutic drugs.
Specific Example: Determination of Bacteroides Protein Antigens for Antibody and Vaccine Development
The selection of Bacteroides protein antigens for vaccine development can be derived from the nucleic acids encoding B. fragilis polypeptides. First, the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985) Biochimica et Biophysica Acta 815, 468–476) for predicting exported and membrane proteins.
Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases. BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database. ORF's with significant homology (e.g. probabilities lower than 1×10−6 that the homology is only due to random chance) to membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to B. fragilis genes based on sequence homology to genes cloned in other organisms.
Discriminant analysis (Klein, et al. supra) can be used to examine the ORF amino acid sequences. This algorithm uses the intrinsic information contained in the ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
Production of Fragments and Analogs of B. Fragilis Nucleic Acids and Polypeptides
Based on the discovery of the B. fragilis gene products of the invention provided in the Sequence Listing, one skilled in the art can alter the disclosed structure of B. fragilis genes, e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind B. fragilis polypeptides. Such screens are useful for the identification of inhibitors of B. fragilis.
Generation of Fragments
Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with “end-nibbling” endonucleases can thus generate DNAs which encode an array of fragments. DNAs which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
Alteration of Nucleic Acids and Polypeptides: Random Methods
Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein).
PCR Mutagenesis
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11–15). The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
Saturation Mutagenesis
Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
Degenerate Oligonucleotides
A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273–289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386–390; Roberts et al. (1992) PNAS 89:2429–2433; Devlin et al. (1990) Science 249: 404–406; Cwirla et al. (1990) PNAS 87: 6378–6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).
Alteration of Nucleic Acids and Polypeptides: Methods for Directed Mutagenesis
Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1–3.
Alanine Scanning Mutagenesis
Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081–1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
Oligonucleotide-Mediated Mutagenesis
Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least about 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765 [1978]).
Cassette Mutagenesis
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315 [1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3′ and 5′ ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.
Combinatorial Mutagenesis
Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630). In this method, the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
Other Modifications of B. Fragilis Nucleic Acids and Polypeptides
It is possible to modify the structure of an B. fragilis polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). A modified B. fragilis protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.
An B. fragilis peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages. In addition, amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.
In order to enhance stability and/or reactivity, an B. fragilis polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non-natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an B. fragilis polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of B. fragilis proteins include reduction/alkylation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton N.J. 155–194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, W H Freeman, San Francisco, Calif. (1980), U.S. Pat. No. 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch. of Allergy and Appl. Immunol., 41: 199–215).
To facilitate purification and potentially increase solubility of an B. fragilis protein or peptide, it is possible to add an amino acid fusion moiety to the peptide backbone. For example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6: 1321–1325). In addition, to facilitate isolation of peptides free of irrelevant sequences, specific endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
To potentially aid proper antigen processing of epitopes within an B. fragilis polypeptide, canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods. For example, charged amino acid pairs, such as KK or RR, can be introduced between regions within a protein or fragment during recombinant construction thereof. The resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes. In addition, such charged amino acid residues can result in an increase in the solubility of the peptide.
Primary Methods for Screening Polypeptides and Analogs
Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to B. fragilis polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
Two Hybrid Systems
Two hybrid assays such as the system described below (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring B. fragilis polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an B. fragilis protein. (The B. fragilis domain is used as the bait protein and the library of variants are expressed as prey fusion proteins.) In an analogous fashion, a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind an B. fragilis polypeptide.
Display Libraries
In one approach to screening assays, the Bacteroides peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a “panning assay”. For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370–1371; and Goward et al. (1992) TIBS 18:136–140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologs which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.
A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 10— phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages, M13, fd., and fl, are most often used in phage display libraries. Either of the phage gIII or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2-terminal end of pIII and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007–16010; Griffiths et al. (1993) EMBO J. 12:725–734; Clackson et al. (1991) Nature 352:624–628; and Barbas et al. (1992) PNAS 89:4457–4461).
A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029–3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387–392), PhoE (Agterberg, et al. (1990) Gene 88, 37–45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369–1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl. Environ. Microbiol. 55, 984–993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080–1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239–4245 and Klauser et al. (1990) EMBO J. 9, 1991–1999).
In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865–1869). This system uses a plasmid containing the LacI gene with an oligonucleotide cloning site at its 3′-end. Under the controlled induction by arabinose, a LacI-peptide fusion protein is produced. This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the LacI-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stablely associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89–1869)
This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pIII and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378–6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The LacI fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233–1251). These particular biases are not a factor in the LacI display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries of 107–109 independent clones are routinely prepared. Libraries as large as 1011 recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3–6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.
In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233–1251), a molecular DNA library encoding 1012 decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ELISA, or for binding specificity in a completion phage ELISA (Barret, et al. (1992) Anal. Biochem 204,357–364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.
Secondary Screening of Polypeptides and Analogs
The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine for one skilled in the art to obtain analogs and fragments.
Peptide Mimetics of B. Fragilis Polypeptides
The invention also provides for reduction of the protein binding domains of the subject B. fragilis polypeptides to generate mimetics, e.g. peptide or non-peptide agents. The peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an B. fragilis polypeptide binding to a naturally occurring ligand. The critical residues of a subject B. fragilis polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate B. fragilis-derived peptidomimetics which competitively or noncompetitively inhibit binding of the B. fragilis polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31,080A).
For example, scanning mutagenesis can be used to map the amino acid residues of a particular B. fragilis polypeptide involved in binding an interacting polypeptide, peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) can be generated which mimic those residues in binding to an interacting polypeptide, and which therefore can inhibit binding of an B. fragilis polypeptide to an interacting polypeptide and thereby interfere with the function of B. fragilis polypeptide. For instance, non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides. Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides. Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and et al. (1986) Biochem Biophys Res Commun 134:71).
Vaccine Formulations for B. Fragilis Nucleic Acids and Polypeptides
This invention also features vaccine compositions for protection against infection by B. fragilis or for treatment of B. fragilis infection. In one embodiment, the vaccine compositions contain one or more immunogenic components such as a surface protein from B. fragilis, or portion thereof, and a pharmaceutically acceptable carrier. Nucleic acids within the scope of the invention are exemplified by the nucleic acids of the invention contained in the Sequence Listing which encode B. fragilis surface proteins. Any nucleic acid encoding an immunogenic B. fragilis protein, or portion thereof, which is capable of expression in a cell, can be used in the present invention. These vaccines have therapeutic and prophylactic utilities.
One aspect of the invention provides a vaccine composition for protection against infection by B. fragilis which contains at least one immunogenic fragment of an B. fragilis protein and a pharmaceutically acceptable carrier. Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10–20 amino acid residues in length, and more preferably about 12–16 amino acid residues in length.
Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length B. fragilis protein. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
In one embodiment, immunogenic components are identified by the ability of the peptide to stimulate T cells. Peptides which stimulate T cells, as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen/T cell interaction, and activation of the B cell cascade, leading to the production of antibodies. A T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.
Screening immunogenic components can be accomplished using one or more of several different assays. For example, in vitro, peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture. Presentation of an immunogenic B. fragilis peptide in association with appropriate MHC molecules to T cells in conjunction with the necessary co-stimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines. For example, any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incorporated herein by reference. A kit for an assay for the production of interferon is also available from Genzyme Corporation (Cambridge, Mass.).
Alternatively, a common assay for T cell proliferation entails measuring tritiated thymidine incorporation. The proliferation of T cells can be measured in vitro by determining the amount of 3H-labeled thymidine incorporated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
Vaccine compositions of the invention containing immunogenic components (e.g., B. fragilis polypeptide or fragment thereof or nucleic acid encoding an B. fragilis polypeptide or fragment thereof) preferably include a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. For vaccines of the invention containing B. fragilis polypeptides, the polypeptide is co-administered with a suitable adjuvant.
It will be apparent to those of skill in the art that the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of antibody administered, whether the protein or DNA is administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or DNA.
Vaccine compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. (1990) Science 247: 1465–1468 and by Sedegah et al. (1994) Immunology 91: 9866–9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by B. fragilis. Cain et. al. (1993) Vaccine 11: 637–642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
The vaccine compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion; and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the B. fragilis polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli, non-B. fragilis bacterial lysates, block polymers or saponins.
Other suitable delivery methods include biodegradable microcapsules or immuno-stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue. The amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 mg to 50 mg, for example 10 mg to 35 mg. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.
Carrier systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including B. fragilis polypeptide in an insoluble form as fusion proteins. Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres. Suitable diluents are 0.2 N NaHCO3 and/or saline.
Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of B. fragilis in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by B. fragilis. The vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art. Thus, for adults a suitable dosage will be in the range of 10 mg to 10 g, preferably 10 mg to 100 mg. A suitable dosage for adults will also be in the range of 5 mg to 500 mg. Similar dosage ranges will be applicable for children. Those skilled in the art will recognize that the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors. Those skilled in the art will also recognize that appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J Urol. 150:917–921 (1993); Boedecker et al., American Gastroenterological Assoc. 999:A-222 (1993)). The number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888:A-222 (1993)).
In a preferred embodiment, a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an B. fragilis protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant.
It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing B. fragilis infection, some are useful only for treating B. fragilis infection, and some are useful for both preventing and treating B. fragilis infection. In a preferred embodiment, the vaccine composition of the invention provides protection against B. fragilis infection by stimulating humoral and/or cell-mediated immunity against B. fragilis. It should be understood that amelioration of any of the symptoms of B. fragilis infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat B. fragilis-caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
Antibodies Reactive with B. Fragilis Polypeptides
The invention also includes antibodies specifically reactive with the subject B. fragilis polypeptide. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies. A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the subject B. fragilis polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the B. fragilis polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least about 95% homologous). In yet a further preferred embodiment of the invention, the anti-B. fragilis antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80% percent homologous to a sequence of the invention contained in the Sequence Listing. By “not substantially cross react”, it is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no cross-reactivity between bacterial and mammalian antigens.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with B. fragilis polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. The antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-B. fragilis portion.
Both monoclonal and polyclonal antibodies (Ab) directed against B. fragilis polypeptides or B. fragilis polypeptide variants, and antibody fragments such as Fab′ and F(ab′)2, can be used to block the action of B. fragilis polypeptide and allow the study of the role of a particular B. fragilis polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the B. fragilis and by microinjection of anti-B. fragilis polypeptide antibodies of the present invention.
Antibodies which specifically bind B. fragilis epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of B. fragilis antigens. Anti-B. fragilis polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate B. fragilis levels in tissue or bodily fluid as part of a clinical testing procedure. Likewise, the ability to monitor B. fragilis polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of an B. fragilis polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy. Diagnostic assays using anti-B. fragilis antibodies can include, for example, immunoassays designed to aid in early diagnosis of B. fragilis infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific B. fragilis antigens.
Another application of anti-B. fragilis polypeptide antibodies of the invention is in the immunological screening of cDNA libraries constructed in expression vectors such as λgt11, λgt18–23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgt11 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject B. fragilis polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-B. fragilis polypeptide antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of B. fragilis gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
Kits Containing Nucleic Acids, Polypeptides or Antibodies of the Invention
The nucleic acid, polypeptides and antibodies of the invention can be combined with other reagents and articles to form kits. Kits for diagnostic purposes typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels. Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like. Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like. Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like. Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular purpose. Kits further can include instructions for use.
Bio Chip Technology
The nucleic acid sequence of the present invention may be used to detect B. fragilis or other species of Bacteroides acid sequence using bio chip technology. Bio chips containing arrays of nucleic acid sequence can also be used to measure expression of genes of B. fragilis or other species of Bacteroides. For example, to diagnose a patient with a B. fragilis or other Bacteroides infection, a sample from a human or animal can be used as a probe on a bio chip containing an array of nucleic acid sequence from the present invention. In addition, a sample from a disease state can be compared to a sample from a non-disease state which would help identify a gene that is up-regulated or expressed in the disease state. This would provide valuable insight as to the mechanism by which the disease manifests. Changes in gene expression can also be used to identify critical pathways involved in drug transport or metabolism, and may enable the identification of novel targets involved in virulence or host cell interactions involved in maintenance of an infection. Procedures using such techniques have been described by Brown et al., 1995, Science 270: 467–470.
Bio chips can also be used to monitor the genetic changes of potential therapeutic compounds including, deletions, insertions or mismatches. Once the therapeutic is added to the patient, changes to the genetic sequence can be evaluated for its efficacy. In addition, the nucleic acid sequence of the present invention can be used to determine essential genes in cell cycling. As described in Iyer et al., 1999 (Science, 283:83–87) genes essential in the cell cycle can be identified using bio chips. Furthermore, the present invention provides nucleic acid sequence which can be used with bio chip technology to understand regulatory networks in bacteria, measure the response to environmental signals or drugs as in drug screening, and study virulence induction. (Mons et al., 1998, Nature Biotechnology, 16: 45–48. Patents teaching this technology include U.S. Pat. Nos. 5,445,934, 5,744,305, and 5,800,992.
Drug Screening Assays Using B. Fragilis Polypeptides
By making available purified and recombinant B. fragilis polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject B. fragilis polypeptides, or of their role in intracellular signaling. Such inhibitors or potentiators may be useful as new therapeutic agents to combat B. fragilis infections in humans. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the person skilled in the art.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as “primary” screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified B. fragilis polypeptide.
Screening assays can be constructed in vitro with a purified B. fragilis polypeptide or fragment thereof, such as an B. fragilis polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive absorption, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated. A variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the B. fragilis polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live B. fragilis cells.
Overexpression Assays
Overexpression assays are based on the premise that overproduction of a protein would lead to a higher level of resistance to compounds that selectively interfere with the function of that protein. Overexpression assays may be used to identify compounds that interfere with the function of virtually any type of protein, including without limitation enzymes, receptors, DNA- or RNA-binding proteins, or any proteins that are directly or indirectly involved in regulating cell growth.
Typically, two bacterial strains are constructed. One contains a single copy of the gene of interest, and a second contains several copies of the same gene. Identification of useful inhibitory compounds of this type of assay is based on a comparison of the activity of a test compound in inhibiting growth and/or viability of the two strains. The method involves constructing a nucleic acid vector that directs high level expression of a particular target nucleic acid. The vectors are then transformed into host cells in single or multiple copies to produce strains that express low to moderate and high levels of protein encoding by the target sequence (strain A and B, respectively). Nucleic acid comprising sequences encoding the target gene can, of course, be directly integrated into the host cell.
Large numbers of compounds (or crude substances which may contain active compounds) are screened for their effect on the growth of the two strains. Agents which interfere with an unrelated target equally inhibit the growth of both strains. Agents which interfere with the function of the target at high concentration should inhibit the growth of both strains. It should be possible, however, to titrate out the inhibitory effect of the compound in the overexpressing strain. That is, if the compound is affecting the particular target that is being tested, it should be possible to inhibit the growth of strain A at a concentration of the compound that allows strain B to grow.
Alternatively, a bacterial strain is constructed that contains the gene of interest under the control of an inducible promoter. Identification of useful inhibitory agents using this type of assay is based on a comparison of the activity of a test compound in inhibiting growth and/or viability of this strain under both inducing and non-inducing conditions. The method involves constructing a nucleic acid vector that directs high-level expression of a particular target nucleic acid. The vector is then transformed into host cells that are grown under both non-inducing and inducing conditions (conditions A and B, respectively).
Large numbers of compounds (or crude substances which may contain active compounds) are screened for their effect on growth under these two conditions. Agents that interfere with the function of the target should inhibit growth under both conditions. It should be possible, however, to titrate out the inhibitory effect of the compound in the overexpressing strain. That is, if the compound is affecting the particular target that is being tested, it should be possible to inhibit growth under condition A at a concentration that allows the strain to grow under condition B.
Ligand-Binding Assays
Many of the targets according to the invention have functions that have not yet been identified. Ligand-binding assays are useful to identify inhibitor compounds that interfere with the function of a particular target, even when that function is unknown. These assays are designed to detect binding of test compounds to particular targets. The detection may involve direct measurement of binding. Alternatively, indirect indications of binding may involve stabilization of protein structure or disruption of a biological function. Non-limiting examples of useful ligand-binding assays are detailed below.
A useful method for the detection and isolation of binding proteins is the Biomolecular Interaction Assay (BIAcore) system developed by Pharmacia Biosensor and described in the manufacturer's protocol (LKB Pharmacia, Sweden). The BIAcore system uses an affinity purified anti-GST antibody to immobilize GST-fusion proteins onto a sensor chip. The sensor utilizes surface plasmon resonance which is an optical phenomenon that detects changes in refractive indices. In accordance with the practice of the invention, a protein of interest is coated onto a chip and test compounds are passed over the chip. Binding is detected by a change in the refractive index (surface plasmon resonance).
A different type of ligand-binding assay involves scintillation proximity assays (SPA, described in U.S. Pat. No. 4,568,649).
Another type of ligand binding assay, also undergoing development, is based on the fact that proteins containing mitochondrial targeting signals are imported into isolated mitochondria in vitro (Hurt et al., 1985, Embo J 4:2061–2068; Eilers and Schatz, Nature, 1986, 322:228–231). In a mitochondrial import assay, expression vectors are constructed in which nucleic acids encoding particular target proteins are inserted downstream of sequences encoding mitochondrial import signals. The chimeric proteins are synthesized and tested for their ability to be imported into isolated mitochondria in the absence and presence of test compounds. A test compound that binds to the target protein should inhibit its uptake into isolated mitochondria in vitro.
Another ligand-binding assay is the yeast two-hybrid system (Fields and Song, 1989, Nature 340:245–246). The yeast two-hybrid system takes advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. The GAL4 protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. This protein consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. The native GAL4 protein, containing both domains, is a potent activator of transcription when yeast are grown on galactose media. The N-terminal domain binds to DNA in a sequence-specific manner but is unable to activate transcription. The C-terminal domain contains the activating regions but cannot activate transcription because it fails to be localized to UASG. In the two-hybrid system, a system of two hybrid proteins containing parts of GAL4: (1) a GAL4 DNA-binding domain fused to a protein ‘X’ and (2) a GAL4 activation region fused to a protein ‘Y’. If X and Y can form a protein—protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. Creation of two hybrid proteins, each containing one of the interacting proteins X and Y, allows the activation region of UASG to be brought to its normal site of action.
The binding assay described in Fodor et al., 1991, Science 251:767–773, which involves testing the binding affinity of test compounds for a plurality of defined polymers synthesized on a solid substrate, may also be useful.
Compounds which bind to the polypeptides of the invention are potentially useful as antibacterial agents for use in therapeutic compositions.
Pharmaceutical formulations suitable for antibacterial therapy comprise the antibacterial agent in conjunction with one or more biologically acceptable carriers. Suitable biologically acceptable carriers include, but are not limited to, phosphate-buffered saline, saline, deionized water, or the like. Preferred biologically acceptable carriers are physiologically or pharmaceutically acceptable carriers.
The antibacterial compositions include an antibacterial effective amount of active agent. Antibacterial effective amounts are those quantities of the antibacterial agents of the present invention that afford prophylactic protection against bacterial infections or which result in amelioration or cure of an existing bacterial infection. This antibacterial effective amount will depend upon the agent, the location and nature of the infection, and the particular host. The amount can be determined by experimentation known in the art, such as by establishing a matrix of dosages and frequencies and comparing a group of experimental units or subjects to each point in the matrix.
The antibacterial active agents or compositions can be formed into dosage unit forms, such as for example, creams, ointments, lotions, powders, liquids, tablets, capsules, suppositories, sprays, aerosols or the like. If the antibacterial composition is formulated into a dosage unit form, the dosage unit form may contain an antibacterial effective amount of active agent. Alternatively, the dosage unit form may include less than such an amount if multiple dosage unit forms or multiple dosages are to be used to administer a total dosage of the active agent. Dosage unit forms can include, in addition, one or more excipient(s), diluent(s), disintegrant(s), lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), absorption enhancer(s), stabilizer(s), bactericide(s), or the like.
For general information concerning formulations, see, e.g., Gilman et al. (eds.), 1990, Goodman and Gilman's. The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack Publishing Co., Easton, Pa.; Avis et al. (eds.), 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, N.Y.; Lieberman et al (eds.), 1990, Pharmaceutical Dosage Forms. Disperse Systems, Dekker, N.Y.
The antibacterial agents and compositions of the present invention are useful for preventing or treating B. fragilis infections. Infection prevention methods incorporate a prophylactically effective amount of an antibacterial agent or composition. A prophylactically effective amount is an amount effective to prevent B. fragilis infection and will depend upon the specific bacterial strain, the agent, and the host. These amounts can be determined experimentally by methods known in the art and as described above.
B. fragilis infection treatment methods incorporate a therapeutically effective amount of an antibacterial agent or composition. A therapeutically effective amount is an amount sufficient to ameliorate or eliminate the infection. The prophylactically and/or therapeutically effective amounts can be administered in one administration or over repeated administrations. Therapeutic administration can be followed by prophylactic administration, once the initial bacterial infection has been resolved.
The antibacterial agents and compositions can be administered topically or systemically. Topical application is typically achieved by administration of creams, ointments, lotions, or sprays as described above. Systemic administration includes both oral and parental routes. Parental routes include, without limitation, subcutaneous, intramuscular, intraperitoneal, intravenous, transdermal, inhalation and intranasal administration.
Exemplification
Cloning and Sequencing B. Fragilis Genomic Sequence
This invention provides nucleotide sequences of the genome of B. fragilis which thus comprises a DNA sequence library of B. fragilis genomic DNA. The detailed description that follows provides nucleotide sequences of B. fragilis, and also describes how the sequences were obtained and how ORFs (Open Reading Frames) and protein-coding sequences can be identified. Also described are methods of using the disclosed B. fragilis sequences in methods including diagnostic and therapeutic applications. Furthermore, the library can be used as a database for identification and comparison of medically important sequences in this and other strains of B. fragilis as well as other species of Bacteroides.
Chromosomal DNA from strain 14062 of B. fragilis was isolated after Zymolyase digestion, sodium dodecyl sulfate lysis, potassium acetate precipitation, phenol:chloroform extraction and ethanol precipitation (Soll, D. R., T. Srikantha and S. R. Lockhart: Characterizing Developmentally Regulated Genes in B. fragilis. In Microbial Genome Methods. K. W. Adolph, editor. CRC Press. New York. p 17–37.). Genomic B. fragilis DNA was hydrodynamically sheared in an HPLC and then separated on a standard 1% agarose gel. Fractions corresponding to 2500–3000 bp in length were excised from the gel and purifed by the GeneClean procedure (Bio101, Inc.).
The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The healed DNA was then ligated to unique BstXI-linker adapters (5′-GTCTTCACCACGGGG-3′ and 5′-GTGGTGAAGAC-3′ in 100–1000 fold molar excess). These linkers are complimentary to the BstXI-cut pGTC vector, while the overhang is not self-complimentary. Therefore, the linkers will not concatermerize nor will the cut-vector religate itself easily. The linker-adapted inserts were separated from the unincorporated linkers on a 1% agarose gel and purified using GeneClean. The linker-adapted inserts were then ligated to BstXI-cut vector to construct a “shotgun” sublclone libraries.
Only major modifications to the protocols are highlighted. Briefly, the library was then transformed into DH5α competent cells (Gibco/BRL, DH5á transformation protocol). It was assessed by plating onto antibiotic plates containing ampicillin and IPTG/Xgal. The plates were incubated overnight at 37° C. Transformants were then used for plating of clones and picking for sequencing. The cultures were grown overnight at 37° C. DNA was purified using a silica bead DNA preparation (Engelstein, 1996) method. In this manner, 25 μg of DNA was obtained per clone.
These purified DNA samples were then sequenced using primarily ABI dye-terminator chemistry. All subsequent steps were based on sequencing by ABI377 automated DNA sequencing methods. The ABI dye terminator sequence reads were run on ABI377 machines and the data was transferred to UNIX machines following lane tracking of the gels. Base calls and quality scores were determined using the program PHRED (Ewing et al., 1998, Genome Res. 8: 175–185; Ewing and Green, 1998, Genome Res. 8: 685–734). Reads were assembled using PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, January 1996, p. 157) with default program parameters and quality scores. The initial assembly was done at 7.8 fold coverage and yielded 223 contigs.
Finishing can follow the initial assembly. Missing mates (sequences from clones that only gave reads from one end of the Bacteroides DNA inserted in the plasmid) can be identified and sequenced with ABI technology to allow the identification of additional overlapping contigs.
End-sequencing of randomly picked genomic lambda was also performed. Sequencing on a both sides was done for all lambda sequences. The lambda library backbone helped to verify the integrity of the assembly and allowed closure of some of the physical gaps. Primers for walking off the ends of contigs would be selected using pick_primer (a GTC program) near the ends of the clones to facilitate gap closure. These walks can be sequenced using the selected clones and primers. These data are then reassembled with PHRAP. Additional sequencing using PCR-generated templates and screened and/or unscreened lambda templates can be done in addition.
To identify B. fragilis polypeptides the complete genomic sequence of B. fragilis were analyzed essentially as follows: First, all possible stop-to-stop open reading frames (ORFS) greater than 180 nucleotides in all six reading frames were translated into amino acid sequences. Second, the identified ORFs were analyzed for homology to known (archeabacter, prokaryotic and eukaryotic) protein sequences. Third, the coding potential of non-homologous sequences were evaluated with the program GENEMARK™ (Borodovsky and Mclninch, 1993, Comp. Chem. 17:123).
Identification, Cloning and Expression of B. Fragilis Nucleic Acids
Expression and purification of the B. fragilis polypeptides of the invention can be performed essentially as outlined below.
To facilitate the cloning, expression and purification of membrane and secreted proteins from B. fragilis, a gene expression system, such as the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, is selected. Also, a DNA sequence encoding a peptide tag, the His-Tag, is fused to the 3′ end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3′ end is selected for fusion in order to avoid alteration of any 5′ terminal signal sequence.
PCR Amplification and Cloning of Nucleic Acids Containing ORF'S Encoding Enzymes
Nucleic acids chosen (for example, from the nucleic acids set forth in SEQ ID NO: 1–SEQ ID NO: 5222 for cloning from the 14062 strain of B. fragilis are prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers specific for the 5/ and 3/ ends of open reading frames (ORFs) are designed and purchased from GibcoBRL Life Technologies (Gaithersburg, Md., USA). All forward primers (specific for the 5/ end of the sequence) are designed to include an NcoI cloning site at the extreme 5/ terminus. These primers are designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native B. fragilis DNA sequence. All reverse primers (specific for the 3/ end of any B. fragilis ORF) include a EcoRI site at the extreme 5/ terminus to permit cloning of each B. fragilis sequence into the reading frame of the pET-28b. The pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids including six histidine residues (at the extreme C-terminus), which comprise the His-Tag.
Genomic DNA prepared from the 14062 strain of B. fragilis is used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing an B. fragilis ORF, genomic DNA (50 nanograms) is introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined B. fragilis ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, N.J., USA) in a final volume of 100 microliters.
Upon completion of thermal cycling reactions, each sample of amplified DNA is washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, Md., USA). All amplified DNA samples are subjected to digestion with the restriction endonucleases, e.g., NcoI and EcoRI (New England BioLabs, Beverly, Mass., USA)(Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples are then subjected to electrophoresis on 1.0% NuSeive (FMC BioProducts, Rockland, Me. USA) agarose gels. DNA is visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel is purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, Calif., USA).
Cloning of B. Fragilis Nucleic Acids into an Expression Vector
The pET-28b vector is prepared for cloning by digestion with restriction endonucleases, e.g., NcoI and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). The pET-28a vector, which encodes a His-Tag that can be fused to the 5/ end of an inserted gene, is prepared by digestion with appropriate restriction endonucleases.
Following digestion, DNA inserts are cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector. Products of the ligation reaction are then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
Transformation of Competent Bacteria with Recombinant Plasmids
Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), are transformed with recombinant pET expression plasmids carrying the cloned B. fragilis sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation reaction is mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples are incubated in 0.45 milliliters SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20, mM glucose) at 37° C. with shaking for 1 hour. Samples are then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 are then picked and analyzed to evaluate cloned inserts as described below.
Identification of Recombinant Expression Vectors with B. Fragilis Nucleic Acids
Individual BL21 clones transformed with recombinant pET-28b B. fragilis ORFs are analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each B. fragilis sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verifies the integration of the B. fragilis sequences in the expression vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
Isolation and Preparation of Nucleic Acids from Transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned B. fragilis ORFs are picked and incubated in 5 mls of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA is isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, Calif., USA).
Expression of Recombinant B. Fragilis Sequences in E. COLI
The pET vector can be propagated in any E. coli K-12 strain e.g. HMS174, HB101, JM109, DH5, etc. for the purpose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lacI gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its gene of interest. Strains used include: BL21(DE3) (Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Meth. Enzymol. 185, 60–89).
To express recombinant B. fragilis sequences, 50 nanograms of plasmid DNA isolated as described above is used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). The lacZ gene (beta-galactosidase) is expressed in the pET-System as described for the B. fragilis recombinant constructions. Transformed cells are cultured in SOC medium for 1 hour, and the culture is then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies are pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O. D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the B. fragilis recombinant DNA constructions.
After induction of gene expression with IPTG, bacteria are pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500×g for 15 minutes at 4° C. Pellets are resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells are then centrifuged at 2000×g for 20 min at 4° C. Wet pellets are weighed and frozen at −80° C. until ready for protein purification.
A variety of methodologies known in the art can be utilized to purify the isolated proteins. (Current Protocols in Protein Science, John Wiley and Sons, Inc., J. E. Coligan et al., eds., 1995). For example, the frozen cells may be thawed, resupended in buffer and ruptured by several passages through a small volume microfluidizer (Model M-110S, Microfluidics International Corporation, Newton, Mass.). The resultant homogenate may be centrifuged to yield a clear supernatant (crude extract) and following filtration the crude extract may be fractionated over columns. Fractions may be monitored by absorbance at OD280 nm. and peak fractions may analyzed by SDS-PAGE
The concentrations of purified protein preparations may be quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S. J. 1986 Eur. J. Biochem. 157, 169–180). Protein concentrations are also measured by the method of Bradford, M. M. (1976) Anal. Biochem. 72, 248–254, and Lowry, O. H., Rosebrough, N., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, pages 265–275, using bovine serum albumin as a standard.
SDS-polyacrylamide gels of various concentrations may be purchased from BioRad (Hercules, Calif., USA), and stained with Coomassie blue. Molecular weight markers may include rabbit skeletal muscle myosin (200 kDa), E. coli (-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. The specific embodiments described herein are offered by way of example only, and the invention is to limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.
Aspergillus oryzae gtaA gene for glutaminase A, complete cds.
Bacillus circulans aman6 gene for alpha-1,6-mannanase, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigen PG87 gene,
Aspergillus oryzae gtaA gene for glutaminase A, complete cds.
Streptomyces coelicolor cosmid 4A7.
Oryza sativa genomic DNA, chromosome 1, clone:P0011D01.
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and
Bacillus subtilis DNA for endo-arabinase, complete cds.
Bacillus circulans aman6 gene for alpha-1,6-mannanase, complete cds.
Lactococcus lactis carB and gpo genes.
Streptococcus pneumoniae rr09 and hk09 genes; two component system09.
Streptomyces coelicolor cosmid F41.
Burkholderia caryophylii PG2982 phosphonate monoester hydrolase (pehA) gene,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Escherichia coli (strain EcoAU9307) infB gene encoding translational initiation factor IF2.
Pseudomonas putida todX, todF, todC1, todC2, todB, todA, todD, todE, todG,
Streptomyces coelicolor cosmid 4A7.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus,
Staphylococcus aureus type B capsule genes, cap8A, cap8B, cap8C, cap8D, cap8E, cap8F,
Pseudomonas aeruginosa Wzz (Rol) (wzz (rol)) gene, partial cds, WbpA (wbpB),
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Staphylococcus aureus capsule gene cluster Cap5A through Cap5P genes, complete cds.
Tachypleus tridentatus mRNA for tachylectin-3, complete cds.
E. coli npl gene or N-acetylneuraminate lyase subunit (EC4.1.3.3).
Leptospira interrogans frb locus, complete sequence.
Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Plesiomonas shigelloides gene for ORF1P, ORF2P, ORF3P, ORF4P, ORF5P, ORF6P,
Pseudomonas aeruginosa Wzz (Rol) (wzz (rol)) gene, partial cds, WbpA (wbpB),
Porphyromonas gingivalis PorR and PorS genes, complete cds.
Bordetella bronchiseptica cosmid BbLPS1.
Bordetella bronchiseptica cosmid BbLPS1.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization
Arabidopsas thaliana chromosome II BAC T20K9 genomic sequence, complete sequence.
E. coli genomic DNA, Kohara clone #347 (44.2–44.5 min.).
Lactococcus lactis aspartate aminotransferase (aspC) gene, complete cds.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Bacillus subtilis (clone pED4) comG-(1,2,3,4,5,6, and 7) proteins incomG operon, complete cds.
Bacillus stearothermophilus lig gene.
Anabaena PCC7120 ORFR1, ORFR2, ORFR3, ORFR4, and ORFR5 genes, complete sequences.
Mycobacterium bovis 63 kDa protein, 47 kDa protein and clpB gene, complete cds.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Bacillus halodurans gene for YFIL, YFIM, YFIN, YHDE, HMP and ARGE, complete cds.
Bacillus stearothermophilus endo-beta-1,4-xylanase (xynA) gene, complete cds.
Pseudomonas aeruginosa gene for OprM, complete cds.
Yersinia pestis 102 kbases unstable region: from 1 to 119443.
Boophilus microplus NADH dehydrogenase subunit 4 (ND4) gene, partial cds;
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Porphyromonas gingivalis high temperature protein HtpG (htpG) gene, complete cds.
Synechococcus elongatus petB gene, petD gene and ORF1.
Streptomyces tendae afp gene and ORF2 (partial), strain Tue901/8c.
Salmonella typhimurium transcriptional
Salmonella typhimurium fragment STMF1.
Legionella pneumophila HelC (helC) gene, complete cds.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Bacillus subtilis (p)ppGpp synthetase (relA) and adeninephosphoribosyltransferase
Porphyromonas gingivalis dnaK operon genes, complete cds.
Bacillus subtilis PhoH (phoH) gene, partial cds, diacylglycerolkinase (dgk)
Homo sapiens DNA helicase homolog (PIF1) mRNA, partial cds.
Streptomyces thermoviolaceus nagB gene for Beta-N-Acetylglucosaminidase, complete cds.
E. coli; the region from 81.5 to 84.5 minutes.
Lactobacillus acidophilus uracil phosphoribosyltransferase (upp) gene,
Anabaena variabilis rbpD gene for RNA-binding protein, complete cds.
C. glutamicum panB, panC & xylB genes.
B. burgdorferi abp gene.
A. haloplanktis amy gene for alpha-amylase 1,4-alpha-D-glucanglucanohydrolase.
Shewanella sp. PT99 single stranded DNA-binding protein (ssb) gene, complete cds.
Lactobacillus reuteri autoaggregation-mediating protein (aggH) gene, complete cds.
S. cerevisiae chromosome IV reading frame ORF YDL057w.
Yersinia enterocolitica WA 314 right arm of the high-pathogenicityisland.
Synechococcus 6301 gene for ss-DNA binding protein 12RNP2, complete cds.
Dermatophagoides farinae mRNA for Mag44, partial cds.
Emericella nidulans gene for branching enzyme, complete cds.
Schizosaccharomyces pombe gene for ATP synthase gamma chain, partial cds, clone:TA25.
Desulfotomaculum thermocisternum
Ruminococcus flavefaciens xylan utilization operon.
Thermoanaerobacter ethanolicus putative TrkG gene, partial cds, and putative
Leuconostoc mesenteroides plasmid pHY30 MesG (mesG) gene, partial cds; and
Burkholderia pseudomallei putative UDP-glucose dehydrogenase (udg), putative ADP-heptose
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Haemophilus ducreyi oxaloacetate decarboxylase gamma chain (oadG) gene,
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Escherichia coli csg cluster, partial sequence.
Streptococcus pneumoniae macrolide-efflux determinant (mefE) gene, complete cds.
Neisseria gonorrhoeae outer membrane protein (omp85) gene, complete cds.
Tetragenococcus halophilus rbsC, rbsB, xylR, xylA, xylB and xylE genes, partial and
Pseudomonas aeruginosa phage phi CTX, complete genome sequence.
Sinorhizobium meliloti protein kinase homolog Thi (thi) and ExoP-like protein genes,
Vibrio cholerae genes for O-antigen synthesis, strain MO45, complete cds.
Moorella thermoacetica rubrerythrin gene, complete cds.
Streptomyces coelicolor cosmid F76.
Escherichia coli strain CFT073 chromosomal hemolysin D (hlyD) gene, partial
Pseudomonas aeruginosa iron-uptake factor (piuC), hydroxamate-type
Pseudomonas aeruginosa atsR, atsB, atsC & atsA genes.
Zymomonas mobilis fosmid clone 43D2, complete sequence.
ERYTHROMYCIN ESTERASE TYPE II
Arabidopsis thaliana DNA chromosome 4, ESSA I AP2 contig fragment No. 2
Streptococcus pneumoniae type 19C Cps19CR (cps19CR) gene, partial cds;
Arabidopsis thaliana BAC T19B17 from chromosome IV, near 19.3 cM, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis DNA for superoxide dismutase, complete cds.
Yersinia pseudotuberculosos alpha-D-glucose−1-phosphatecytidylyltransferase (ascA) gene,
Salmonella typhimurium ThiH (thiH) gene, complete cds.
Leptospira interrogans rfb locus, complete sequence.
Yersinia pestis strain EV76 hemH gene (partial) and O-antigen gene cluster
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Porphyromonas gingivalis strain W50 immunoreactive 50kD antigen PG53 gene,
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Enterococcus faecalis strain OG1RF polysaccharide biosynthetic gene cluster, partial sequence.
Nicotiana tabacum chaperone GrpE type 2 (GrpE2) mRNA, nuclear gene coding
S. cerevisiae 10.6 kbp fragment from chromosome XV.
Escherichia coli RhsE gentic element; defective RhsE core protein, complete
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Synechococus PCC7942 moeB gene.
Vibrio anguillarum rfb region, partial sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Serratia marcescens putative glycosyltransferase, putative glycosyltransferase, putative
Flavobacterium johnsoniae gliding motility protein (gldA) gene, complete cds; and
Salmonella typhimurium fragment STMF1.
Bacillus megaterium 16kb genomic sequence, cobalamin operon.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Flavobacterium johnsoniae gliding motility protein (gldA) gene, complete cds; and
Brachydanio rerio growth-associated protein, complete cds.
Leptospira interrogans rfb locus, complete sequence.
Proteus mirabilis regulator of swarming behavior precursor (rsbA) and RcsB
Bacillus megaterium 16kb genomic sequence, cobalamin operon.
Bacteroides fragilis batI operon, complete sequence.
Bacteroides fragilis batI operon, complete sequence.
Bacteroides fragilis batI operon, complete sequence.
Bacteroides fragilis batI operon, complete sequence.
Bacteroides fragilis gyrA gene for DNA gyrase A subunit, complete cds.
Bacteroides fragilis batI operon, complete sequence.
Bacteroides fragilis batI operon, complete sequence.
Bombus pascuorum cytochrome b (cytb) gene, mitochondrial gene encoding mitochondrial
Porphyromonas gingivalis strain W50 immunoreactive 36 kDa antigen PG14 gene,
Thermoplasma acidophilum GTP-binding protein and tricorn protease (TRI) genes,
Pseudomonas aeruginosa two component sensor (lemA) gene, partial cds.
Salmonella typhimurium alpha-ribazole-5′-phosphate phospatase CobC (cobC)
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Nostoc punctiforme histidine kinase (hepK) gene, complete cds.
E. coli chromosomal region from 89.2 to 92.8 minutes.
Plasmodium falciparum MAL3Ps, complete sequence.
Synechocystis sp. (clone pSYN411) glycinamide ribonucleotide transformylase (purT), Orf134
Helicobacter pylori hypothetical protein (HP0395), hypothetical protein (HP0394),
Buchnera aphidicola genomic fragment containing (chaperone Hsp60) groEL, DNA
Psuedomonas putida CumA precursor (cumA) and CumB (cumB) genes, complete cds; and
Bordetella pertussis lep operon, complete sequence.
Staphylococcus aureus strain ISP3 ferric uptake regulator homolog (furB) gene, complete cds.
Cytophaga sp. raw starch digesting amylase precursor, gene, complete cds.
Arabidopsis thaliana chromosome I BAC F28O16 genomic sequence, complete sequence.
Cellulomonas fimi Man26A (man26A) gene, complete cds.
E. coli genomic DNA, Kohara clone #280 (33.7–34.1 min.).
Haemophilus influenzae Rd section 60 of 163 of the complete genome.
Methanosarcina barkeri dimethylamine corrinoid protein MtbC
Cellulomonas fimi Man26A (man26A) gene, complete cds.
Pyrococcus furiosus beta-glucosidase (celB) gene, complete cds; adh-lam
Streptomyces sp. alpha-1,3/4-fucosidase precursor gene, complete cds.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Methanosarcina barkeri methylcobamide:CoM methyltransferase isozyme A (mtbA),
E. coli DNA sequence of nuo operon.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Thauera aromatica utilizing regulatory protein tutC (tutC), utilizing regulatory protein
Methylbacterium sp. CM4, cobD, metF, cmuB, omuC, partial cobC and cobQ,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and
T. brockii cglF, cglG, xglS and cglT genes.
Caulobacter crescentus GTP-binding protein (cgtA) gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Vibrio vulnificus nucleotide sugar epimerase gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Pseudomonas fluorescens activator protein (mtlR) gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Malonomonas rubra putative IS-element gene, partial cds, and malonate
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Streptococcus mutans YtqB (ytqB) gene, partial cds; ABC transporter (abcX),
Porphyromonas gingivalis strain W50 immunogenic 75 kDa protein PG4 gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Arabidopsis thaliana gr I gene, exons 1-3.
Pasteurella haemolytica methyltransferase (mod) and restriction endonuclease (res) genes,
Porphyromonas gingivalis strain W50 immunoreactive 47 kD antigen PG120 gene,
Synechococcus PCC7942 phoV gene for alkaline phosphatase.
B. cereus DNA for ORF1, ORF2 and ORF3 (2402 bp).
Burkholderia pseudomallei putative UDP-glucose dehydrogenase (udg), putative ADP-heptose
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Pseudomonas aeruginosa strain IATS O6 RpsA (rpsA) gene, partial cds;
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanse, and
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Acinetobacter sp. ADP1 alkR & alkM genes, ORF1 & ORF4.
Pseudomonas putida todX, todF, todC1, todC2, todB, todA, todD, todE, todG,
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Escherichia coli GalF (galF) gene, partial cds; O-antigen repeat unit
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Alicyclobacillus acidocaldarius maltose/maltodextrine transport gene region (malEFGR genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanse, and mannanase genes,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Aeromonas hydrophila FK506 binding protein (fkpA) gene, complete cds in 3.9 kb fragment.
Homo sapiens mRNA for KIAA0879 protein, complete cds.
A. franciscana Scr gene (homologue of Drosophila Sex combs reduced).
Arabidopsis thaliana hypothetical protein, clone EMG9a29.
Vibrio cholerae glutamyl tRNA synthetase (gltX) gene, partial cds; putative
Bacteroides thetaiotao micron 30S ribosomal protein S16-like protein, fucose
Ateline herpes virus 3 complete genome.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Human fibronectin gene type III homology unit corresponding to the cell-binding domain,
Pseudomonas fluorescens PpsA (ppsA) gene, partial cds; EstX (estX), MenG
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
E. coli genomic DNA, Kohara clone #303 (34.3–34.6 min.).
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Homo sapiens cell cycle progression restoration 8 protein (CPR8) mRNA, complete cds.
Methanobacterium thermoautotrophicum ehbA, B, C, D, E, F, G, H, I, J, K, L,
Bacillus subtilis (p) ppGpp synthetase (relA) and adeninephosphoribosyltransferase (apt) genes,
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Pseudomonas fluorescens activator protein (mtlR) gene, complete cds.
Streptococcus pneumoniae LicD1 (licD1) and LicD2 (licD2) genes, complete cds; and
Amycolatopsis orientalis cosmid pCZA382.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Pseudomonas aeruginosa phage phi CTX, complete genome sequence.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Cyanidioschyzon merolae gene or FtsH2, complete cds.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Yersinia pestsis 102 kbases unstable region: from 1 to 119443.
Streptococcus agalactiae DNA, cps (capsular polysaccharide) genes, partial and complete cds.
Bacillus halodurans C-125 mamX, yjdA, ykoK and yvfK genes, partial and complete cds.
Amycolatopsis orientalis cosmid PCZA361.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence; and
Streptococcus pneumoniae D-glutamic acid adding enzyme MurD (murD),
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Gallus gallus slow myosin heavy chain 2 (SM2) mRNA, partial cds.
Zymomonas mobilis strain ZM4 fosmid clone 42B3, complete sequence.
Yersinia pestis 102 kbases unstable region: from 1 to 119443.
Proteus mirabilis NrpS (nrpS) gene, partial cds, NrpU (nrpU), NrpT (nrpT),
Porphyromonas gingivalis DNA for 60 kDa protein, complete cds.
Aulonocara hansbaenschi MHC class II alpha chain MHC-Auha-DAA1 mRNA
Pseudomonas sp. pobA, pobR, pcaQ, pcaH and pcaG genes.
L. ivanovii 23S rRNA, 5S rRNA, tRNA-Asn, tRNA-Thr, ORF Z, in1D, anc in1C genes.
Porphyromonas gingivalis DNA for 60 kDa protein, complete cds.
Staphylococcus epidermidis lipase precursor (geh1) gene, complete cds.
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Bacteroides fragilis nanH gene for neuraminidase, complete cds.
Flavobacterium johnsoniae LeuS (leuS) gene, partial cds; and Fjo12 (fjo12),
Trypanosoma cruzi ATPase subunit 6 mRNA, complete cds.
Flavobacterium johnsoniae LeuS (leuS) gene, partial cds; and Fjo12 (fjo12),
Sinorhizobium meliloti bet operon, complete sequence.
Streptomyces coelicolor cosmid F81.
Flavobacterium johnsoniae LeuS (leuS) gene, partial cds; and Fjo12 (fjo12),
Haemophilus influenzae strain Rd KW20 HI0454 gene, partial cds.
Streptococcus pneumoniae rr09 and hk09 genes; two component system 09.
Mus musculus WW domain binding protein 5 mRNA, partial cds.
Listeria monocytogenes transcription-repair coupling factor (mfdL), low
Clostridium perfringens hyp27, bacH, ptp, cpd genes for hypothetical protein, bacterial
Streptomyces coelicolor cosmid 6D7.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane sensor
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
S. cerevisiae chromosome XIII cosmid 9745.
Porphyromonas gingivalis gene or DDP IV, complete cds.
Streptomyces coelicolor cosmid F91.
Agrobacterium radiobacter putative dihydrolipoamideS-acetyltransferase
Pseudoalteromonas sp. S9 putative glucosyl hydrolase precursor and adaptive
Clostridium pasteurianum 1,3-propanediol dehydrogenase (dhaT) gene, complete cds.
Bacteroides fragilis transposon Tn4555 TnpA (tnpA), integrase (int), TnpC
Bacteroides uniformis insertion element NBU1 fragment, integrase IntN1 gene,
Wolinella succinogenes aspA, dcuA genes and partial ansA gene.
Pseudomonas aeruginosa AlgR-cognate sensor AlgZ (algZ) gene, complete cds.
Bacillus subtilis rrnB-dnaB genomic region.
E. coli aspA gene for aspartase (L-aspartate ammonia-lyase_ (EC4.3.1.1).
Escherichia coli K30 capsule biosynthesis cluster, partial sequence.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization
Porphyromonas gingivalis orf1, orf2 and orf3 genes, complete cds.
Streptomyces sp. alpha-1,¾-fucosidase precursor gene, complete cds.
Lactobacillus crispatus silent surface layer protein (cbsB) gene, partial cds.
Arabidopsis thaliana chromosome I BAC T23K23 genomic sequence, complete sequence.
Prevotella albensis ftsQ (partial), ftsA and ftsZ genes and ORF-fts (partial).
Porphyromonas gingivalis genes for FtsQ, FtsA, FtsZ, comlete cds.
Bacteroides fragilis gyrB gene for DNA gyrase B subunit, complete cds.
Saccharomyces cerevisiae replicative mitochondrial DNA polymerasecatalytic
Enterococcus faecalis strain A24836 cell wall/cell division gene cluster,
Prevotella albensis ftsQ (partial), ftsA and ftsZ genes and ORF-fts (partial).
Prevotella melaninogenica hemolysin A (phyA) gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Mus musculus P4 (21)n mRNA, partial cds.
Rattus norvegicus RING finger protein mRNA, complete cds.
Haemophilus influenzae opacity associated proteins OapA and OapB (oapA and oapB) genes,
Escherichia coli plasmid pB171 DNA, complete sequence.
Clostridium sticklandii proC gene and 5′ flanking region.
Bacillus stearothermophilus argR gene and partial recN gene.
Heamophilus paragallinarum IS-like putative transposase gene, complete cds.
Bacteroides fragilis mobilization protein B (mobB) gene, complete cds.
Pseudomonas aeruginosa pigACDE operon, complete sequence; hypothetical PigB (pigB)
Bacillus thuringiensis plasmid pTX14-1, MOB, REP, and collagen-like protein genes,
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Porphyromonas gangavalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Bacteroides fragilis mobilization protein MbpA (mbpA), MbpB (mbpB) and MbpC (mbpC)
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Streptomyces coelicolor cosmid F1.
Sphingomonas aromaticivorans plasmid pNL1, complete plasmid sequence.
Porphyromonas gingivalis strain W50 immunogenic 23 KDa lipoprotein PG3 gene,
Vibrio cholerae glutamyl tRNA synthetase (gltX) gene, partial cds; putative
Bacteroides fragilis mobilization protein A (mobA) gene, complete cds.
Listeria monocytogenes LemA (lemA) gene, complete cds, and LemB (lemB) gene, partial cds.
Myxococcus xanthus response regulator FrzZ (frzZ) gene, partial cds; alanine
Pseudomonas sp. KC hypothetical proteins, methallothionein-like protein, MoeB-like protein,
Hydra vulgaris HT2 mRNA for collagen, partial cds.
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Mus musculus glucosidase II beta-subunit gene, alternatively spliced products, partial cds.
Bacteroides fragilis levanase (scrL) gene, complete cds.
C. perfringens gene encoding N-acetylneuraminate lyase and two partial open reading frames.
Porphyromonas gingivalis strain W50 immunoreactive 52 kD antigen PG41 gene,
Porphyromonas gingivalis DNA for 60 kDa protein, complete cds.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Porphyromonas gingivalis strain W50 immunoreactive 51 kD antigen PG52 gene,
Mycobacterium smegmatis garA-containing gene cluster, partial sequence.
Salmonella typhimurium cytochrome d oxidase subunit I (cydA) and cytochrome
Toxoplasma gondii chloroplast, complete genome.
Actinobacillus pleuropneumoniae transcriptional regulator (apuR) gene,
Streptomyces coelicolor cosmid F81.
Agrobacterium tumefaciens plasmid pTi-SAKURA, complete sequence.
P. falciparum gene for STARP antigen.
P. gingivalis rnhB & pgaA genes & orfs 150, 197, 202 & 199.
Bacillus megaterium 2,3-bisphosphoglycerate-independentphosphoglycerate mutase (pgm)
Streptomyces coelicolor cosmid F12.
Plasmodium falciparum MAL3P7, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Porphyromonas gingivalis strain W50 immunoreactive 43 kD antigen PG32 gene,
Streptococcus suis strain 6555 Cps1E (cps1E) gene, partial cds; Cps2F
Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis
Pseudomonas aeruginosa phage phi CTX, complete genome sequence.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence;
Manduca sexta lacunin mRNA, complete cds.
Propionigenium modestum mmdD, mmdC, mmdB genes and partial mmdA gene.
Saccharomyces cerevisiae putative transmembrane protein (PTM1) gene, complete cds.
Clostridium perfringens virR gene for positive regulator for virulence factors, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Clostridium perfringens hyp27, bacH, ptp, cpd genes for hypothetical
Campylobacter lari GlyA (glyA) gene, partial cds.
Streptococcus pneumoniae type 8 capsular gene cluster.
Streptococcus pneumoniae type 33F DNA, capsular gene cluster.
Bordetella pertussis partial gene for putative thioesterase, tRNA-Gly, murB,
Salmonella typhimurium YjgF (yjgF) gene, complete cds; and unknown gene.
S. pneumoniae cps14 locus.
Vibrio cholerae genes for o-antigen synthesis, strain O22, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Legionella pneumophila EnhA (enhA), EnhB (enhB), and enhanced entry protein
Agrobacterium tumefaciens plasmid pTi-SAKURA, complete sequence.
Homo sapiens ES/130 mRNA, complete cds.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Anabaena PCC7120 ORFR1, ORFR2, ORFR3, ORFR4, and ORFR5 genes, complete sequences.
Streptococcus pneumoniae type 33F DNA, capsular gene cluster.
Bacteroides thetaiotaomicron rteA and rtaB genes involved inproduction of plasmid-like
Homo sapiens actin binding protein MAYVEN mRNA, complete cds.
Bacteroides fragilis (bctA) gene, complete cds.
Arabidopsis thaliana chromosome II BAC T26I20 genomic sequence, complete sequence
Leptospira interrogans rfb locus, complete sequence.
Bacteroides fragilis mobilization protein C (mobC) gene, comlete cds.
Streptomyces coelicolor cosmid E46.
Mus musculus glucosidase II beta-subunit gene, alternatively spliced products, partial cds.
P. falciparum gene for 41 kd antigen, clone 41-7.
A. brasilense acpF gene.
Arabictopsis thaliana mRNA tor phosphoribosylglycinamideformyltransferase encoded
Phytophthora infestans mitochondrion, complete genome.
Pseudomonas aeruginosa stress tactor A (psfA), ECF sigma factor(fiuI), transmembrane
Staphylococcus aureus O-acetyl transferase (cap5H) gene, complete cds.
Pyrococcus furiosus gene tor DNA polymerase II subunit 1, DNA polymerase II subunit 2,
Staphylococcus aureus genes for orf1, orfX, orf2, orf3, partial and complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Phytomonas serpens 12S Large subunit ribosomal RNA and 9S small subunit
Legionella pneumophilia serogroup 1 lipopolysaccharide biosynthesisgene cluster.
Leptospira interrogans rfb locus, complete sequence.
Streptococcus pneumoniae strain SP264 alpha, 1-6-glucosidase(deXB) gene, complete cds;
Streptococcus suis putative glycosyltransterase Cps7E (cps7E) gene, partial
Streptococcus pneumoniae DexB (dexB) gene, partial cds; putative transposase
Plasmodium falciparum STARP antigen (STARP) gene, complete cds.
Legionella pneumophila serogroup 1 lipopolysaccharide biosynthesis gene cluster.
Myxococcus xanthus response regulator FrzZ (frzZ) gene, partial cds; alanine
Legionella pneumophila serogroup 1 lipopolysaccharide biosynthesis gene cluster.
Oryza sativa water-stress inducible protein (WSI) mRNA, complete cds.
Shigella sonnei O-antigen gene cluster tor ORF6S, ORF7S, ORF8S, ORF9S,
Vibrio proteolyticus alanine dehydrogenase (ald) gene, complete cds.
Neisseria meningitidis strain Z2491, genomic sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Burkholderia pseudomallei putative dihydroorotase (pyrC) gene, partial cds; putative
Campylobacter jejuni polysaccharide biosynthesis protein homolog gene,
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Leptospira interrogans S10-spc-alpha locus, complete sequence.
Leptospira interrogans S10-spc-alpha locus, complete sequence.
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Leptospira interrogans S10-spc-alpha locus, complete sequence.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigen PG87 gene,
Thermotoga neapolitana galactose utilization operon, complete sequence.
Pseudomonas sp. pobA, pobR, pcaQ, pcaH and pcaG genes.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigen PG87 gene,
Aquifex pyrophilus alanyl-tRNA synthetase (alaS) gene, complete cds; and
Streptomyces sp. lacto-N-biosidase precursor gene, complete cds.
Streptomyces sp. alpha-1,3/4-fucosidase precursor gene, complete cds.
Streptomyces coelicolor cosmid C75A.
Rhizobium leguminosarum positive regulator of pobA (pobR) gene, complete
Flavobacterium jojnsoniae gliding motility protein (gldA) gene, complete cds; and
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Porphyromonas gingivalis tonB-linked receptor Tlr (tlr) gene, complete cds.
Pseudomonas aeruginosa oprC gene for outer membrane protein C, complete cds.
Streptomyces coelicolor cosmid M10.
Streptomyces coelicolor cosmid M10.
Bartonella bacilliformis citrate synthase (gltA) gene, partial cds.
Methanosarcina barkeri O-acetylserine(thiol)-lyase-A related protein (cysK) gene, complete cds.
Porphyromonas gingivalis DNA for collagenase, complete cds.
Pseudomonas aureofaciens partial bolA gene and ORF1 DNA.
Streptomyces coelicolor cosmid M10.
S. sciuri mecA2 gene, strain K3 (MM2).
S. pyogenes fcrA2 gene for Ig-Fc-binding protein.
Lactococcus lactis extracellular protein Exp4 precursor, gene, partial cds.
Selenomonas ruminantium gene for Phosphoenolpyruvate carboxykinase, complete cds.
Bacillus halodurans C-125 genomic DNA, 9A/3S′ fragment, clone ALBAC001.
Mycoplasma genitalium repetitive sequence element mgp-r4.
Streptomyces coelicolor cosmid F11.
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Bacillus halodurans C-125 genomic DNA, 32 kb fragment, complete cds.
Caenorhabditis elegans cosmid Y39C12A, complete sequence.
Streptococcus pneumoniae cap37 locus.
Yersinia enterocolitica lipopolysaccharide O-side chainbiosynthesis genes.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Leptospira borgpetersenii lipopolysaccharide o-antigen biosyntheticlocus, complete sequence.
Shewanella hanedai single stranded DNA-binding protein (ssb) gene, complete cds.
Canine herpesvirus DNA for capsid and host shut off virion protein genes.
Klebsiella pneumoniae dTDP-D-glucose 4,6 dehydratase (rmlB), glucose-1-phosphate
Phytophthora infestans mitochondrion, complete genome.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Lactococcus lactis nisZ gene and 3 ORF's.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Fugu rubripes genes encoding carbamoyl phosphate synthetase III, myosin light chain, MAP2.
Streptococcus suis strain 5218 Cps9D (cps9D) gene, partial cds; Cps9E (cps9E), Cps9F
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Moritella marina genes, complete cds, similar to eicosapentaenoicacid synthesis gene cluster.
A. ambivalens doxA gene locus with doxD and doxA genes.
Vibrio cholerae polyphosphate kinase (ppk) and exopolyphosphatase (ppx) genes, complete cds.
Caenorhabditis elegans cosmid B0454.
Porphyromonas gingivalis strain W50 immunoreactive 36 kDa antigen PG14 gene, complete cds.
Pseudomonas putida todX, todF, todC1, todC2, todB, todA, todD, todE, todG, todI, todH,
Pseudomonas putida pobR gene for PobR protein and pobA gene for PobA protein.
Escherichia coli genomic DNA. (13.6–14.0 min).
Desulfitobacterium dehalogenans putative oxygen-independent coproporphyrinogen III
Bacteroides fragilis mobilization protein C (mobC) gene, complete cds.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Bacteroides fragilis unknown gene.
Streptococcus sp. (group G) emm gene or M protein.
B. stearothermophilus hemN gene (partial) and hrcA gene.
Porphyromonas gingivalis strain W50 50 kD antigen PG1 gene, complete cds.
Vibrio harveyi Na+-translocating NADH-quinone oxidoreductase complex operon,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Yersinia pestis plasmid pPCP1.
Aeromonas caviae polar flagella locus, complete sequence.
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Salmonella dublin regulatory protein CopR (copR), histidine kinase (copS), SPI-4 pathogenicity
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane sensor
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Caenorhabditis elegans cosmid Y111B2C, complete sequence.
Homo sapiens mRNA for KIAA0850 protein, complete cds.
Legionella spiritensis macrophage infectivity potentiator (mip) gene, complete cds.
Clostridium histolyticum genes for hypoxanthine-guaninephosphoribosyl-transferase
Homo sapiens PRO1914 mRNA, complete cds.
Pseudomonas sp. DNA for low specificity L-threonine aldolase, complete cds.
Pseudomonas syringae pv. syringae AlgT (algT) gene, complete cds.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Synechococcus PCC7942 ribosomal protein S1 of 30S ribosome (rps1), ORF271,
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Neisseria gonorrhoeae thymidylate synthase (thyA) gene, complete cds.
Mus musculus mRNA for UBE-1a, complete cds.
Lactococcus lactis subsp. lactis citrate synthase, aconitatehydratase, and truncated isocitrate
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Mycobacterium leprae cosmid B22.
Rattus norvegicus mRNA for dipeptidyl peptidase III, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigen PG87 gene,
Streptomyces coelicolor cosmid M11.
Vibrio cholerae heme receptor (hutA) gene, complete cds.
Streptococcus pneumoniae rr07 and hk07 genes; two component system07.
Bacteroides fragilis beta-glucosidase gene, complete cds.
Dictyostelium discoideum hybrid histidine kinase (dhkD) mRNA, complete cds.
Asarina barclaiana sucrose transporter 1 (SUT1) mRNA, complete cds.
Bacillus subtilis mycosubtilin operon, complete sequence.
H. influenzae predicted coding region HI1127
Haemophilus influenzae Rd section 107 of 163 of the complete genome.
Porphyromonas gingivalis strain W50 immunoreactive 52 kD antigen PG41 gene,
Bacillus subtilis mycosubtilin operon, complete sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Porphyromonas gingivalis strain W50 immunoreactive 42 kD antigen PG33 gene,
B. megaterium ATP synthase i, a, c, b, delta, alpha, gamma, beta and epsilon subunit genes,
Gallus gallus vitellogenin gene coding for phosvitin, exons 23 and 24.
Haemophilus influenzae outer membrane protein (OMPP1) gene, complete CDS.
Porphyromonas gingivalis strain W50 immunoreactive 42kD antigen PG33 gene,
Dictyostelium discoideum PksB (pksB) mRNA, complete cds.
Pseudomonas aeruginosa ATP sulfurylase small subunit (cysD) and
Pseudomonas aeruginosa ATP sulfurylase small subunit (cysD) and
Sus scrofa complement C7 mRNA, complete cds.
Listeria monocytogenes putative transcriptional attenuator leader peptide
Bacillus megaterium Arac (araC) gene, partial cds; gas vesicle proteins GvpU
Bacteroides fragilis transposon Tn4555 TnpA (tnpA), integrase (int), TnpC
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
Pseudomonas aeruginosa MigA (migA) gene, complete cds.
Klebsiella pneumoniae waa gene cluster.
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
Serratia marcescens DNA tor integrase,
Streptomyces cyanogenus landomycin biosynthetic gene cluster, complete sequence.
Thermus thermophilus genes for DnaK, GrpE, DnaJ, DafA, ClpB, complete cds.
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
Pseudomonas aeruginosa genomic DNA, partial sequence, strain:PAO1.
Poeciliopsis occidentalis occidentalis MHC class II protein gene, partial exon II,
Campylobacter jejuni kpsM, kpsT genes.
Flavobacterium johnsoniae hypothetical protein gene, partial cds; GldB (gldB), GldC (gldC),
Caenorhabditis elegans cosmid Y51H4A, complete sequence.
Borrelia burgdorferi (sectin 11 of 70) of the complete genome.
Oryza sativa mRNA for MADS box-like protein, complete cds, clone:E20969.
Porphyromonas gingivalis strain W50 immunoreactive 52kD antigen PG41 gene,
Azotobacter vinelandii mannuronan C-5-epimerase (algG) gene, partial cds;
Enterococcus faecalis plasmid pPD1 bacA, bacB, bacC, bacD, bacE, bacF, bacG,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Bacillus cereus bc297a, alkD genes and partial glyS gene.
Rattus norvegicus alpha 1,2 fucosyltransferase mRNA, partial cds.
Streptococcus pneumoniae DexB (dexB) gene, partial cds; putative transposase
Tetrahymena pyriformis mitochondrial DNA, complete genome.
Plasmodium falciparum MAL3P8, complete sequence.
Pseudomonas putida glucose−binding protein (gltB) gene, complete cds.
Neisseria gonorrhoeae GlylORF1 and Gly1ORF2 genes, complete cds.
E. coli genomic DNA, Kohara clone #414 (53.8–54.2 min.).
Pseudomonas sp. BG33R strain BG33R octylprenyl diphosphate synthase−like
Thodothermus obamensis DNA polymerase I (polA) gene, complete cds.
Yersinia pestis strain EV76 hemH gene (partial) and O-antigen gene cluster
Streptococcus pneumoniae strain SP-264 alpha, 1-6-glucosidase(dexB) gene,
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Lactococcus lactis plasmid pSRQ802 abortive intection protein K(abiK) gene,
E. coli genomic DNA, Kohara clone #273 (32.5–32.8 min.).
Salmonella typhimurium ThiJ and Orf1 genes, partial cds, and PhnX, PhnW,
Enterococcus faecalis gph, ydjH, ydjG, ydjI, pbp4 and ydiC, ORF2 and ORF3 genes, partial.
S. cerevisiae chromosome IV reading frame ORF YDL057w.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Prevotella ruminicola genes for polyA polymerase, D-alanineglycinepermease and cellulase,
Pseudomonas aeruginosa 2-phosphonoacetaldehyde hydrolase gene, complete cds.
Drosophila melanogaster sperm-specific protein component (dj) mRNA, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 106 kDa antigenPG115 gene,
Staphylococcus aureus toxic shock syndrome toxin-1 (tst), enterotoxin (ent), and
U. maydis crg1 gene.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Xenopus laevis middle molecular weight neurofilament protein NF-M (1) mRNA,
Vibrio cholerae lipoprotein (nlpD) gene, partial cds, sigma S (rpoS) gene,
Pseudomonas syringae pv. syringae AlgT (algT) gene, complete cds.
Streptococcus pneumoniae type 8 capsular gene cluster.
Haemophilus ducreyi putative
E. agglomerans pKLH272 incomplete unit of mosaic mercury resistancetransposon.
Streptomyces coelicolor cosmid AH10.
Porphyromonas gingivalis PorT, upp, and prtQ genes, complete cds.
Bacteroides fragilis transposon Tn5520 transposase (bipH) andmobilization
Porphyromonas gingivalis DNA for Fimbrilin, ORF1-4, complete cds.
Enterococcus faecalis plasmid pPD1 bacA, bacB, bacC, bacD, bacE, bacF, bacG,
Porphyromonas gingivalis strain W50 immunoreactive 21 kD antigenPG10 gene,
Nocardia corallina NcoI DNA modification methyltransferase (ncoIM) and NcoI
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major
Timarcha metallica mitochondrial tRNA-Leu and partial COII genes, isolate Forest d′Anlier.
Dictyostelium discoideum hybrid histidine kinase (dhkD) mRNA, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigenPG123 gene,
Bacillus subtilis CzcD (czcD) gene, partial cds, TrkA (trkA) gene, complete cds.
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-likeprotein, fucose
Legionella adelaidensis macrophage infectivity potentiator (mip) gene, complete cds.
S. cerevisiae chromosome IV reading frame ORF YDL057w.
Pseudomonas aeruginosa AlgR-cognate sensor AlgZ (algZ) gene, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 52 kD antigenPG41 gene,
Porphyromonas gingivalis olpA and rbfA genes and ORF3 (partial), strain ATCC33277.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence;
Homo sapiens otoferlin (OTOF) mRNA, complete cds.
Plasmodium falciparum MAL3P7, complete sequence.
Enterococcus faecium entI and entJ genes and two open reading frames.
Bacteroides uniformis insertion element NBU1 fragment, integrase IntN1 gene,
Bos taurus immunoglobulin rearranged heavy chain variable region mRNA, partial cds.
Rhodothermus marinus dnaB gene.
Acinetobacter lwoffii wzc, wzb, wza, weeA, weeB, wceC, wzx, wzy, weeD, weeE,
Streptomyces coelicolor cosmid F12.
Staphylococcus aureus tyrosine recombinase XerD (xerD) gene, complete cds.
Porphyromonas gingivalis olpA and rbfA genes and ORF3 (partial), strain ATCC33277.
Escherichia coli GalF (galF) gene, partial cds; O-antigen repeat unit
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Mycobacterium tuberculosis H37Rv hypothetical protein Jv0534 (Jv0534) mRNA,
Homo sapiens clone 23856 unknown mRNA, partial cds.
Arabidopsis thaliana chromosome II BAC F24D13 genomic sequence, complete sequence.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and
Pseudomonas aeruginosa strain PAO1 penicillin-binding protein 2 (pbpA),
Zymomonas mobilis ZM4 fosmid clone 41A4, complete sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major
Haemophilus ducreyi OapA (oapA), OapB (oapB), RfaF (rfaF), 30S ribosomal protein S12, and
Mycobacterium tuberculosis H37Rv hypothetical protein Jv0166c (Jv0166c) mRNA,
Porphyromonas gingivalis gene for EF-G, complete cds, strain:SUNY1021.
Leptospira interrogans S10-spc-alpha locus, complete sequence.
Listeria monocytogenes unidentified gene and partial rpoB gene.
Synechococcus sp. PCC 7942 RNA-binding protein (rbpA) gene, complete cds.
P. gingivalis gpdxJ, put, and yhbG-pg genes.
Mus musculus mRNA for CMP-N-acetylneuraminic acid synthetase.
B. subtilis genomic sequence 89009bp.
B. subtilis clpX gene.
Myxococcus xanthus ATP-dependent protease proteolytic subunit ClpP (clpP),
Bacillus halodurans gene for TNPA, ERMK, YCBJ, YHCG, YHCF and YHCE, complete cds.
Bacteroides thetaiotaomicron rteA and rtaB genes involved in production of plasmid-like
Butyrivibrio fibrisolvens bytyrivibriocin OR79 (bvi79) gene, complete cds; and unknown genes.
Pseudomonas putida ruvB, tolQ, tolR, tolA, tolB and oprL genes.
Neisseria meningitidis outer membrane protein Omp85 (omp85) gene, complete cds.
Vibrio parahaemolyticus gene for NorM, complete cds.
Pseudomonas aeruginosa strain PAO1 penicillin-binding protein 2 (pbpA),
Porphyromonas gingivalis gene for PepO, complete cds.
Vibrio parahaemolyticus gene for NorM, complete cds.
Vibrio cholerae TolR (tolR), ExbB2 (exbB2), ExbD2 (exbD2), and TonB2 (tonB2)
Streptococcus mutans ABC transporter MutF (mutF), membrane spanning protein
Marinococcus halophilus plasmid pPL1, complete sequence.
Homo sapiens CGI-124 protein mRNA, complete cds.
Pseudomonas fluorescens hybrid histidine kinase homolog (styS) and response
P. falciparum gene for STARP antigen.
Porphyromonas gingivalis strain W50 immunoreactive 52kD antigen PG41 gene,
Pseudomonas aeruginosa (strain PAK) putative fleR kinase (fleS) and transcriptional
Bacteroides fragilis transposon Tn4555 TnpA (tnpA), integrase (int), TnpC (tnpC), excisionase
Streptomyces coelicolor cosmid M11.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization
Porphyromonas gingivalis strain W50 immunoreactive 42kD antigen PG33 gene,
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
H. vulgaris mRNA for Hyp1 protein.
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigenPG123 gene, complete cds.
Arabidopsis thaliana chromosome II BAC F14M4 genomic sequence, complete sequence.
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanse, and mannanase genes,
Porphyromonas gingivalis strain W50 immunoreactive 36 kDa antigen PG14 gene,
Cydia pomonella granulovirus late expression factor 2 homolog lef-2gene, complete cds.
Bacteroides thetaiotaomicron neopullulanse (susA) and alpha-glucosidase (susB) genes,
Flavobacterium johnsoniae gliding motility protein (gldA) gene, complete cds; and
Bacteroides thetaiotamicron 30S ribosomal protein S16-like protein, fucose
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Arabidopsis thaliana DNA chromosome 4, ESSA I FCA contig fragment No. 5.
Mycobacterium bovis 63 kDa protein, 47 kDa protein and clpB gene, complete cds.
Enterococcus seriolicida SA2F01-1, -2, -3 genes, partial and complete cds.
Bacteroides thetaiotaomicron 30S ribosomal protien S16-like protein, fucose
Homo sapiens mRNA for KIAA0636 protein, complete cds.
Synechococcus PCC7002 SigG (sigG) and hypothetical protein genes, complete cds.
Streptomyces coelicolor cosmid 4A7.
Bacteriodes thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Bacteroides fragilis mobilization protein C (mobC) gene, complete cds.
Bacteroides fragilis mobilization protein C (mobC) gene, complete cds.
Bacteroides fragilis mobilization protein B (mobB) gene, complete cds.
Plasmodium falciparum MAL1P3, complete sequence.
Bacillus subtilis 42.7 kB DNA fragment from yvsA to yvqA.
Bacillus anthracis plasmid pXO2, complete sequence.
Vibrio cholerae ADP-L-glycero-D-mannoheptose−6-epimerase (rfaD) gene, complete cds.
Treponema denticola DNA gyrase subunit B (gyrB) and chromosomalreplication
Rhodothermus marinus strain ITI278 laminarinase (lamR) gene, complete cds.
Streptomyces lavendulae LinA homolog, cytochrome P450 hydroxylaseORF4,
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigenPG87 gene, complete cds.
Homo sapiens mRNA for beta-1,4-galactosyltransferase IV, complete cds.
Bacillus cereus strain Ae10 enterotoxin mRNA, complete cds.
P. gingivalis rnhB & pgaA genes & orfs 150, 197, 202 & 199.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigenPG87 gene, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigenPG87 gene, complete cds.
Neisseria gonorrhoeae UvrA (uvrA) and ORF259 genes, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 89 kD antigenPG87 gene, complete cds.
Enterococcus faecalis vic operon and flanking genes.
M. mazei surface antigen genes orf492, orf375 and orf783.
P. vivax pval gene.
Porphyromonas gingivalis strain W50 immunoreactive 106 kDa antigenPG115
Escherichia coli genomic DNA. (22.0–22.3 min).
Arabidopsis thaliana chromosome II BAC F24H14 genomic sequence, complete sequence.
Porphyromonas gingivalis heme uptake protein A and B gene, complete cds.
Streptococcus pneumoniae Vexp1 (vex1), Vexp2 (vex2), Vexp3 (vex3), and P28
Zymomonas mobilis fosmid clone 43D2, complete sequence.
Arabidopsis thaliana chromosome II BAC F24H14 genomic sequence, complete sequence.
S. cerevisiae chromosome XI reading frame ORF YKL202w.
Pseudoalteromonas sp. S9 beta-N-acetylglucosaminidase (chiQ) gene, complete cds.
Wolinella succinogenes hydD, hydE, panD and ispA genes; orf102 and orf341.
Rhizobium leguminosarum positive regulator of pobA (pobR) gene, complete
Pseudomonas aeruginosa gene for OprM, complete cds.
Homo sapiens mRNA for KIAA0738 protein, complete cds.
Vibrio cholerae lipoprotein (vlpA) and unknown proteins genes, complete cds.
Lactococcus lactis cremoris phosphopentomutase (deoB) and purine nucleoside
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Escherichia coli 0157 DNA, map position at 46 min., complete cds.
Streptomyces coelicolor cosmid C75A.
Vibrio alginolyticus rpoN gene for RNA polymerase sigma factor N, partial and complete cds.
Drosophila melanogaster Daminopep-p gene, partial.
Serratia marcescens putative glycosyltransferase, putative glycosyltransferase, putative heptosyl
Staphylococcus aureus tyrosine recombinase XerD (xerD) gene, complete cds.
coli
Salmonella typhimurium fragment STMF1.
Legionella pneumophila RNA polymerase B-subunit (rpoB) gene, complete cds;
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and
Streptomyces coelicolor cosmid M11.
Bacteroides ovatus arabinosidase (asdII) gene, complete cds and putative transketolase,
P. fluorescens arbA gene or endo-a1,5-arabinanase.
Klebsiella pneumoniae waa gene cluster.
Prevotella albensis ORF1, isolate M384.
Pseudomonas putida todX, todF, todC1, todC2, todB, todA, todD, todE, todG, todI,
Vibrio anguillarum rfb region, partial sequence.
Myxococcus xanthus lipoic acid synthetase precursor, lipoamideacyltransferase,
Azotobacter chroococcum 4-hydroxybenzoate hydroxylase (pobA) gene, partial cds; and
Triticum aestivum chitinase IV precursor (Cht4) mRNA, complete cds.
Porphyromonas gingivalis DNA for Fimbrilin, ORF1-4, complete cds.
L. lactis predicted coding region ORF00061
Lactococcus lactis DPC3147 plasmid pMRC01, complete plasmid sequence.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Pseudomonas stutzeri pilT, pilU, ORF1 (partial) and ORF2 (partial) genes.
Myxococcus xanthus MxppcB gene for propionyl-CoA carboxylase, complete cds.
Dictyostelium discoideum mitochondrial DNA, complete sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Pseudomonas sp. pobA, pobR, pcaQ, pcaH and pcaG genes.
Yersinia pestis 102 kbases unstable region: from 1 to 119443.
Homo sapiens CGI-32 protein mRNA, complete cds.
Streptomyces coelicolor cosmid 51A.
Thermus thermophilus genes, Operon or Vacuolar type ATP syntase subunit, complete cds.
Enterococcus faecium strain N97-330 vanD glycopeptide resistance gene cluster, complete
Litomosoides sigmodontis microfilarial sheath protein SHP3a (shp3a) and
Burkholderia cepacia CeoA (ceoA) and CeoB (ceoB) genes, complete cds.
Stenotrophomonas maltophilia multidrug efflux system SmeR (smeR), SmeS
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein
Zymomonas mobilis cosmid clone 65G3, partial sequence.
Prevotella intermedia hemolysin hemolytic protein, hemolysinerythrocyte
Prevotella ruminicola genes for polygalacturonase, xylosidase, protein-export
Zymomonas mobilis fosmid clone 42C11, complete sequence.
Streptococcus pneumoniae type 33F DNA, capsular gene cluster.
Staphylococcus aureus glmM gene cluster.
Canis familiaris triadin isoform 3 mRNA, complete cds.
Danio rerio mRNA for interphotoreceptor retinoid-binding protein.
Neisseria meningitidis outer membrane protein Omp85 (omp85) gene, complete cds.
Pseudomonas aeruginosa two component sensor (lemA) gene, partialcds.
Porphyromonas gingivalis strain W50 immunoreactive 30 kD antigenPG44 gene, complete cds.
Arabidopsis thaliana putative transmembrane protein Glp (AtG1), putative nuclear DNA-binding
Plectonema boryanum ORF270, proline iminopeptidase, ferredoxin andamidase enhancer
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Thermoanaerobacterium thermosaccharolyticum ptaA and ackA genes, orf1, orf2, orf3, orf4.
Staphylococcus aureus norA23 gene for NorA, complete cds.
Haemophilus influenzae lic3 locus, containing galE and adk genesfor UDP-galactose-4-
S. thermophilus proB and proA genes.
Wolinella succinogenes mreB gene (partial), nrfH, nrfA, nrfI, and nrfJ genes.
Lipolexis gracilis NADH dehydrogenase 1 gene, mitochondrial gene encoding mitochondrial
Y. enterocolitica ompH gene.
Photorhabdus luminescens yaeL (partial), firA (partial), oma and ompH genes.
Y. enterocolitica hemS, hemT, hemU and hemV genes.
Neisseria gonorrhoeae strain FA19 putative efflux pump component MtrF (mtrF) gene,
Porphyromonas gingivalis DNA for Fimbrilin, ORF1–4, complete cds.
Alteromonas phage, complete genome.
Lactobacillus sake putative response regulator (rrp1) and putative histidine kinase (hpk1)
Zymomonas mobilis cosmid clone 65G3, partial sequence.
Methanococcus jannaschii section 73 of 150 of the complete genome.
Porphyromonas gingivalis DNA for 53 kDa major outer membrane protein, complete cds.
Listeria monocytogenes lisR/lisK gene locus, complete sequence.
Agrobacterium tumefaciens plasmid pTiA6NC VirM (virM) and VirL (virL) genes,
Flavobacterium johnsoniae gliding motility protein (gldA) gene, complete cds; and
Escherichia coli genomic DNA. (19.4–19.8 min).
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacillus halodurans C-125 comGB and bsaA genes and tRNA-His, Ala, Arg, Gly and Tyr
Staphylococcus aureus lytS and lytR genes, complete cds.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Burkholderia pseudomallei strain 1026b DbhB (dbhB), general secretory
Yersinia pestis 102 kbases unstable region: from 1 to 119443.
Porphyromonas gingivalis orf1, orf2 and orf3 genes, complete cds.
Drosophila virilis sodium channel protein (para) gene, exons 1, 2, 3, 4, and optional
Exoneurella eremophila cytochrome oxidase I gene, mitochondrial gene encoding
Staphylococcus aureus trxA and uvrC genes and partial mutS and dhsC genes.
Porphyromonas gingivalis orf1, orf2 and orf3 genes, complete cds.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Borrelia burgdorferi strain CA12 putative outer membrane protein (ospE)
Pseudomonas aeruginosa czrR, czrC, czrB, czrA genes, ORF5 and partial ORF6.
Burkholderia cepacia principal sigma factor (sigA), phthalate dioxygenase
Saccharopolyspora erythraea alpha galactosidase precursor (melA) gene, complete cds.
Bacillus caldolyticus cdd gene for cytidine deaminase.
Pseudomonas aeruginosa enoyl-CoA hydratase gene, partial cds;
Pseudomonas putida pobR gene for PobR protein and pobA gene for PobA protein
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Prevotella intermedia hemagglutinin (phg) gene, complete cds.
Bacillus subtilis 42.7 kB DNA fragment from yvsA to yvqA.
Streptomyces coelicolor cosmid F56.
Melanoplus sanguinipes entomopoxvirus, complete genome.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Flavobacterium keratolyticus endo-beta-galactosidase gene, complete cds.
Enterococcus faecalis plasmid pYI17 genes for BacA, BacB, ORF3, ORF4, ORF5, ORF6,
Streptomyces lincolnensis putative regulatory protein WdlA (wdLA) gene, complete cds;
Shigella flexneri aminopeptidase (pepP) gene, complete cds.
Trichomonas vaginalis calcium motive P-type ATPase (CA-2) gene, partial cds.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization Protein BmpH
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides thetaiotaomicron neopullulanase (susA) and alpha-glucosidase (susB) genes,
Porphyromonas gingivalis strain W50 immunoreactive 42 kD antigen PG33 gene, complete cds.
Porphyromonas gingivalis orf1, orf2 and orf3 genes, complete cds.
Tetrahymena pyriformis mitochondrial DNA, complete genome.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Pseudomonas fluorescens fbsC, fbsE, fbsA and fbsB genes.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuf), transmembrane
Bacteroides fragilis alkyl hydroperoxide reductase subunit C (ahpC) and alkyl hydroperoxide
Staphylococcus carnosus N5,N10-methylenetetrahydromethanopterin reductase
E. coli mor gene.
E. coli ampG3 gene.
Porphyromonas gingivalis gene for Dps, complete cds.
Drosophila melanogaster HCG-1 protein (HCG-1) mRNA, complete cds.
Arabidopsis thaliana chromosome I BAC F28O16 genomic sequence, complete sequence.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization BmpH (bmpH)
H. vulgaris mRNA for Hyp1 protein.
E. coli genomic DNA, Kohara clone #373 (49.5–49.9 min.).
Bacteroides fragilis alkyl hydroperoxide reductase subunit C (aphC) and alkyl
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Homo sapiens intracellular hyaluronic acid binding protein (IHABP) mRNA, complete cds.
Bacillus subtilis complete genome (section 17 of 21): from 3197001 to 3414420.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Streptomyces coelicolor cosmid E20.
Staphylococcus aureus genes, mec region, partial and complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Escherichia coli genomic DNA. (17.6–18.0 min).
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immundominatn
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Clostradium perfringens DNA for butyrate kinase and hydrogenase, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 42 kD antigen PG33 gene,
Porphyromonas gingivalis strain W50 immunogenic 75 kDa protein PG4 gene,
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Enterococcus faecium enterocin A (entA), EntI (entI), EntF (entF), EntK
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Enterococcus faecium strain BM4339 vanD glycopeptide resistance gene cluster,
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, sequence;
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Pseudomonas putida strain S-313 sulfate ester desulfurization gene locus, complete sequence.
Escherichia coli 0157 DNA, map position at 46 min., complete cds.
Arabidopsis thaliana chromosome 1 BAC F23A5 sequence, complete sequence.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesis locus, complete sequence;
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Staphylococcus aureus type 8 capsule genes, cap8A, cap8B, cap8C, cap8D, cap8E, cap8F,
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Brucella melitensis strain 16M lipopolysaccharide O side chain biosynthesis gene cluster,
Streptomyces coelicolor cosmid F11.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Dictyostelium discoicteum AX3 glutamine-asparagine rich protein gene, partial cds.
Streptococcus pneumoniae rr03 and hk03 genes; two component system03.
Bacillus megaterium DSM319 spoIV operon, 5′ flanking region, 3′ flanking region.
H. triserialis Lzf1 gene.
Drosophila melanogaster clone GH11973 BcDNA.GH11973 (BcDNA.GH11973) mRNA,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Pseudomonas aeruginosa stress factor A (pstA), ECF sigma factor (fiuI), transmembrane
Bacteroides fragilis nanH gene for neuraminidase, complete cds.
Prevotella bryantii putative polygalacturonase,B-1,4-endogluconase, and mannanase genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Mus musculus lysosomal sialic acid O-acetylesterase mRNA, complete cds.
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Enterococcus faecium strain BM4339 vanD glycopeptide resistance gene cluster,
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Legionella pneumophila serogroup 1 lipopolysaccharide biosynthesis gene cluster.
Pseudomonas putida strain S-313 sulfate ester desulfurization genelocus, complete sequence.
Staphylococcus aureus capsule gene cluster Cap5A through Cap5P genes, complete cds.
Pseudomonas putida OCT plasmid alk genes cluster and flanking DNA, strain TF4-1L.
Sinorhizobium meliloti insertion sequence ISRm14, complete sequence.
Staphylococcus capitis epr gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Porphyromonas gingivalis strain W50 immunoreactive 53 kD antigen PG123 gene,
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Salmonella typhimurium transcriptional
Salmonella typhimurium fragment STMF1.
Listeria monocytogenes DNA for DnaK operon, complete cds.
Prevotella bryantii putative polygalacturonase, B-1,4-endoglucanase, and mannanase genes,
Aureobacterium esteraromaticum gene for prolidase, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 50 kD antigen PG53 gene,
Myxococcus xanthus MxppcB gene for propionyl-CoA carboxylase, complete cds.
Piromyces sp. E2 mRNA for ketol-acid reductoisomerase.
Gossypium hirsutum palmitoyl-acyl carrier protein thioesterase (FatB1) mRNA, partial cds.
Bacillus israeli isocitrate dehydrogenase gene.
Prevotella ruminicola genes for polyA polymerase, D-alanineglycinepermease and cellulase,
Z. mays cell wall protein mRNA, 3′ end.
P. gingivalis gpdxJ, put, and yhbG-pg genes.
Clostridium acetobutylicum glyceraldehyde−3-phosphate dehydrogenase (gap),
P. gingivalis gpdxJ, put, and yhbG-pg genes.
Pseudomonas stutzeri hyp1 and comA genes and putative tolQ, exbB, tolR and exbD genes.
Pseudomonas aeruginosa alginate gene cluster AlgI (algI), AlgJ (algJ) and AlgF (algF) genes,
Homo sapiens mRNA for KIAA1275 protein, partial cds.
Bacteroides thetaiotaomicron outer membrane protein (susD) gene, complete cds.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Populus nigra PnCytPGK1 mRNA for cytosolic phosphoglycerate kinase1, complete cds.
Bacteroides thetaiotaomicron pullulanase (pulI) gene, complete cds.
Microcystis aeruginosa mcyA, mcyB and mcyC genes, complete cds.
Vibrio cholerae efflux gene A (vceA) and efflux gene B (bceB) multidrug resistance pump
Myxococcus xanthus Dog (dog), isocitrate lyase (ic1), Mis (mls), Ufo (ufo),
Bacteroides thetaiotaomicron neopullulanase (susA) and alpha-glucosidase (susB) genes,
Bacteroides thetaiotaomicron 30S ribosomal protein S16-likeprotein, fucose gene
Flavobacterium keratolyticus endo-beta-galactosidase gene, completecds.
Acidaminococcus fermentans methylmalonyl-CoA decarboxylase alphasubunit
Bacteroides thetaiotaomicron outer membrane protein (susE) gene, complete cds.
Streptococcus equi M-protein (seM) gene, complete cds.
E. coli colicin I receptor gene, complete cds.
Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene, complete cds; and
Rhodothermus sp. ‘ITI 518’ thymidine kinase (tdk) gene, completecds.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Streptomyces collinus ansatrienin AHBA biosynthetic gene cluster region 2, complete sequence.
Pseudoalteromonas sp. S9 putative glucosyl hydrolase precursor and adaptive response
Streptococcus pyogenes MsmR (msmR) gene, partial cds; LepA (lepA), Cpa
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
M. musculus mRNA for sialic acid-specific 9-O-acetylesterase.
Bacteroides thetaiotaomicron neopullulanase (susA) and alpha-glucosidase (susB) genes,
Porphyromonas gingivalis strain W50 immunoreactive 87 kD antigen PG92 gene, complete cds.
Streptomyces coelicolor cosmid F81.
Porphyromonas gingivalis strain W50 immunoreactive heat shock protein DnaJ gene,
Bacteroides thetaiotaomicron outer membrane protein (susD) gene, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 87 kD antigen PG92 gene, complete cds.
Bacteroides fragilis transposon Tn4555 TnpA (tnpA), integrase (int), TnpC
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Bacillus pumilus plasmid pSH1452, Rep gene, complete cds.
Bacteroides fragilis transposon Tn4555 TnpA (tnpA), integrase (int), TnpC
Alicyclobacillus acidocaldarius maltose/maltodextrine transport gene region
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Lactococcus lactis plasmid pNP40, abortive infection locus, AbiEi, AbiEii, RecA(LP),
Porphyromonas gingivalis dnaK operon genes, complete cds.
Porphyromonas gingivalis dnaK operon genes, complete cds.
Columba livia mRNA for 5′-nucleotidase.
Heliothis virescens Cry1A toxin receptor A mRNA, complete cds.
Pseudomonas fluorescens hypothetical metabolite transport protein, positive
Azotobacter vinelandii histidine protein kinase homolog GacS (gacS) gene, complete cds.
Zymomonas mobilis strain ZM4 clone 43F4, complete sequence.
Bacteroides fragilis 638R polysaccharide B (PS B2) biosynthesislocus, complete sequence;
Agrobacterium tumefaciens plasmid pTi-SAKURA, complete sequence.
Porphyromonas gingivalis TonB-dependent receptor HmuR (hmuR) gene, complete cds.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-likeprotein, fucose
Sulfolobus solfataricus 281 kb genomic DNA fragment, strain P2.
Porphyromonas gingivalis TonB-dependent receptor HmuR (hmuR) gene, complete cds.
Cydia pomonella granulosis virus ORF13L gene, partial cds, ORF15L, ORF15R,
Bacteroides thetaiotaomicron 30S ribosomal protein S16-likeprotein, fucose
Rhizobium leguminosarum bv. trifolii plasmid PR1e162Y10C rspDEFoperon, partial sequence.
Arabidopsis thaliana chromosome III BAC T7M13 genomic sequence, complete sequence.
Pseudomonas sp. DNA for styrene catabolism genes.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major immunodominant
Flavobacterium johnsoniae gliding motility protein (gldA) gene, complete cds; and
Bacillus sp. GL1 genes for orf and unsaturated glucuronylhydrolase, complete cds.
Anabaena sp. cyaC gene for adenylate cyclase, complete cds.
Globodera pallida mitochondrial COII, ND4, COIII, ND6, ND1, ND3 and cytb genes.
Pediococcus pentosaceus strain ATCC43200 plasmid pMD136, complete plasmid sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encoding a major
Arabidopsis thaliana chromosome I BAC F12B7 genomic sequence, complete sequence.
Yersinia pseudotuberculosis group
Staphylococcus aureus genes, mec region, partial and complete cds.
Toxocara canis TcH SLdT.460 mRNA, complete cds.
Anabaena sp. L-31 kdp operon, complete sequence.
Alicyclobacillus acidocaldarius kdpA gene.
Streptomyces coelicolor cosmid F41.
S. sciuri mecA1 gene, strain K3 (MM2).
Enterococcus faecalis vic operon and flanking genes.
Staphylococcus carnosus response regulator-like protein (orfx) gene, partial cds.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-like protein, fucose
Arabidopsis thaliana chromosome III BAC F28J7 genomic sequence, complete sequence.
Pseudomonas putida strain S-313 sulfate ester desulfurization genelocus, complete sequence.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Edwardsiella ictaluri D-3 phosphoglycerate dehydrogenase (serA) gene,
Streptomyces lividans tripeptidyl aminopeptidase gene, complete cds.
Vibrio cholerae heme receptor (hutA) gene, complete cds.
Comamonas acidovorans omp21 gene.
Escherichia coli GalF (galF) gene, partial cds; O-antigen repeat unit
Salmonella typhimurium putative operon regulated by PmrAB, necessary for
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Bacteroides fragilis (bctA) gene, complete cds.
Bacteroides fragilis mobiliation protein C (mobC) gene, complete cds.
Homo sapiens mRNA for KIAA0775 protein, complete cds.
C. burnetii put. genes for encoding glucose inhibited division protein A and B.
Macaca mulatta rhadinovirus 17577 R1, dihydrofolate reductase, complement
Brugia malayi beta-D-galactosidase (lacZ) mRNA, partial cds.
Bacteroides fragilis transposon Tn5520 transposase (bipH) and mobilization protein BmpH
Desulfurolobus ambivalens tnpA, tnpB, rfbD and sor genes and ORF2, ORF3, ORF4 and ORF5.
Porphyromonas gingivalis DNA for Fimbrilin, ORF1–4, complete cds.
Vibrio cholerae z47f gene.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Streptomyces thermoviolaceus nagA gene forbeta-N-Acetylglucosaminidase, complete cds.
Bacteroides thetaiotaomicron 30S ribosomal protein S16-liKe protein, fucose
Streptomyces thermoviolaceus nagA gene forbeta-N-Acetylglucosaminidase, complete cds.
Pseudomonas aeruginosa stress factor A (psfA), ECF sigma factor (fiuI), transmembrane
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Bacillus megaterium glk gene.
Porphyromonas gingivalis strain W50 immunoreactive 51 kD antigen PG52 gene, complete cds.
T. ethanolicus melA and lacA genes.
Alicyclobacillus acidocaldarius maltose/maltodextrine transport gene region (malEFGR genes,
Alicyclobacillus acidocaldarius maltose/maltodextrine transport gene region (malEFGR genes,
M. mazei surface antigen genes orf492, orf375 and orf783.
Prevotella bryantii putative polygalacturonase,B-1,4-endoglucanase, and mannanase genes,
Bacteroides fragilis beta-glucosidase gene, complete cds.
Zymomonas mobilis ZM4 fosmid clone 41A4, complete sequence.
Porphyromonas gingivalis W50 receptor antigen (rag) locus encodinga major immunodominant
Bacteroides fragilis mobilization protein A (mobA) gene, complete cds.
Chlamydophila pneumoniae pmp_3.1 gene for polymorphic outermembrane protein G family,
Synechococcus PCC7942 ribosomal protein S1 of 30S ribosome (rps1), ORF271,
Pseudomonas aeruginosa two component sensor (lemA) gene, partial cds.
Porphyromonas gingivalis TonB-dependent receptor HmuR (hmuR) gene, complete cds.
Lactococcus lactis DPC3147 plasmid pMRC01, complete plasmid sequence.
Dictyostelium discoideum ras interacting protein RIPA (ripA) mRNA, complete cds.
Porphyromonas gingivalis strain W50 immunoreactive 42 kD antigen PG33 gene, complete cds.
Phaeodactylum tricornutum ribulose-1,5-bisphosphatecarboxylase/oxygenase
Aegilops tauschii high molecular weight glutenin subunit (Glu-1-2) gene, complete cds.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Bacteroides fragilis capsular polysaccharide biosynthesis operon, complete sequence.
Mycobacterium smegmatis putative HSP20 (hsp) gene, complete cds.
Melanoplus sanguinipes entomopoxvirus, complete genome.
Lactococcus lactis BusAA (busAA) and glycine-betaine binding permease protein (busAB)
Dictyostelium discoideum hybrid histidine kinase (dhkD) mRNA, complete cds.
Listeria monocytogenes ATPase homolog GbuA (gbuA), putative glycine betaine membrane
Clostridium pasteurianum genes encoding putative pyruvate ferredoxin oxidoreductase
Porphyromonas gingivalis TonB-dependent receptor HmuR (hmuR) gene, complete cds.
Porphyromonas gingivalis W50 receptor antigen (rag) Locus encodling a major
Oenococcus oeni histidine decarboxylase (hdcA) gene, complete cds; and HdcB (hdcB) gene,
Acetabularia mediterranea mRNA for inorganic pyrophosphatase, complete cds.
Bacteroides forsythus groEL gene, strain ATCC 43037.
Bacteroides fragilis (bctA) gene, complete cds.
Melanoplus sanguinipes entomopoxvirus, complete genome.
Bacteroides uniformis insertion element NBU1 fragment, integrase IntN1 gene, complete cds.
Streptococcus pneumoniae methyl transferase gene cluster, complete sequence.
Ruminococcus albus beta-glucosidase (gluA) mRNA, complete cds.
Pseudomonas putida todX, todF, todC1, todC2, todB, todA, todD, todE, todG, todI, todH,
Arabidopsis thaliana chromosome II BAC T3B23 genomic sequence, complete sequence.
Nostoc sp. ATCC 29133 oxidoreductase (hrmU) and HrmA (hrmA) genes, complete cds.
Bradyrhizobium japonicum putative arylsulfatase (arsA), putative soluble
Pseudomonas aeruginosa gene for OprM, complete cds.
Thermotoga neapolitana galactose utilization operon, complete sequence.
Clostridium perfringens hyp27, bacH, ptp, cpd genes for hypothetical
Nostoc punctiforme histidine kinase (hepK) gene, complete cds.
Bacillus stearothermophilus
Homo sapiens chromosome 16, cosmid clone 444B9 (LANL), complete sequence.
Bacteroides fragilis 7-alpha-hydroxysteroid dehydrogenase (hdhA) gene, complete cds.
Klebsiella pneumoniae (strain SD8) ompK37 gene.
E. coli colicin I receptor gene, complete cds.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/128,705, filed Apr. 9, 1999, the entire teachings of which are incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5571693 | Rasmussen et al. | Nov 1996 | A |
| 5606022 | Rasmussen et al. | Feb 1997 | A |
| 5679540 | Rasmussen et al. | Oct 1997 | A |
| 5792642 | Rasmussen et al. | Aug 1998 | A |
| 6110705 | Ratti et al. | Aug 2000 | A |
| Number | Date | Country | |
|---|---|---|---|
| 60128705 | Apr 1999 | US |
