The invention provides gidB polypeptides and polynucleotides encoding gidB polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing gidB polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION 
This invention relates to newly identified polynucleotides and 
polypeptides, and their production and uses, as well as their variants, 
agonists and antagonists, and their uses. In particular, the invention 
relates to polynucleotides and polypeptides of the gidB family, as well as 
their variants, hereinafter referred to as "gidB," "gidB 
polynucleotide(s)," and "gidB polypeptide(s)" as the case may be. 
BACKGROUND OF THE INVENTION 
It is particularly preferred to employ Staphylococcal genes and gene 
products as targets for the development of antibiotics. The Staphylococci 
make up a medically important genera of microbes. They are known to 
produce two types of disease, invasive and toxigenic. Invasive infections 
are characterized generally by abscess formation effecting both skin 
surfaces and deep tissues. S. aureus is the second leading cause of 
bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic 
thrombophlebitis and acute bacterial endocarditis are also relatively 
common. There are at least three clinical conditions resulting from the 
toxigenic properties of Staphylococci. The manifestation of these diseases 
result from the actions of exotoxins as opposed to tissue invasion and 
bacteremia. These conditions include: Staphylococcal food poisoning, 
scalded skin syndrome and toxic shock syndrome. 
The frequency of Staphylococcus aureus infections has risen dramatically in 
the past few decades. This has been attributed to the emergence of 
multiply antibiotic resistant strains and an increasing population of 
people with weakened immune systems. It is no longer uncommon to isolate 
Staphylococcus aureus strains which are resistant to some or all of the 
standard antibiotics. This phenomenon has created an unmet medical need 
and demand for new anti-microbial agents, vaccines, drug screening 
methods, and diagnostic tests for this organism. 
Moreover, the drug discovery process is currently undergoing a fundamental 
revolution as it embraces "functional genomics," that is, high throughput 
genome- or gene-based biology. This approach is rapidly superseding 
earlier approaches based on "positional cloning" and other methods. 
Functional genomics relies heavily on the various tools of bioinformatics 
to identify gene sequences of potential interest from the many molecular 
biology databases now available as well as from other sources. There is a 
continuing and significant need to identify and characterize further genes 
and other polynucleotides sequences and their related polypeptides, as 
targets for drug discovery. 
Clearly, there exists a need for polynucleotides and polypeptides, such as 
the gidB embodiments of the invention, that have a present benefit of, 
among other things, being useful to screen compounds for antibiotic 
activity. Such factors are also useful to determine their role in 
pathogenesis of infection, dysfunction and disease. There is also a need 
for identification and characterization of such factors and their 
antagonists and agonists to find ways to prevent, ameliorate or correct 
such infection, dysfunction and disease. 
Certain of the polypeptides of the invention possess significant amino acid 
sequence homology to a known gidB protein. 
SUMMARY OF THE INVENTION 
The present invention relates to gidB, in particular gidB polypeptides and 
gidB polynucleotides, recombinant materials and methods for their 
production. In another aspect, the invention relates to methods for using 
such polypeptides and polynucleotides, including the treatment of 
microbial diseases, amongst others. In a further aspect, the invention 
relates to methods for identifying agonists and antagonists using the 
materials provided by the invention, and for treating microbial infections 
and conditions associated with such infections with the identified 
compounds. In a still further aspect, the invention relates to diagnostic 
assays for detecting diseases associated with microbial infections and 
conditions associated with such infections, such as assays for detecting 
gidB expression or activity. 
Various changes and modifications within the spirit and scope of the 
disclosed invention will become readily apparent to those skilled in the 
art from reading the following descriptions and from reading the other 
parts of the present disclosure. 
DESCRIPTION OF THE INVENTION 
The invention relates to gidB polypeptides and polynucleotides as described 
in greater detail below. In particular, the invention relates to 
polypeptides and polynucleotides of a gidB of Staphylococcus aureus, which 
is related by amino acid sequence homology to gidB polypeptide. The 
invention relates especially to gidB having the nucleotide and amino acid 
sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4 
respectively. 
TABLE 1 
__________________________________________________________________________ 
gidB Polynucleotide and Polypeptide Sequences 
__________________________________________________________________________ 
(A) Staphylococcus aureus gidB polynucleotide sequence [SEQ ID NO: 1]. 
5'- 
TCTATATTATTGATTTACTTAGAACAAGGTAAACTCCAAAGGGTGAGTGACTAATGACTGTAGAATGGTTAG 
- CAGAACAA 
- TTAAAAGAACATAATATTGAATTAACTGAGACTCAAAAACAACAGTTTCAAACATATTATCGTTTACTTG 
TT 
- GAATGGAA 
- TGAAAAGATGAATTTGACAAGTATTACAGATGAACACGATGTATATTTGAAACATTTTTATGATTCCATT 
GC 
- ACCTAGTT 
- TTTATTTTGATTTTAATCAGCCTATAAGTATATGTGATGTAGGCGCTGGAGCTGGTTTTCCAAGTATTCC 
GT 
- TAAAAATA 
- ATGTTTCCGCAGTTAAAAGTGACGATTGTTGATTCATTAAATAAGCGTATTCAATTTTTAAACCATTTAG 
CG 
- TCAGAATT 
- ACAATTACAGGATGTCAGCTTTATACACGATAGAGCAGAAACATTTGGTAAGGGTGTCTACAGGGAGTCT 
TA 
- TGATGTTG 
- TTACTGCAAGAGCAGTAGCTAGATTATCCGTGTTGAGTGAATTGTGTTTACCGCTAATTAAAAAAGGTGG 
AC 
- AGTTTGTT 
- GCATTAAAATCTTCAAAAGGTGAAGAAGAATTAGAAGAAGCAAAATTTGCAATTAGTGTGTTAGGTGGTA 
AC 
- GTTACAGA 
- AACACATACCTTTAAATTGCCAGAAGATGCTGGAGAGCGCCAGATGTTCATTATTGATAAAAAAAGACAG 
AC 
- GCCGAAAA 
- AGTACCCAAGAAAACCAGGGACGCCTAATAAGACTCCTTTACTTGAAAAATAATGCATAATCCTTTACAA 
CT 
- AACATAAA 
- AGGAGCGAAT-3' 
(B) Staphylococcus aureus gidB polypeptide sequence deduced from a 
polynucleotide sequence in this table [SEQ ID NO: 2]. 
NH.sub.2 - 
MTVEWLAEQLKEHNIELTETQKQQFQTYYRLLVEWNEKMNLTSITDEHDVYLKHFYDSIAPSFYFDFNQPIS 
- ICDVGAGA 
- GFPSIPLKIMFPQLKVTIVDSLNKRIQFLNHLASELQLQDVSFIHDRAETFGKGVYRESYDVVTARAVAR 
LS 
- VLSELCLP 
- LIKKGGQFVALKSSKGEEELEEAKFAISVLGGNVTETHTFKLPEDAGERQMFIIDKKRQTPKKYPRKPGT 
PN 
- KTPLLEK-COOH 
- (C) Staphylococcus aureus gidB ORF sequence [SEQ ID NO: 3]. 
5'- 
GTAAATCCAGCAGACATATCTATATTATTGATTTACTTAGAACAAGGTAAACTCCAAAGGGTGAGTGACTAA 
- TGACTGTA 
- GAATGGTTAGCAGAACAATTAAAAGAACATAATATTGAATTAACTGAGACTCAAAAACAACAGTTTCAAA 
CA 
- TATTATCG 
- TTTACTTGTTGAATGGAATGAAAAGATGAATTTGACAAGTATTACAGATGAACACGATGTATATTTGAAA 
CA 
- TTTTTATG 
- ATTCCATTGCACCTAGTTTTTATTTTGATTTTAATCAGCCTATAAGTATATGTGATGTAGGCGCTGGAGC 
TG 
- GTTTTCCA 
- AGTATTCCGTTAAAAATAATGTTTCCGCAGTTAAAAGTGACGATTGTTGATTCATTAAATAAGCGTATTC 
AA 
- TTTTTAAA 
- CCATTTAGCGTCAGAATTACAATTACAGGATGTCAGCTTTATACACGATAGAGCAAAAACATNTGGTAAG 
GG 
- TGTCTACA 
- NGGAGTCTTATGATGTTGTTACTGCAAGAGCAGTANCTAAATTATCCGTGTTGAGTGAATTGTGTTTACC 
GC 
- TAATTAAA 
- AAAGGTGGACAGTNTGTTGCATTAAAATCTTCAAAAGGTGAAGAAAAATTANAAAAAACANAATTTGCAA 
TT 
- AGTGTGTT 
- AGGTGGTAACGTTACAGAAACACATACCTTTAAATTGCCAGAAGATGCTGGAGAGCGCCAGATGTTCATT 
AT 
- TGATAAAA 
- AAAGACAGACGCCGAAAAAGTACCCAAGAAAACCAGGGACGCCTAATAAGACTCCTTTACTTGAAAAATA 
AT 
- GCATAATC 
- CTTTACAACTAACATAAAAGGAGCGAATGGATAATGAAAAAACCTTTTTCAAAATTATTTGGTTTGAAAA 
AC 
- AAAGATGA 
- CATCATTGGACATATTGAAG-3' 
- (D) Staphylococcus aureus gidB polypeptide sequence deduced from 
a polynudeotide ORF sequence in this table [SEQ ID NO: 4]. 
NH.sub.2 - 
MTVEWLAEQLKEHNIELTETQKQQFQTYYRLLVEWNEKMNLTSITDEHDVYLKHFYDSIAPSFYFDFNQPIS 
- ICDVGAGA 
- GFPSIPLKIMFPQLKVTIVDSLNKRIQFLNHLASELQLQDVSFIHDRAKTXGKGVYXESYDVVTARAVXK 
LS 
- VLSELCLP 
- LIKKGGQXVALKSSKGEEKLXKTXFAISVLGGNVTETHTFKLPEDAGERQMFIIDKKRQTPKKYPRKPGT 
PN 
- KTPLLEK-COOH 
__________________________________________________________________________ 
Deposited materials 
A deposit containing a Staphylococcus aureus WCUH 29 strain has been 
deposited with the National Collections of Industrial and Marine Bacteria 
Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 
Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and referred to as 
Staphylococcus aureus WCUH29 on deposit. The Staphylococcus aureus strain 
deposit is referred to herein as "the deposited strain" or as "the DNA of 
the deposited strain." 
The deposited strain contains the full length gidB gene. The sequence of 
the polynucleotides contained in the deposited strain, as well as the 
amino acid sequence of any polypeptide encoded thereby, are controlling in 
the event of any conflict with any description of sequences herein. 
The deposit of the deposited strain has been made under the terms of the 
Budapest Treaty on the International Recognition of the Deposit of 
Micro-organisms for Purposes of Patent Procedure. The strain will be 
irrevocably and without restriction or condition released to the public 
upon the issuance of a patent. The deposited strain is provided merely as 
convenience to those of skill in the art and is not an admission that a 
deposit is required for enablement, such as that required under 35 U.S.C. 
.sctn.112. 
A license may be required to make, use or sell the deposited strain, and 
compounds derived therefrom, and no such license is hereby granted. 
In one aspect of the invention there is provided an isolated nucleic acid 
molecule encoding a mature polypeptide expressible by the Staphylococcus 
aureus WCUH 29 strain, which polypeptide is contained in the deposited 
strain. Further provided by the invention are gidB polynucleotide 
sequences in the deposited strain, such as DNA and RNA, and amino acid 
sequences encoded thereby. Also provided by the invention are gidB 
polypeptide and polynucleotide sequences isolated from the deposited 
strain. 
Polypeptides 
gidB polypeptide of the invention is substantially phylogenetically related 
to other proteins of the gidB family. 
In one aspect of the invention there are provided polypeptides of 
Staphylococcus aureus referred to herein as "gidB" and "gidB polypeptides" 
as well as biologically, diagnostically, prophylactically, clinically or 
therapeutically useful variants thereof, and compositions comprising the 
same. 
Among the particularly preferred embodiments of the invention are variants 
of gidB polypeptide encoded by naturally occurring alleles of the gidB 
gene. 
The present invention further provides for an isolated polypeptide which: 
(a) comprises or consists of an amino acid sequence which has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, most preferably at 
least 97-99% or exact identity, to that of SEQ ID NO:2 over the entire 
length of SEQ ID NO:2; 
(b) a polypeptide encoded by an isolated polynucleotide comprising or 
consisting of a polynucleotide sequence which has at least 70% identity, 
preferably at least 80% identity, more preferably at least 90% identity, 
yet more preferably at least 95% identity, even more preferably at least 
97-99% or exact identity to SEQ ID NO: 1 over the entire length of SEQ ID 
NO: 1; 
(c) a polypeptide encoded by an isolated polynucleotide comprising or 
consisting of a polynucleotide sequence encoding a polypeptide which has 
at least 70% identity, preferably at least 80% identity, more preferably 
at least 90% identity, yet more preferably at least 95% identity, even 
more preferably at least 97-99% or exact identity, to the amino acid 
sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; or 
(d) a polypeptide encoded by an isolated polynucleotide comprising or 
consisting of a polynucleotide sequence which has at least 70% identity, 
preferably at least 80% identity, more preferably at least 90% identity, 
yet more preferably at least 95% identity, even more preferably at least 
97-99% or exact identity, to SEQ ID NO: 1 over the entire length of SEQ ID 
NO:3; 
(e) a polypeptide encoded by an isolated polynucleotide comprising or 
consisting of a polynucleotide sequence which has at least 70% identity, 
preferably at least 80% identity, more preferably at least 90% identity, 
yet more preferably at least 95% identity, even more preferably at least 
97-99% or exact identity to SEQ ID NO:3 over the entire length of SEQ ID 
NO:3; or 
(f) a polypeptide encoded by an isolated polynucleotide comprising or 
consisting of a polynucleotide sequence encoding a polypeptide which has 
at least 70% identity, preferably at least 80% identity, more preferably 
at least 90% identity, yet more preferably at least 95% identity, even 
more preferably at least 97-99% or exact identity, to the amino acid 
sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4; 
(g) comprises or consists of an amino acid sequence which has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, most preferably at 
least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO:2 
over the entire length of SEQ ID NO:4. 
The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID 
NO:2 or 4] (in particular the mature polypeptide) as well as polypeptides 
and fragments, particularly those which have the biological activity of 
gidB, and also those which have at least 70% identity to a polypeptide of 
Table 1 [SEQ ID NO: 1 or 3] or the relevant portion, preferably at least 
80% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4 and more 
preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO:2 
or 4] and still more preferably at least 95% identity to a polypeptide of 
Table 1 [SEQ ID NO:2 or 4] and also include portions of such polypeptides 
with such portion of the polypeptide generally containing at least 30 
amino acids and more preferably at least 50 amino acids. 
The invention also includes a polypeptide consisting of or comprising a 
polypeptide of the formula: 
EQU X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y 
wherein, at the amino terminus, X is hydrogen, a metal or any other moiety 
described herein for modified polypeptides, and at the carboxyl terminus, 
Y is hydrogen, a metal or any other moiety described herein for modified 
polypeptides, R.sub.1 and R.sub.3 are any amino acid residue or modified 
amino acid residue, m is an integer between 1 and 1000 or zero, n is an 
integer between 1 and 1000 or zero, and R.sub.2 is an amino acid sequence 
of the invention, particularly an amino acid sequence selected from Table 
1 or modified forms thereof. In the formula above, R.sub.2 is oriented so 
that its amino terminal amino acid residue is at the left, covalently 
bound to R.sub.1, and its carboxy terminal amino acid residue is at the 
right, covalently bound to R.sub.3. Any stretch of amino acid residues 
denoted by either R.sub.1 or R.sub.3, where m and/or n is greater than 1, 
may be either a heteropolymer or a homopolymer, preferably a 
heteropolymer. Other preferred embodiments of the invention are provided 
where m is an integer between 1 and 50, 100 or 500, and n is an integer 
between 1 and 50, 100, or 500. 
It is most preferred that a polypeptide of the invention is derived from 
Staphylococcus aureus, however, it may preferably be obtained from other 
organisms of the same taxonomic genus. A polypeptide of the invention may 
also be obtained, for example, from organisms of the same taxonomic family 
or order. 
A fragment is a variant polypeptide having an amino acid sequence that is 
entirely the same as part but not all of any amino acid sequence of any 
polypeptide of the invention. As with gidB polypeptides, fragments may be 
"free-standing," or comprised within a larger polypeptide of which they 
form a part or region, most preferably as a single continuous region in a 
single larger polypeptide. 
Preferred fragments include, for example, truncation polypeptides having a 
portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or of 
variants thereof, such as a continuous series of residues that includes an 
amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of 
the polypeptides of the invention produced by or in a host cell, 
particularly a Staphylococcus aureus, are also preferred. Further 
preferred are fragments characterized by structural or functional 
attributes such as fragments that comprise alpha-helix and alpha-helix 
forming regions, beta-sheet and beta-sheet-forming regions, turn and 
turn-forming regions, coil and coil-forming regions, hydrophilic regions, 
hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, 
flexible regions, surface-forming regions, substrate binding region, and 
high antigenic index regions. 
Further preferred fragments include an isolated polypeptide comprising an 
amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous 
amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated 
polypeptide comprising an amino acid sequence having at least 15, 20, 30, 
40, 50 or 100 contiguous amino acids truncated or deleted from the amino 
acid sequence of SEQ ID NO: 2. 
Also preferred are biologically active fragments which are those fragments 
that mediate activities of gidB, including those with a similar activity 
or an improved activity, or with a decreased undesirable activity. Also 
included are those fragments that are antigenic or immunogenic in an 
animal, especially in a human. Particularly preferred are fragments 
comprising receptors or domains of enzymes that confer a function 
essential for viability of Staphylococcus aureus or the ability to 
initiate, or maintain cause Disease in an individual, particularly a 
human. 
Fragments of the polypeptides of the invention may be employed for 
producing the corresponding full-length polypeptide by peptide synthesis; 
therefore, these variants may be employed as intermediates for producing 
the full-length polypeptides of the invention. 
In addition to the standard single and triple letter representations for 
amino acids, the term "X" or "Xaa" may also be used in describing certain 
polypeptides of the invention. "X" and "Xaa" mean that any of the twenty 
naturally occurring amino acids may appear at such a designated position 
in the polypeptide sequence. 
Polynucleotides 
It is an object of the invention to provide polynucleotides that encode 
gidB polypeptides, particularly polynucleotides that encode the 
polypeptide herein designated gidB. 
In a particularly preferred embodiment of the invention the polynucleotide 
comprises a region encoding gidB polypeptides comprising a sequence set 
out in Table 1 [SEQ ID NO: 1 or 3] which includes a full length gene, or a 
variant thereof. The Applicants believe that this full length gene is 
essential to the growth and/or survival of an organism which possesses it, 
such as Staphylococcus aureus. 
As a further aspect of the invention there are provided isolated nucleic 
acid molecules encoding and/or expressing gidB polypeptides and 
polynucleotides, particularly Staphylococcus aureus gidB polypeptides and 
polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, 
mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the 
invention include biologically, diagnostically, prophylactically, 
clinically or therapeutically useful polynucleotides and polypeptides, and 
variants thereof, and compositions comprising the same. 
Another aspect of the invention relates to isolated polynucleotides, 
including at least one full length gene, that encodes a gidB polypeptide 
having a deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4] and 
polynucleotides closely related thereto and variants thereof. 
In another particularly preferred embodiment of the invention there is a 
gidB polypeptide from Staphylococcus aureus comprising or consisting of an 
amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof. 
Using the information provided herein, such as a polynucleotide sequence 
set out in Table 1 [SEQ ID NO: 1 or 3], a polynucleotide of the invention 
encoding gidB polypeptide may be obtained using standard cloning and 
screening methods, such as those for cloning and sequencing chromosomal 
DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as 
starting material, followed by obtaining a full length clone. For example, 
to obtain a polynucleotide sequence of the invention, such as a 
polynucleotide sequence given in Table 1 [SEQ ID NO: 1 or 3], typically a 
library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in 
E.coli or some other suitable host is probed with a radiolabeled 
oligonucleotide, preferably a 17-mer or longer, derived from a partial 
sequence. Clones carrying DNA identical to that of the probe can then be 
distinguished using stringent hybridization conditions. By sequencing the 
individual clones thus identified by hybridization with sequencing primers 
designed from the original polypeptide or polynucleotide sequence it is 
then possible to extend the polynucleotide sequence in both directions to 
determine a full length gene sequence. Conveniently, such sequencing is 
performed, for example, using denatured double stranded DNA prepared from 
a plasmid clone. Suitable techniques are described by Maniatis, T., 
Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
(1989). (see in particular Screening By Hybridization 1.90 and Sequencing 
Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA 
sequencing may also be performed to obtain a full length gene sequence. 
Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ 
ID NO: 1 or 3] was discovered in a DNA library derived from Staphylococcus 
aureus WCUH 29. 
Moreover, each DNA sequence set out in Table 1 [SEQ ID NO: 1 or 3] contains 
an open reading frame encoding a protein having about the number of amino 
acid residues set forth in Table 1 [SEQ ID NO:2 or 4] with a deduced 
molecular weight that can be calculated using amino acid residue molecular 
weight values well known to those skilled in the art. The polynucleotide 
of SEQ ID NO: 1, between nucleotide number 54 and the stop codon which 
begins at nucleotide number 771 of SEQ ID NO: 1, encodes the polypeptide 
of SEQ ID NO:2. 
In a further aspect, the present invention provides for an isolated 
polynucleotide comprising or consisting of: 
(a) a polynucleotide sequence which has at least 70% identity, preferably 
at least 80% identity, more preferably at least 90% identity, yet more 
preferably at least 95% identity, even more preferably at least 97-99% or 
exact identity to SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, or 
the entire length of SEQ ID NO: 1 encoding SEQ ID NO:2; 
(b) a polynucleotide sequence encoding a polypeptide which has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, even more preferably 
at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2, 
over the entire length of SEQ ID NO:2; or 
(c) a nucleotide sequence which has at least 70% identity, preferably at 
least 80% identity, more preferably at least 90% identity, yet more 
preferably at least 95% identity, even more preferably at least 97-99% or 
100% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO:3; 
(d) a nucleotide sequence which has at least 70% identity, preferably at 
least 80% identity, more preferably at least 90% identity, yet more 
preferably at least 95% identity, even more preferably at least 97-99% or 
exact identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3; or 
(e) a polynucleotide sequence encoding a polypeptide which has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, even more preferably 
at least 97-99% or exact identity, to the amino acid sequence of SEQ ID 
NO:4, over the entire length of SEQ ED NO:4. 
A polynucleotide encoding a polypeptide of the present invention, including 
homologs and orthologs from species other than Staphylococcus aureus, may 
be obtained by a process which comprises the steps of screening an 
appropriate library under stringent hybridization conditions with a 
labeled or detectable probe consisting of or comprising the sequence of 
SEQ ID NO: 1 or 3 or a fragment thereof; and isolating a full-length gene 
and/or genomic clones containing said polynucleotide sequence. 
The invention provides a polynucleotide sequence identical over its entire 
length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO: 1 
or 3]. Also provided by the invention is a coding sequence for a mature 
polypeptide or a fragment thereof, by itself as well as a coding sequence 
for a mature polypeptide or a fragment in reading frame with another 
coding sequence, such as a sequence encoding a leader or secretory 
sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide 
of the invention may also contain at least one non-coding sequence, 
including for example, but not limited to at least one non-coding 5' and 
3' sequence, such as the transcribed but non-translated sequences, 
termination signals (such as rho-dependent and rho-independent termination 
signals), ribosome binding sites, Kozak sequences, sequences that 
stabilize mRNA, introns, and polyadenylation signals. The polynucleotide 
sequence may also comprise additional coding sequence encoding additional 
amino acids. For example, a marker sequence that facilitates purification 
of the fused polypeptide can be encoded. In certain embodiments of the 
invention, the marker sequence is a hexa-histidine peptide, as provided in 
the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. 
Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al., 
Cell 37: 767 (1984), both of which may be useful in purifying polypeptide 
sequence fused to them. Polynucleotides of the invention also include, but 
are not limited to, polynucleotides comprising a structural gene and its 
naturally associated sequences that control gene expression. 
A preferred embodiment of the invention is a polynucleotide of consisting 
of or comprising nucleotide 54 to the nucleotide immediately upstream of 
or including nucleotide 771 set forth in SEQ ID NO: 1 of Table 1, both of 
which encode the gidB polypeptide. 
The invention also includes a polynucleotide consisting of or comprising a 
polynucleotide of the formula: 
EQU X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y 
wherein, at the 5' end of the molecule, X is hydrogen, a metal or a 
modified nucleotide residue, or together with Y defines a covalent bond, 
and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified 
nucleotide residue, or together with X defines the covalent bond, each 
occurrence of R.sub.1 and R.sub.3 is independently any nucleic acid 
residue or modified nucleic acid residue, m is an integer between 1 and 
3000 or zero , n is an integer between 1 and 3000 or zero, and R.sub.2 is 
a nucleic acid sequence or modified nucleic acid sequence of the 
invention, particularly a nucleic acid sequence selected from Table 1 or a 
modified nucleic acid sequence thereof. In the polynucleotide formula 
above, R.sub.2 is oriented so that its 5' end nucleic acid residue is at 
the left, bound to R.sub.1, and its 3' end nucleic acid residue is at the 
right, bound to R.sub.3. Any stretch of nucleic acid residues denoted by 
either R.sub.1 and/or R.sub.2, where m and/or n is greater than 1, may be 
either a heteropolymer or a homopolymer, preferably a heteropolymer. 
Where, in a preferred embodiment, X and Y together define a covalent bond, 
the polynucleotide of the above formula is a closed, circular 
polynucleotide, which can be a double-stranded polynucleotide wherein the 
formula shows a first strand to which the second strand is complementary. 
In another preferred embodiment m and/or n is an integer between 1 and 
1000. Other preferred embodiments of the invention are provided where m is 
an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 
50, 100, or 500. 
It is most preferred that a polynucleotide of the invention is derived from 
Staphylococcus aureus, however, it may preferably be obtained from other 
organisms of the same taxonomic genus. A polynucleotide of the invention 
may also be obtained, for example, from organisms of the same taxonomic 
family or order. 
The term "polynucleotide encoding a polypeptide" as used herein encompasses 
polynucleotides that include a sequence encoding a polypeptide of the 
invention, particularly a bacterial polypeptide and more particularly a 
polypeptide of the Staphylococcus aureus gidB having an amino acid 
sequence set out in Table 1 [SEQ ID NO:2 or 4]. The term also encompasses 
polynucleotides that include a single continuous region or discontinuous 
regions encoding the polypeptide (for example, polynucleotides interrupted 
by integrated phage, an integrated insertion sequence, an integrated 
vector sequence, an integrated transposon sequence, or due to RNA editing 
or genomic DNA reorganization) together with additional regions, that also 
may contain coding and/or non-coding sequences. 
The invention further relates to variants of the polynucleotides described 
herein that encode variants of a polypeptide having a deduced amino acid 
sequence of Table 1 [SEQ ID NO:2 or 4]. Fragments of a polynucleotides of 
the invention may be used, for example, to synthesize full-length 
polynucleotides of the invention. 
Further particularly preferred embodiments are polynucleotides encoding 
gidB variants, that have the amino acid sequence of gidB polypeptide of 
Table 1 [SEQ ID NO:2 or 4] in which several, a few, 5 to 10, 1 to 5, 1 to 
3, 2, 1 or no amino acid residues are substituted, modified, deleted 
and/or added, in any combination. Especially preferred among these are 
silent substitutions, additions and deletions, that do not alter the 
properties and activities of gidB polypeptide. 
Further preferred embodiments of the invention are polynucleotides that are 
at least 70% identical over their entire length to a polynucleotide 
encoding gidB polypeptide having an amino acid sequence set out in Table 1 
[SEQ ID NO:2 or 4], and polynucleotides that are complementary to such 
polynucleotides. Alternatively, most highly preferred are polynucleotides 
that comprise a region that is at least 80% identical over its entire 
length to a polynucleotide encoding gidB polypeptide and polynucleotides 
complementary thereto. In this regard, polynucleotides at least 90% 
identical over their entire length to the same are particularly preferred, 
and among these particularly preferred polynucleotides, those with at 
least 95% are especially preferred. Furthermore, those with at least 97% 
are highly preferred among those with at least 95%, and among these those 
with at least 98% and at least 99% are particularly highly preferred, with 
at least 99% being the more preferred. 
Preferred embodiments are polynucleotides encoding polypeptides that retain 
substantially the same biological function or activity as the mature 
polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1 or 3]. 
In accordance with certain preferred embodiments of this invention there 
are provided polynucleotides that hybridize, particularly under stringent 
conditions, to gidB polynucleotide sequences, such as those 
polynucleotides in Table 1. 
The invention further relates to polynucleotides that hybridize to the 
polynucleotide sequences provided herein. In this regard, the invention 
especially relates to polynucleotides that hybridize under stringent 
conditions to the polynucleotides described herein. As herein used, the 
terms "stringent conditions" and "stringent hybridization conditions" mean 
hybridization occurring only if there is at least 95% and preferably at 
least 97% identity between the sequences. A specific example of stringent 
hybridization conditions is overnight incubation at 42.degree. C. in a 
solution comprising: 50% formamide, 5.times. SSC (150 mM NaCl, 15 mM 
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's 
solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared 
salmon sperm DNA, followed by washing the hybridization support in 
0.1.times. SSC at about 65.degree. C. Hybridization and wash conditions 
are well known and exemplified in Sambrook, et al., Molecular Cloning: A 
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), 
particularly Chapter 11 therein. Solution hybridization may also be used 
with the polynucleotide sequences provided by the invention. 
The invention also provides a polynucleotide consisting of or comprising a 
polynucleotide sequence obtained by screening an appropriate library 
containing the complete gene for a polynucleotide sequence set forth in 
SEQ ID NO: 1 or 3 under stringent hybridization conditions with a probe 
having the sequence of said polynucleotide sequence set forth in SEQ ID 
NO: 1 or 3 or a fragment thereof; and isolating said polynucleotide 
sequence. Fragments useful for obtaining such a polynucleotide include, 
for example, probes and primers fully described elsewhere herein. 
As discussed elsewhere herein regarding polynucleotide assays of the 
invention, for instance, the polynucleotides of the invention, may be used 
as a hybridization probe for RNA, cDNA and genomic DNA to isolate 
full-length cDNAs and genomic clones encoding gidB and to isolate cDNA and 
genomic clones of other genes that have a high identity, particularly high 
sequence identity, to the gidB gene. Such probes generally will comprise 
at least 15 nucleotide residues or base pairs. Preferably, such probes 
will have at least 30 nucleotide residues or base pairs and may have at 
least 50 nucleotide residues or base pairs. Particularly preferred probes 
will have at least 20 nucleotide residues or base pairs and will have lee 
than 30 nucleotide residues or base pairs. 
A coding region of a gidB gene may be isolated by screening using a DNA 
sequence provided in Table 1 [SEQ ID NO: 1 or 3] to synthesize an 
oligonucleotide probe. A labeled oligonucleotide having a sequence 
complementary to that of a gene of the invention is then used to screen a 
library of cDNA, genomic DNA or mRNA to determine which members of the 
library the probe hybridizes to. 
There are several methods available and well known to those skilled in the 
art to obtain full-length DNAs, or extend short DNAs, for example those 
based on the method of Rapid Amplification of cDNA ends (RACE) (see, for 
example, Frohman, et al., PNAS USA 85. 8998-9002, 1988). Recent 
modifications of the technique, exemplified by the Marathon.TM. technology 
(Clontech Laboratories Inc.) for example, have significantly simplified 
the search for longer cDNAs. In the Marathon.TM. technology, cDNAs have 
been prepared from mRNA extracted from a chosen tissue and an `adaptor` 
sequence ligated onto each end. Nucleic acid amplification (PCR) is then 
carried out to amplify the "missing" 5' end of the DNA using a combination 
of gene specific and adaptor specific oligonucleotide primers. The PCR 
reaction is then repeated using "nested" primers, that is, primers 
designed to anneal within the amplified product (typically an adaptor 
specific primer that anneals further 3' in the adaptor sequence and a gene 
specific primer that anneals further 5' in the known gene sequence). The 
products of this reaction can then be analyzed by DNA sequencing and a 
full-length DNA constructed either by joining the product directly to the 
existing DNA to give a complete sequence, or carrying out a separate 
full-length PCR using the new sequence information for the design of the 
5' primer. 
The polynucleotides and polypeptides of the invention may be employed, for 
example, as research reagents and materials for discovery of treatments of 
and diagnostics for diseases, particularly human diseases, as further 
discussed herein relating to polynucleotide assays. 
The polynucleotides of the invention that are oligonucleotides derived from 
a sequence of Table 1 [SEQ ID NOS: 1 or 2 or 3 or 4] may be used in the 
processes herein as described, but preferably for PCR, to determine 
whether or not the polynucleotides identified herein in whole or in part 
are transcribed in bacteria in infected tissue. It is recognized that such 
sequences will also have utility in diagnosis of the stage of infection 
and type of infection the pathogen has attained. 
The invention also provides polynucleotides that encode a polypeptide that 
is the mature protein plus additional amino or carboxyl-terminal amino 
acids, or amino acids interior to the mature polypeptide (when the mature 
form has more than one polypeptide chain, for instance). Such sequences 
may play a role in processing of a protein from precursor to a mature 
form, may allow protein transport, may lengthen or shorten protein 
half-life or may facilitate manipulation of a protein for assay or 
production, among other things. As generally is the case in vivo, the 
additional amino acids may be processed away from the mature protein by 
cellular enzymes. 
For each and every polynucleotide of the invention there is provided a 
polynucleotide complementary to it. It is preferred that these 
complementary polynucleotides are fully complementary to each 
polynucleotide with which they are complementary. 
A precursor protein, having a mature form of the polypeptide fused to one 
or more prosequences may be an inactive form of the polypeptide. When 
prosequences are removed such inactive precursors generally are activated. 
Some or all of the prosequences may be removed before activation. 
Generally, such precursors are called proproteins. 
In addition to the standard A, G, C, T/U representations for nucleotides, 
the term "N" may also be used in describing certain polynucleotides of the 
invention. "N" means that any of the four DNA or RNA nucleotides may 
appear at such a designated position in the DNA or RNA sequence, except it 
is preferred that N is not a nucleic acid that when taken in combination 
with adjacent nucleotide positions, when read in the correct reading 
frame, would have the effect of generating a premature termination codon 
in such reading frame. 
In sum, a polynucleotide of the invention may encode a mature protein, a 
mature protein plus a leader sequence (which may be referred to as a 
preprotein), a precursor of a mature protein having one or more 
prosequences that are not the leader sequences of a preprotein, or a 
preproprotein, which is a precursor to a proprotein, having a leader 
sequence and one or more prosequences, which generally are removed during 
processing steps that produce active and mature forms of the polypeptide. 
Vectors, Host Cells, Expression Systems 
The invention also relates to vectors that comprise a polynucleotide or 
polynucleotides of the invention, host cells that are genetically 
engineered with vectors of the invention and the production of 
polypeptides of the invention by recombinant techniques. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the invention. 
Recombinant polypeptides of the present invention may be prepared by 
processes well known in those skilled in the art from genetically 
engineered host cells comprising expression systems. Accordingly, in a 
further aspect, the present invention relates to expression systems which 
comprise a polynucleotide or polynucleotides of the present invention, to 
host cells which are genetically engineered with such expression systems, 
and to the production of polypeptides of the invention by recombinant 
techniques. 
For recombinant production of the polypeptides of the invention, host cells 
can be genetically engineered to incorporate expression systems or 
portions thereof or polynucleotides of the invention. Introduction of a 
polynucleotide into the host cell can be effected by methods described in 
many standard laboratory manuals, such as Davis, et al., BASIC METHODS IN 
MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: A 
LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold 
Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, 
DEAE-dextran mediated transfection, transvection, microinjection, cationic 
lipid-mediated transfection, electroporation, transduction, scrape 
loading, ballistic introduction and infection. 
Representative examples of appropriate hosts include bacterial cells, such 
as cells of streptococci, staphylococci, enterococci E. coli, 
streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcus aureus; 
fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a 
basidiomycete, Candida albicans and Aspergillus; insect cells such as 
cells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, 
HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, 
such as cells of a gymnosperm or angiosperm. 
A great variety of expression systems can be used to produce the 
polypeptides of the invention. Such vectors include, among others, 
chromosomal-, episomal- and virus-derived vectors, for example, vectors 
derived from bacterial plasmids, from bacteriophage, from transposons, 
from yeast episomes, from insertion elements, from yeast chromosomal 
elements, from viruses such as baculoviruses, papova viruses, such as 
SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies 
viruses, picornaviruses and retroviruses, and vectors derived from 
combinations thereof, such as those derived from plasmid and bacteriophage 
genetic elements, such as cosmids and phagemids. The expression system 
constructs may contain control regions that regulate as well as engender 
expression. Generally, any system or vector suitable to maintain, 
propagate or express polynucleotides and/or to express a polypeptide in a 
host may be used for expression in this regard. The appropriate DNA 
sequence may be inserted into the expression system by any of a variety of 
well-known and routine techniques, such as, for example, those set forth 
in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra). 
In recombinant expression systems in eukaryotes, for secretion of a 
translated protein into the lumen of the endoplasmic reticulum, into the 
periplasmic space or into the extracellular environment, appropriate 
secretion signals may be incorporated into the expressed polypeptide. 
These signals may be endogenous to the polypeptide or they may be 
heterologous signals. 
Polypeptides of the invention can be recovered and purified from 
recombinant cell cultures by well-known methods including ammonium sulfate 
or ethanol precipitation, acid extraction, anion or cation exchange 
chromatography, phosphocellulose chromatography, hydrophobic interaction 
chromatography, affinity chromatography, hydroxylapatite chromatography, 
and lectin chromatography. Most preferably, high performance liquid 
chromatography is employed for purification. Well known techniques for 
refolding protein may be employed to regenerate active conformation when 
the polypeptide is denatured during isolation and or purification. 
Diagnostic, Prognostic, Serotyping and Mutation Assays 
This invention is also related to the use of gidB polynucleotides and 
polypeptides of the invention for use as diagnostic reagents. Detection of 
gidB polynucleotides and/or polypeptides in a eukaryote, particularly a 
mammal, and especially a human, will provide a diagnostic method for 
diagnosis of disease, staging of disease or response of an infectious 
organism to drugs. Eukaryotes, particularly mammals, and especially 
humans, particularly those infected or suspected to be infected with an 
organism comprising the gidB gene or protein, may be detected at the 
nucleic acid or amino acid level by a variety of well known techniques as 
well as by methods provided herein. 
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis 
may be obtained from a putatively infected and/or infected individual's 
bodily materials. Polynucleotides from any of these sources, particularly 
DNA or RNA, may be used directly for detection or may be amplified 
enzymatically by using PCR or any other amplification technique prior to 
analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in 
the same ways. Using amplification, characterization of the species and 
strain of infectious or resident organism present in an individual, may be 
made by an analysis of the genotype of a selected polynucleotide of the 
organism. Deletions and insertions can be detected by a change in size of 
the amplified product in comparison to a genotype of a reference sequence 
selected from a related organism, preferably a different species of the 
same genus or a different strain of the same species. Point mutations can 
be identified by hybridizing amplified DNA to labeled gidB polynucleotide 
sequences. Perfectly or significantly matched sequences can be 
distinguished from imperfectly or more significantly mismatched duplexes 
by DNase or RNase digestion, for DNA or RNA respectively, or by detecting 
differences in melting temperatures or renaturation kinetics. 
Polynucleotide sequence differences may also be detected by alterations in 
the electrophoretic mobility of polynucleotide fragments in gels as 
compared to a reference sequence. This may be carried out with or without 
denaturing agents. Polynucleotide differences may also be detected by 
direct DNA or RNA sequencing. See, for example, Myers et al., Science, 
230: 1242 (1985). Sequence changes at specific locations also may be 
revealed by nuclease protection assays, such as RNase, V1 and S1 
protection assay or a chemical cleavage method. See, for example, Cotton 
et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985). 
In another embodiment, an array of oligonucleotides probes comprising gidB 
nucleotide sequence or fragments thereof can be constructed to conduct 
efficient screening of, for example, genetic mutations, serotype, 
taxonomic classification or identification. Array technology methods are 
well known and have general applicability and can be used to address a 
variety of questions in molecular genetics including gene expression, 
genetic linkage, and genetic variability (see, for example, Chee et al., 
Science, 274. 610 (1996)). 
Thus in another aspect, the present invention relates to a diagnostic kit 
which comprises: 
(a) a polynucleotide of the present invention, preferably the nucleotide 
sequence of SEQ ID NO: 1 or 3, or a fragment thereof; 
(b) a nucleotide sequence complementary to that of (a); 
(c) a polypeptide of the present invention, preferably the polypeptide of 
SEQ ID NO:2 or 4 or a fragment thereof; or 
(d) an antibody to a polypeptide of the present invention, preferably to 
the polypeptide of SEQ ID NO:2 or 4. 
It will be appreciated that in any such kit, (a), (b), (c) or (d) may 
comprise a substantial component. Such a kit will be of use in diagnosing 
a disease or susceptibility to a Disease, among others. 
This invention also relates to the use of polynucleotides of the present 
invention as diagnostic reagents. Detection of a mutated form of a 
polynucleotide of the invention, preferable, SEQ ID NO: 1 or 3, which is 
associated with a disease or pathogenicity will provide a diagnostic tool 
that can add to, or define, a diagnosis of a disease, a prognosis of a 
course of disease, a determination of a stage of disease, or a 
susceptibility to a disease, which results from under-expression, 
over-expression or altered expression of the polynucleotide. Organisms, 
particularly infectious organisms, carrying mutations in such 
polynucleotide may be detected at the polynucleotide level by a variety of 
techniques, such as those described elsewhere herein. 
The nucleotide sequences of the present invention are also valuable for 
organism chromosome identification. The sequence is specifically targeted 
to, and can hybridize with, a particular location on an organism's 
chromosome, particularly to a Staphylococcus aureus chromosome. The 
mapping of relevant sequences to chromosomes according to the present 
invention may be an important step in correlating those sequences with 
pathogenic potential and/or an ecological niche of an organism and/or drug 
resistance of an organism, as well as the essentiality of the gene to the 
organism. Once a sequence has been mapped to a precise chromosomal 
location, the physical position of the sequence on the chromosome can be 
correlated with genetic map data. Such data may be found on-line in a 
sequence database. The relationship between genes and diseases that have 
been mapped to the same chromosomal region are then identified through 
known genetic methods, for example, through linkage analysis 
(coinheritance of physically adjacent genes) or mating studies, such as by 
conjugation. 
The differences in a polynucleotide and/or polypeptide sequence between 
organisms possessing a first phenotype and organisms possessing a 
different, second different phenotype can also be determined. If a 
mutation is observed in some or all organisms possessing the first 
phenotype but not in any organisms possessing the second phenotype, then 
the mutation is likely to be the causative agent of the first phenotype. 
Cells from an organism carrying mutations or polymorphisms (allelic 
variations) in a polynucleotide and/or polypeptide of the invention may 
also be detected at the polynucleotide or polypeptide level by a variety 
of techniques, to allow for serotyping, for example. For example, RT-PCR 
can be used to detect mutations in the RNA. It is particularly preferred 
to use RT-PCR in conjunction with automated detection systems, such as, 
for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the 
same purpose, PCR. As an example, PCR primers complementary to a 
polynucleotide encoding gidB polypeptide can be used to identify and 
analyze mutations. Examples of representative primers are shown below in 
Table 
TABLE 2 
______________________________________ 
Primers for amplification of gidB polynucleotides 
SEQ ID NO PRIMER SEQUENCE 
______________________________________ 
5 5'-ATGACTGTAGAATGGTTAGC-3' 
6 5'-TTATTTTTCAAGTAAAGGAG-3' 
______________________________________ 
The invention also includes primers of the formula: 
EQU X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y 
wherein, at the 5' end of the molecule, X is hydrogen, a metal or a 
modified nucleotide residue, and at the 3' end of the molecule, Y is 
hydrogen, a metal or a modified nucleotide residue, R.sub.1 and R.sub.3 
are any nucleic acid residue or modified nucleotide residue, m is an 
integer between 1 and 20 or zero , n is an integer between 1 and 20 or 
zero, and R.sub.2 is a primer sequence of the invention, particularly a 
primer sequence selected from Table 2. In the polynucleotide formula above 
R.sub.2 is oriented so that its 5' end nucleotide residue is at the left, 
bound to R.sub.1, and its 3' end nucleotide residue is at the right, bound 
to R.sub.3. Any stretch of nucleic acid residues denoted by either R 
group, where m and/or n is greater than 1, may be either a heteropolymer 
or a homopolymer, preferably a heteropolymer being complementary to a 
region of a polynucleotide of Table 1. In a preferred embodiment m and/or 
n is an integer between 1 and 10. 
The invention further provides these primers with 1, 2, 3 or 4 nucleotides 
removed from the 5' and/or the 3' end. These primers may be used for, 
among other things, amplifying gidB DNA and/or RNA isolated from a sample 
derived from an individual, such as a bodily material. The primers may be 
used to amplify a polynucleotide isolated from an infected individual, 
such that the polynucleotide may then be subject to various techniques for 
elucidation of the polynucleotide sequence. In this way, mutations in the 
polynucleotide sequence may be detected and used to diagnose and/or 
prognose the infection or its stage or course, or to serotype and/or 
classify the infectious agent. 
The invention further provides a process for diagnosing, disease, 
preferably bacterial infections, more preferably infections caused by 
Staphylococcus aureus, comprising determining from a sample derived from 
an individual, such as a bodily material, an increased level of expression 
of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1 or 3]. 
Increased or decreased expression of a gidB polynucleotide can be measured 
using any on of the methods well known in the art for the quantitation of 
polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase 
protection, Northern blotting, spectrometry and other hybridization 
methods. 
In addition, a diagnostic assay in accordance with the invention for 
detecting over-expression of gidB polypeptide compared to normal control 
tissue samples may be used to detect the presence of an infection, for 
example. Assay techniques that can be used to determine levels of a gidB 
polypeptide, in a sample derived from a host, such as a bodily material, 
are well-known to those of skill in the art. Such assay methods include 
radioimmunoassays, competitive-binding assays, Western Blot analysis, 
antibody sandwich assays, antibody detection and ELISA assays. 
Differential Expression 
The polynucleotides and polynucleotides of the invention may be used as 
reagents for differential screening methods. There are many differential 
screening and differential display methods known in the art in which the 
polynucleotides and polypeptides of the invention may be used. For 
example, the differential display technique is described by Chuang et al., 
J. Bacreriol. 175:2026-2036 (1993). This method identifies those genes 
which are expressed in an organism by identifying mRNA present using 
randomly-primed RT-PCR. By comparing pre-infection and post infection 
profiles, genes up and down regulated during infection can be identified 
and the RT-PCR product sequenced and matched to ORF "unknowns." 
In Vivo Expression Technology (IVET) is described by Camilli et al., Proc. 
Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genes 
up-regulated during infection when compared to laboratory cultivation, 
implying an important role in infection. ORFs identified by this technique 
are implied to have a significant role in infection establishment and/or 
maintenance. In this technique random chromosomal fragments of target 
organism are cloned upstream of a promoter-less recombinase gene in a 
plasmid vector. This construct is introduced into the target organism 
which carries an antibiotic resistance gene flanked by resolvase sites. 
Growth in the presence of the antibiotic removes from the population those 
fragments cloned into the plasmid vector capable of supporting 
transcription of the recombinase gene and therefore have caused loss of 
antibiotic resistance. The resistant pool is introduced into a host and at 
various times after infection bacteria may be recovered and assessed for 
the presence of antibiotic resistance. The chromosomal fragment carried by 
each antibiotic sensitive bacterium should carry a promoter or portion of 
a gene normally upregulated during infection. Sequencing upstream of the 
recombinase gene allows identification of the up regulated gene. 
RT-PCR may also be used to analyze gene expression patterns. For RT PCR 
using the polynucleotides of the invention, messenger RNA is isolated from 
bacterial infected tissue, e.g., 48 hour murine lung infections, and the 
amount of each mRNA species assessed by reverse transcription of the RNA 
sample primed with random hexanucleotides followed by PCR with gene 
specific primer pairs. The determination of the presence and amount of a 
particular mRNA species by quantification of the resultant PCR product 
provides information on the bacterial genes which are transcribed in the 
infected tissue. Analysis of gene transcription can be carried out at 
different times of infection to gain a detailed knowledge of gene 
regulation in bacterial pathogenesis allowing for a clearer understanding 
of which gene products represent targets for screens for antibacterials. 
Because of the gene specific nature of the PCR primers employed it should 
be understood that the bacterial mRNA preparation need not be free of 
mammalian RNA. This allows the investigator to carry out a simple and 
quick RNA preparation from infected tissue to obtain bacterial mRNA 
species which are very short lived in the bacterium (in the order of 2 
minute halflives). Optimally the bacterial mRNA is prepared from infected 
murine lung tissue by mechanical disruption in the presence of TRIzole 
(GIBCO-BRL) for very short periods of time, subsequent processing 
according to the manufacturers of TRizole reagent and DNAase treatment to 
remove contaminating DNA. Preferably the process is optimized by finding 
those conditions which give a maximum amount of Staphylococcus aureus 16S 
ribosomal RNA as detected by probing Northerns with a suitably labeled 
sequence specific oligonucleotide probe. Typically a 5' dye labeled primer 
is used in each PCR primer pair in a PCR reaction which is terminated 
optimally between 8 and 25 cycles. The PCR products are separated on 6% 
polyacrylamide gels with detection and quantification using GeneScanner 
(manufactured by ABI). 
Gridding and Polynucleotide Subtraction 
Methods have been described for obtaining information about gene expression 
and identity using so called "high density DNA arrays" or grids. See, 
e.g., M. Chee et al., Science, 274:610-614 (1996) and other references 
cited therein. Such gridding assays have been employed to identify certain 
novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams 
et a., Science, 252:1651-1656 (1991)). A variety of techniques have also 
been described for identifying particular gene sequences on the basis of 
their gene products. For example, see International Patent Application No. 
WO91/07087, published May 30, 1991. In addition, methods have been 
described for the amplification of desired sequences. For example, see 
International Patent Application No. WO91/17271, published Nov. 14, 1991. 
The polynucleotides of the invention may be used as components of 
polynucleotide arrays, preferably high density arrays or grids. These high 
density arrays are particularly useful for diagnostic and prognostic 
purposes. For example, a set of spots each comprising a different gene, 
and further comprising a polynucleotide or polynucleotides of the 
invention, may be used for probing, such as using hybridization or nucleic 
acid amplification, using a probes obtained or derived from a bodily 
sample, to determine the presence of a particular polynucleotide sequence 
or related sequence in an individual. Such a presence may indicate the 
presence of a pathogen, particularly Staphylococcus aureus, and may be 
useful in diagnosing and/or prognosing disease or a course of disease. A 
grid comprising a number of variants of the polynucleotide sequence of SEQ 
ID NO: 1 or 3 are preferred. Also preferred is a comprising a number of 
variants of a polynucleotide sequence encoding the polypeptide sequence of 
SEQ ID NO:2 or 4. 
Antibodies 
The polypeptides and polynucleotides of the invention or variants thereof, 
or cells expressing the same can be used as immunogens to produce 
antibodies immunospecific for such polypeptides or polynucleotides 
respectively. 
In certain preferred embodiments of the invention there are provided 
antibodies against gidB polypeptides or polynucleotides. 
Antibodies generated against the polypeptides or polynucleotides of the 
invention can be obtained by administering the polypeptides and/or 
polynucleotides of the invention, or epitope-bearing fragments of either 
or both, analogues of either or both, or cells expressing either or both, 
to an animal, preferably a nonhuman, using routine protocols. For 
preparation of monoclonal antibodies, any technique known in the art that 
provides antibodies produced by continuous cell line cultures can be used. 
Examples include various techniques, such as those in Kohler, G. and 
Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 
4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER 
THERAPY, Alan R. Liss, Inc. (1985). 
Techniques for the production of single chain antibodies (U.S. Pat. No. 
4,946,778) can be adapted to produce single chain antibodies to 
polypeptides or polynucleotides of this invention. Also, transgenic mice, 
or other organisms such as other mammals, may be used to express humanized 
antibodies immunospecific to the polypeptides or polynucleotides of the 
invention. 
Alternatively, phage display technology may be utilized to select antibody 
genes with binding activities towards a polypeptide of the invention 
either from repertoires of PCR amplified v-genes of lymphocytes from 
humans screened for possessing anti-gidB or from naive libraries 
(McCafferty, et al., (1990), Nature 348, 552-554; Marks, et al., (1992) 
Biotechnology 10, 779-783). The affinity of these antibodies can also be 
improved by, for example, chain shuffling (Clackson et al., (1991) Nature 
352: 628). 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptides or polynucleotides of the invention to 
purify the polypeptides or polynucleotides by, for example, affinity 
chromatography. 
Thus, among others, antibodies against gidB-polypeptide or 
gidB-polynucleotide may be employed to treat infections, particularly 
bacterial infections. 
Polypeptide variants include antigenically, epitopically or immunologically 
equivalent variants form a particular aspect of this invention. 
A polypeptide or polynucleotide of the invention, such as an antigenically 
or immunologically equivalent derivative or a fusion protein of the 
polypeptide is used as an antigen to immunize a mouse or other animal such 
as a rat or chicken. The fusion protein may provide stability to the 
polypeptide. The antigen may be associated, for example by conjugation, 
with an immunogenic carrier protein for example bovine serum albumin, 
keyhole limpet haemocyanin or tetanus toxoid. Alternatively, a multiple 
antigenic polypeptide comprising multiple copies of the polypeptide, or an 
antigenically or immunologically equivalent polypeptide thereof may be 
sufficiently antigenic to improve immunogenicity so as to obviate the use 
of a carrier. 
Preferably, the antibody or variant thereof is modified to make it less 
immunogenic in the individual. For example, if the individual is human the 
antibody may most preferably be "humanized," where the complimentarity 
determining region or regions of the hybridoma-derived antibody has been 
transplanted into a human monoclonal antibody, for example as described in 
Jones et al. (1986), Nature 321, 522-525 or Tempest et al., (1991) 
Biotechnology 9, 266-273. 
In accordance with an aspect of the invention, there is provided the use of 
a polynucleotide of the invention for therapeutic or prophylactic 
purposes, in particular genetic immunization. Among the particularly 
preferred embodiments of the invention are naturally occurring allelic 
variants of gidB polynucleotides and polypeptides encoded thereby. 
The use of a polynucleotide of the invention in genetic immunization will 
preferably employ a suitable delivery method such as direct injection of 
plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, 
Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery of DNA 
complexed with specific protein carriers (Wu et al., J Biol Chem. (1989) 
264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & 
Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms 
of liposomes (Kaneda et al., Science (1989) 243:375), particle bombardment 
(Tang et al., Nature (1992) 356:152, Eisenbraun et al., DNA Cell Biol 
(1993) 12: 791) and in vivo infection using cloned retroviral vectors 
(Seeger et al., PNAS USA (1984) 81: 5849). 
Antagonists and Agonists--Assays and Molecules 
Polypeptides and polynucleotides of the invention may also be used to 
assess the binding of small molecule substrates and ligands in, for 
example, cells, cell-free preparations, chemical libraries, and natural 
product mixtures. These substrates and ligands may be natural substrates 
and ligands or may be structural or functional mimetics. See, e.g., 
Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991). 
Polypeptides and polynucleotides of the present invention are responsible 
for many biological functions, including many disease states, in 
particular the Diseases hereinbefore mentioned. It is therefore desirable 
to devise screening methods to identify compounds which stimulate or which 
inhibit the function of the polypeptide or polynucleotide. Accordingly, in 
a further aspect, the present invention provides for a method of screening 
compounds to identify those which stimulate or which inhibit the function 
of a polypeptide or polynucleotide of the invention, as well as related 
polypeptides and polynucleotides. In general, agonists or antagonists may 
be employed for therapeutic and prophylactic purposes for such Diseases as 
hereinbefore mentioned. Compounds may be identified from a variety of 
sources, for example, cells, cell-free preparations, chemical libraries, 
and natural product mixtures. Such agonists, antagonists or inhibitors 
so-identified may be natural or modified substrates, ligands, receptors, 
enzymes, etc., as the case may be, of gidB polypeptides and 
polynucleotides; or may be structural or functional mimetics thereof (see 
Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). 
The screening methods may simply measure the binding of a candidate 
compound to the polypeptide or polynucleotide, or to cells or membranes 
bearing the polypeptide or polynucleotide, or a fusion protein of the 
polypeptide by means of a label directly or indirectly associated with the 
candidate compound. Alternatively, the screening method may involve 
competition with a labeled competitor. Further, these screening methods 
may test whether the candidate compound results in a signal generated by 
activation or inhibition of the polypeptide or polynucleotide, using 
detection systems appropriate to the cells comprising the polypeptide or 
polynucleotide. Inhibitors of activation are generally assayed in the 
presence of a known agonist and the effect on activation by the agonist by 
the presence of the candidate compound is observed. Constitutively active 
polypeptide and/or constitutively expressed polypeptides and 
polynucleotides may be employed in screening methods for inverse agonists 
or inhibitors, in the absence of an agonist or inhibitor, by testing 
whether the candidate compound results in inhibition of activation of the 
polypeptide or polynucleotide, as the case may be. Further, the screening 
methods may simply comprise the steps of mixing a candidate compound with 
a solution containing a polypeptide or polynucleotide of the present 
invention, to form a mixture, measuring gidB polypeptide and/or 
polynucleotide activity in the mixture, and comparing the gidB polypeptide 
and/or polynucleotide activity of the mixture to a standard. Fusion 
proteins, such as those made from Fc portion and gidB polypeptide, as 
hereinbefore described, can also be used for high-throughput screening 
assays to identify antagonists of the polypeptide of the present 
invention, as well as of phylogenetically and and/or functionally related 
polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); 
and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)). 
The polynucleotides, polypeptides and antibodies that bind to and/or 
interact with a polypeptide of the present invention may also be used to 
configure screening methods for detecting the effect of added compounds on 
the production of mRNA and/or polypeptide in cells. For example, an ELISA 
assay may be constructed for measuring secreted or cell associated levels 
of polypeptide using monoclonal and polyclonal antibodies by standard 
methods known in the art. This can be used to discover agents which may 
inhibit or enhance the production of polypeptide (also called antagonist 
or agonist, respectively) from suitably manipulated cells or tissues. 
The invention also provides a method of screening compounds to identify 
those which enhance (agonist) or block (antagonist) the action of gidB 
polypeptides or polynucleotides, particularly those compounds that are 
bacteristatic and/or bactericidal. The method of screening may involve 
high-throughput techniques. For example, to screen for agonists or 
antagonists, a synthetic reaction mix, a cellular compartment, such as a 
membrane, cell envelope or cell wall, or a preparation of any thereof, 
comprising gidB polypeptide and a labeled substrate or ligand of such 
polypeptide is incubated in the absence or the presence of a candidate 
molecule that may be a gidB agonist or antagonist. The ability of the 
candidate molecule to agonize or antagonize the gidB polypeptide is 
reflected in decreased binding of the labeled ligand or decreased 
production of product from such substrate. Molecules that bind 
gratuitously, i.e., without inducing the effects of gidB polypeptide are 
most likely to be good antagonists. Molecules that bind well and, as the 
case may be, increase the rate of product production from substrate, 
increase signal transduction, or increase chemical channel activity are 
agonists. Detection of the rate or level of, as the case may be, 
production of product from substrate, signal transduction, or chemical 
channel activity may be enhanced by using a reporter system. Reporter 
systems that may be useful in this regard include but are not limited to 
colorimetric, labeled substrate converted into product, a reporter gene 
that is responsive to changes in gidB polynucleotide or polypeptide 
activity, and binding assays known in the art. 
Polypeptides of the invention may be used to identify membrane bound or 
soluble receptors, if any, for such polypeptide, through standard receptor 
binding techniques known in the art. These techniques include, but are not 
limited to, ligand binding and crosslinking assays in which the 
polypeptide is labeled with a radioactive isotope (for instance, .sup.125 
I), chemically modified (for instance, biotinylated), or fused to a 
peptide sequence suitable for detection or purification, and incubated 
with a source of the putative receptor (e.g., cells, cell membranes, cell 
supernatants, tissue extracts, bodily materials). Other methods include 
biophysical techniques such as surface plasmon resonance and spectroscopy. 
These screening methods may also be used to identify agonists and 
antagonists of the polypeptide which compete with the binding of the 
polypeptide to its receptor(s), if any. Standard methods for conducting 
such assays are well understood in the art. 
The fluorescence polarization value for a fluorescently-tagged molecule 
depends on the rotational correlation time or tumbling rate. Protein 
complexes, such as formed by gidB polypeptide associating with another 
gidB polypeptide or other polypeptide, labeled to comprise a 
fluorescently-labeled molecule will have higher polarization values than a 
fluorescently labeled monomeric protein. It is preferred that this method 
be used to characterize small molecules that disrupt polypeptide 
complexes. 
Fluorescence energy transfer may also be used characterize small molecules 
that interfere with the formation of gidB polypeptide dimers, trimers, 
tetramers or higher order structures, or structures formed by gidB 
polypeptide bound to another polypeptide. gidB polypeptide can be labeled 
with both a donor and acceptor fluorophore. Upon mixing of the two labeled 
species and excitation of the donor fluorophore, fluorescence energy 
transfer can be detected by observing fluorescence of the acceptor. 
Compounds that block dimerization will inhibit fluorescence energy 
transfer. 
Surface plasmon resonance can be used to monitor the effect of small 
molecules on gidB polypeptide self-association as well as an association 
of gidB polypeptide and another polypeptide or small molecule. gidB 
polypeptide can be coupled to a sensor chip at low site density such that 
covalently bound molecules will be monomeric. Solution protein can then 
passed over the gidB polypeptide -coated surface and specific binding can 
be detected in real-time by monitoring the change in resonance angle 
caused by a change in local refractive index. This technique can be used 
to characterize the effect of small molecules on kinetic rates and 
equilibrium binding constants for gidB polypeptide self-association as 
well as an association of gidB polypeptide and another polypeptide or 
small molecule. 
A scintillation proximity assay may be used to characterize the interaction 
between an association of gidB polypeptide with another gidB polypeptide 
or a different polypeptide . gidB polypeptide can be coupled to a 
scintillation-filled bead. Addition of radio-labeled gidB polypeptide 
results in binding where the radioactive source molecule is in close 
proximity to the scintillation fluid. Thus, signal is emitted upon gidB 
polypeptide binding and compounds that prevent gidB polypeptide 
self-association or an association of gidB polypeptide and another 
polypeptide or small molecule will diminish signal. 
ICS biosensors have been described by AMBR.sub.1 (Australian Membrane 
Biotechnology Research Institute). They couple the self-association of 
macromolecules to the closing of gramacidin-facilitated ion channels in 
suspended membrane bilayers and hence to a measurable change in the 
admittance (similar to impedence) of the biosensor. This approach is 
linear over six decades of admittance change and is ideally suited for 
large scale, high through-put screening of small molecule combinatorial 
libraries. 
In other embodiments of the invention there are provided methods for 
identifying compounds which bind to or otherwise interact with and inhibit 
or activate an activity or expression of a polypeptide and/or 
polynucleotide of the invention comprising: contacting a polypeptide 
and/or polynucleotide of the invention with a compound to be screened 
under conditions to permit binding to or other interaction between the 
compound and the polypeptide and/or polynucleotide to assess the binding 
to or other interaction with the compound, such binding or interaction 
preferably being associated with a second component capable of providing a 
detectable signal in response to the binding or interaction of the 
polypeptide and/or polynucleotide with the compound; and determining 
whether the compound binds to or otherwise interacts with and activates or 
inhibits an activity or expression of the polypeptide and/or 
polynucleotide by detecting the presence or absence of a signal generated 
from the binding or interaction of the compound with the polypeptide 
and/or polynucleotide. 
Another example of an assay for gidB agonists is a competitive assay that 
combines gidB and a potential agonist with gidB-binding molecules, 
recombinant gidB binding molecules, natural substrates or ligands, or 
substrate or ligand mimetics, under appropriate conditions for a 
competitive inhibition assay. gidB can be labeled, such as by 
radioactivity or a colorimetric compound, such that the number of gidB 
molecules bound to a binding molecule or converted to product can be 
determined accurately to assess the effectiveness of the potential 
antagonist. 
Potential antagonists include, among others, small organic molecules, 
peptides, polypeptides and antibodies that bind to a polynucleotide and/or 
polypeptide of the invention and thereby inhibit or extinguish its 
activity or expression. Potential antagonists also may be small organic 
molecules, a peptide, a polypeptide such as a closely related protein or 
antibody that binds the same sites on a binding molecule, such as a 
binding molecule, without inducing gidB-induced activities, thereby 
preventing the action or expression of gidB polypeptides and/or 
polynucleotides by excluding gidB polypeptides and/or polynucleotides from 
binding. 
Potential antagonists include a small molecule that binds to and occupies 
the binding site of the polypeptide thereby preventing binding to cellular 
binding molecules, such that normal biological activity is prevented. 
Examples of small molecules include but are not limited to small organic 
molecules, peptides or peptide-like molecules. Other potential antagonists 
include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); 
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC 
Press, Boca Raton, Fla. (1988), for a description of these molecules). 
Preferred potential antagonists include compounds related to and variants 
of gidB. 
Other examples of potential polypeptide antagonists include antibodies or, 
in some cases, oligonucleotides or proteins which are closely related to 
the ligands, substrates, receptors, enzymes, etc., as the case may be, of 
the polypeptide, e.g., a fragment of the ligands, substrates, receptors, 
enzymes, etc.; or small molecules which bind to the polypeptide of the 
present invention but do not elicit a response, so that the activity of 
the polypeptide is prevented. 
Certain of the polypeptides of the invention are biomimetics, functional 
mimetics of the natural gidB polypeptide. These functional mimetics may be 
used for, among other things, antagonizing the activity of gidB 
polypeptide or as a antigen or immunogen in a manner described elsewhere 
herein. Functional mimetics of the polypeptides of the invention include 
but are not limited to truncated polypeptides. For example, preferred 
functional mimetics include, a polypeptide comprising the polypeptide 
sequence set forth in SEQ ID NO:2 lacking 20, 30, 40, 50, 60, 70 or 80 
amino- or carboxy-terminal amino acid residues, including fusion proteins 
comprising one or more of these truncated sequences. Polynucleotides 
encoding each of these functional mimetics may be used as expression 
cassettes to express each mimetic polypeptide. It is preferred that these 
cassettes comprise 5' and 3' restriction sites to allow for a convenient 
means to ligate the cassettes together when desired. It is further 
preferred that these cassettes comprise gene expression signals known in 
the art or described elsewhere herein. 
Thus, in another aspect, the present invention relates to a screening kit 
for identifying agonists, antagonists, ligands, receptors, substrates, 
enzymes, etc. for a polypeptide and/or polynucleotide of the present 
invention; or compounds which decrease or enhance the production of such 
polypeptides and/or polynucleotides , which comprises: 
(a) a polypeptide and/or a polynucleotide of the present invention; 
(b) a recombinant cell expressing a polypeptide and/or polynucleotide of 
the present invention; 
(c) a cell membrane expressing a polypeptide and/or polynucleotide of the 
present invention; or 
(d) antibody to a polypeptide and/or polynucleotide of the present 
invention; 
which polypeptide is preferably that of SEQ ID NO:2, and which 
polynucleotide is preferably that of SEQ ID NO: 1. 
It will be appreciated that in any such kit, (a), (b), (c) or (d) may 
comprise a substantial component. 
It will be readily appreciated by the skilled artisan that a polypeptide 
and/or polynucleotide of the present invention may also be used in a 
method for the structure-based design of an agonist, antagonist or 
inhibitor of the polypeptide and/or polynucleotide, by: 
(a) determining in the first instance the three-dimensional structure of 
the polypeptide and/or polynucleotide, or complexes thereof; 
(b) deducing the three-dimensional structure for the likely reactive 
site(s), binding site(s) or motif(s) of an agonist, antagonist or 
inhibitor; 
(c) synthesizing candidate compounds that are predicted to bind to or react 
with the deduced binding site(s), reactive site(s), and/or motif(s); and 
(d) testing whether the candidate compounds are indeed agonists, 
antagonists or inhibitors. 
It will be further appreciated that this will normally be an iterative 
process, and this iterative process may be performed using automated and 
computer-controlled steps. 
In a further aspect, the present invention provides methods of treating 
abnormal conditions such as, for instance, a Disease, related to either an 
excess of, an under-expression of, an elevated activity of, or a decreased 
activity of gidB polypeptide and/or polynucleotide. 
If the expression and/or activity of the polypeptide and/or polynucleotide 
is in excess, several approaches are available. One approach comprises 
administering to an individual in need thereof an inhibitor compound 
(antagonist) as herein described, optionally in combination with a 
pharmaceutically acceptable carrier, in an amount effective to inhibit the 
function and/or expression of the polypeptide and/or polynucleotide, such 
as, for example, by blocking the binding of ligands, substrates, 
receptors, enzymes, etc., or by inhibiting a second signal, and thereby 
alleviating the abnormal condition. In another approach, soluble forms of 
the polypeptides still capable of binding the ligand, substrate, enzymes, 
receptors, etc. in competition with endogenous polypeptide and/or 
polynucleotide may be administered. Typical examples of such competitors 
include fragments of the gidB polypeptide and/or polypeptide. 
In a further aspect, the present invention relates to genetically 
engineered soluble fusion proteins comprising a polypeptide of the present 
invention, or a fragment thereof, and various portions of the constant 
regions of heavy or light chains of immunoglobulins of various subclasses 
(IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part 
of the heavy chain of human IgG, particularly IgG1, where fusion takes 
place at the hinge region. In a particular embodiment, the Fc part can be 
removed simply by incorporation of a cleavage sequence which can be 
cleaved with blood clotting factor Xa. Furthermore, this invention relates 
to processes for the preparation of these fusion proteins by genetic 
engineering, and to the use thereof for drug screening, diagnosis and 
therapy. A further aspect of the invention also relates to polynucleotides 
encoding such fusion proteins. Examples of fusion protein technology can 
be found in International Patent Application Nos. WO94/29458 and 
WO94/22914. 
In still another approach, expression of the gene encoding endogenous gidB 
polypeptide can be inhibited using expression blocking techniques. This 
blocking may be targeted against any step in gene expression, but is 
preferably targeted against transcription and/or translation. 
An examples of a known technique of this sort involve the use of antisense 
sequences, either internally generated or separately administered (see, 
for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides 
as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. 
(1988)). Alternatively, oligonucleotides which form triple helices with 
the gene can be supplied (see, for example, Lee et al., Nucleic Acids Res 
(1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., 
Science (1991) 251:1360). These oligomers can be administered per se or 
the relevant oligomers can be expressed in vivo. 
Each of the polynucleotide sequences provided herein may be used in the 
discovery and development of antibacterial compounds. The encoded protein, 
upon expression, can be used as a target for the screening of 
antibacterial drugs. Additionally, the polynucleotide sequences encoding 
the amino terminal regions of the encoded protein or Shine-Delgarno or 
other translation facilitating sequences of the respective mRNA can be 
used to construct antisense sequences to control the expression of the 
coding sequence of interest. 
The invention also provides the use of the polypeptide, polynucleotide, 
agonist or antagonist of the invention to interfere with the initial 
physical interaction between a pathogen or pathogens and a eukaryotic, 
preferably mammalian, host responsible for sequelae of infection. In 
particular, the molecules of the invention may be used: in the prevention 
of adhesion of bacteria, in particular gram positive and/or gram negative 
bacteria, to eukaryotic, preferably mammalian, extracellular matrix 
proteins on in-dwelling devices or to extracellular matrix proteins in 
wounds; to block gidB protein-mediated mammalian cell invasion by, for 
example, initiating phosphorylation of mammalian tyrosine kinases 
(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterial 
adhesion between eukaryotic, preferably mammalian, extracellular matrix 
proteins and bacterial gidB proteins that mediate tissue damage and/or; to 
block the normal progression of pathogenesis in infections initiated other 
than by the implantation of in-dwelling devices or by other surgical 
techniques. 
In accordance with yet another aspect of the invention, there are provided 
gidB agonists and antagonists, preferably bacteristatic or bactericidal 
agonists and antagonists. 
The antagonists and agonists of the invention may be employed, for 
instance, to prevent, inhibit and/or treat diseases. 
Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs of 
over one-third of the world's population causing stomach cancer, ulcers, 
and gastritis (International Agency for Research on Cancer (1994) 
Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency 
for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). 
Moreover, the International Agency for Research on Cancer recently 
recognized a cause-and-effect relationship between H. pylori and gastric 
adenocarcinoma, classifying the bacterium as a Group I (definite) 
carcinogen. Preferred antimicrobial compounds of the invention (agonists 
and antagonists of gidB polypeptides and/or polynucleotides) found using 
screens provided by the invention, or known in the art, particularly 
narrow-spectrum antibiotics, should be useful in the treatment of H. 
pylori infection. Such treatment should decrease the advent of H. 
pylori-induced cancers, such as gastrointestinal carcinoma. Such treatment 
should also prevent, inhibit and/or cure gastric ulcers and gastritis. 
Vaccines 
There are provided by the invention, products, compositions and methods for 
assessing gidB expression, treating disease, assaying genetic variation, 
and administering a gidB polypeptide and/or polynucleotide to an organism 
to raise an immunological response against a bacteria, especially a 
Staphylococcus aureus bacteria. 
Another aspect of the invention relates to a method for inducing an 
immunological response in an individual, particularly a mammal which 
comprises inoculating the individual with gidB polynucleotide and/or 
polypeptide, or a fragment or variant thereof, adequate to produce 
antibody and/or T cell immune response to protect said individual from 
infection, particularly bacterial infection and most particularly 
Staphylococcus aureus infection. Also provided are methods whereby such 
immunological response slows bacterial replication. Yet another aspect of 
the invention relates to a method of inducing immunological response in an 
individual which comprises delivering to such individual a nucleic acid 
vector, sequence or ribozyme to direct expression of gidB polynucleotide 
and/or polypeptide, or a fragment or a variant thereof, for expressing 
gidB polynucleotide and/or polypeptide, or a fragment or a variant thereof 
in vivo in order to induce an immunological response, such as, to produce 
antibody and/ or T cell immune response, including, for example, 
cytokine-producing T cells or cytotoxic T cells, to protect said 
individual, preferably a human, from disease, whether that disease is 
already established within the individual or not. One example of 
administering the gene is by accelerating it into the desired cells as a 
coating on particles or otherwise. Such nucleic acid vector may comprise 
DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a 
DNA-protein complex or an RNA-protein complex. 
A further aspect of the invention relates to an immunological composition 
that when introduced into an individual, preferably a human, capable of 
having induced within it an immunological response, induces an 
immunological response in such individual to a gidB polynucleotide and/or 
polypeptide encoded therefrom, wherein the composition comprises a 
recombinant gidB polynucleotide and/or polypeptide encoded therefrom 
and/or comprises DNA and/or RNA which encodes and expresses an antigen of 
said gidB polynucleotide, polypeptide encoded therefrom, or other 
polypeptide of the invention. The immunological response may be used 
therapeutically or prophylactically and may take the form of antibody 
immunity and/or cellular immunity, such as cellular immunity arising from 
CTL or CD4+ T cells. 
A gidB polypeptide or a fragment thereof may be fused with co-protein or 
chemical moiety which may or may not by itself produce antibodies, but 
which is capable of stabilizing the first protein and producing a fused or 
modified protein which will have antigenic and/or immunogenic properties, 
and preferably protective properties. Thus fused recombinant protein, 
preferably further comprises an antigenic co-protein, such as lipoprotein 
D from Hemophilus influenzae, Glutathione-S-transferase (GST) or 
beta-galactosidase, or any other relatively large co-protein which 
solubilizes the protein and facilitates production and purification 
thereof. Moreover, the co-protein may act as an adjuvant in the sense of 
providing a generalized stimulation of the immune system of the organism 
receiving the protein. The co-protein may be attached to either the amino- 
or carboxy-terminus of the first protein. 
Provided by this invention are compositions, particularly vaccine 
compositions, and methods comprising the polypeptides and/or 
polynucleotides of the invention and immunostimulatory DNA sequences, such 
as those described in Sato, Y. et al. Science 273: 352 (1996). 
Also, provided by this invention are methods using the described 
polynucleotide or particular fragments thereof, which have been shown to 
encode non-variable regions of bacterial cell surface proteins, in 
polynucleotide constructs used in such genetic immunization experiments in 
animal models of infection with Staphylococcus aureus. Such experiments 
will be particularly useful for identifying protein epitopes able to 
provoke a prophylactic or therapeutic immune response. It is believed that 
this approach will allow for the subsequent preparation of monoclonal 
antibodies of particular value, derived from the requisite organ of the 
animal successfully resisting or clearing infection, for the development 
of prophylactic agents or therapeutic treatments of bacterial infection, 
particularly Staphylococcus aureus infection, in mammals, particularly 
humans. 
A polypeptide of the invention may be used as an antigen for vaccination of 
a host to produce specific antibodies which protect against invasion of 
bacteria, for example by blocking adherence of bacteria to damaged tissue. 
Examples of tissue damage include wounds in skin or connective tissue 
caused, for example, by mechanical, chemical, thermal or radiation damage 
or by implantation of indwelling devices, or wounds in the mucous 
membranes, such as the mouth, throat, mammary glands, urethra or vagina. 
The invention also includes a vaccine formulation which comprises an 
immunogenic recombinant polypeptide and/or polynucleotide of the invention 
together with a suitable carrier, such as a pharmaceutically acceptable 
carrier. Since the polypeptides and polynucleotides may be broken down in 
the stomach, each is preferably administered parenterally, including, for 
example, administration that is subcutaneous, intramuscular, intravenous, 
or intradermal. Formulations suitable for parenteral administration 
include aqueous and non-aqueous sterile injection solutions which may 
contain anti-oxidants, buffers, bacteristatic compounds and solutes which 
render the formulation isotonic with the bodily fluid, preferably the 
blood, of the individual; and aqueous and non-aqueous sterile suspensions 
which may include suspending agents or thickening agents. The formulations 
may be presented in unit-dose or multi-dose containers, for example, 
sealed ampoules and vials and may be stored in a freeze-dried condition 
requiring only the addition of the sterile liquid carrier immediately 
prior to use. The vaccine formulation may also include adjuvant systems 
for enhancing the immunogenicity of the formulation, such as oil-in water 
systems and other systems known in the art. The dosage will depend on the 
specific activity of the vaccine and can be readily determined by routine 
experimentation. 
While the invention has been described with reference to certain gidB 
polypeptides and polynucleotides, it is to be understood that this covers 
fragments of the naturally occurring polypeptides and polynucleotides, and 
similar polypeptides and polynucleotides with additions, deletions or 
substitutions which do not substantially affect the immunogenic properties 
of the recombinant polypeptides or polynucleotides. 
Compositions, kits and administration 
In a further aspect of the invention there are provided compositions 
comprising a gidB polynucleotide and/or a gidB polypeptide for 
administration to a cell or to a multicellular organism. 
The invention also relates to compositions comprising a polynucleotide 
and/or a polypeptides discussed herein or their agonists or antagonists. 
The polypeptides and polynucleotides of the invention may be employed in 
combination with a non-sterile or sterile carrier or carriers for use with 
cells, tissues or organisms, such as a pharmaceutical carrier suitable for 
administration to an individual. Such compositions comprise, for instance, 
a media additive or a therapeutically effective amount of a polypeptide 
and/or polynucleotide of the invention and a pharmaceutically acceptable 
carrier or excipient. Such carriers may include, but are not limited to, 
saline, buffered saline, dextrose, water, glycerol, ethanol and 
combinations thereof. The formulation should suit the mode of 
administration. The invention further relates to diagnostic and 
pharmaceutical packs and kits comprising one or more containers filled 
with one or more of the ingredients of the aforementioned compositions of 
the invention. 
Polypeptides, polynucleotides and other compounds of the invention may be 
employed alone or in conjunction with other compounds, such as therapeutic 
compounds. 
The pharmaceutical compositions may be administered in any effective, 
convenient manner including, for instance, administration by topical, 
oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, 
subcutaneous, intranasal or intradermal routes among others. 
In therapy or as a prophylactic, the active agent may be administered to an 
individual as an injectable composition, for example as a sterile aqueous 
dispersion, preferably isotonic. 
Alternatively the composition may be formulated for topical application for 
example in the form of ointments, creams, lotions, eye ointments, eye 
drops, ear drops, mouthwash, impregnated dressings and sutures and 
aerosols, and may contain appropriate conventional additives, including, 
for example, preservatives, solvents to assist drug penetration, and 
emollients in ointments and creams. Such topical formulations may also 
contain compatible conventional carriers, for example cream or ointment 
bases, and ethanol or oleyl alcohol for lotions. Such carriers may 
constitute from about 1% to about 98% by weight of the formulation; more 
usually they will constitute up to about 80% by weight of the formulation. 
In a further aspect, the present invention provides for pharmaceutical 
compositions comprising a therapeutically effective amount of a 
polypeptide and/or polynucleotide, such as the soluble form of a 
polypeptide and/or polynucleotide of the present invention, agonist or 
antagonist peptide or small molecule compound, in combination with a 
pharmaceutically acceptable carrier or excipient. Such carriers include, 
but are not limited to, saline, buffered saline, dextrose, water, 
glycerol, ethanol, and combinations thereof. The invention further relates 
to pharmaceutical packs and kits comprising one or more containers filled 
with one or more of the ingredients of the aforementioned compositions of 
the invention. Polypeptides, polynucleotides and other compounds of the 
present invention may be employed alone or in conjunction with other 
compounds, such as therapeutic compounds. 
The composition will be adapted to the route of administration, for 
instance by a systemic or an oral route. Preferred forms of systemic 
administration include injection, typically by intravenous injection. 
Other injection routes, such as subcutaneous, intramuscular, or 
intraperitoneal, can be used. Alternative means for systemic 
administration include transmucosal and transdermal administration using 
penetrants such as bile salts or fusidic acids or other detergents. In 
addition, if a polypeptide or other compounds of the present invention can 
be formulated in an enteric or an encapsulated formulation, oral 
administration may also be possible. Administration of these compounds may 
also be topical and/or localized, in the form of salves, pastes, gels, and 
the like. 
For administration to mammals, and particularly humans, it is expected that 
the daily dosage level of the active agent will be from 0.01 mg/kg to 10 
mg/kg, typically around 1 mg/kg. The physician in any event will determine 
the actual dosage which will be most suitable for an individual and will 
vary with the age, weight and response of the particular individual. The 
above dosages are exemplary of the average case. There can, of course, be 
individual instances where higher or lower dosage ranges are merited, and 
such are within the scope of this invention. 
In-dwelling devices include surgical implants, prosthetic devices and 
catheters, i.e., devices that are introduced to the body of an individual 
and remain in position for an extended time. Such devices include, for 
example, artificial joints, heart valves, pacemakers, vascular grafts, 
vascular catheters, cerebrospinal fluid shunts, urinary catheters, 
continuous ambulatory peritoneal dialysis (CAPD) catheters. 
The composition of the invention may be administered by injection to 
achieve a systemic effect against relevant bacteria shortly before 
insertion of an in-dwelling device. Treatment may be continued after 
surgery during the in-body time of the device. In addition, the 
composition could also be used to broaden perioperative cover for any 
surgical technique to prevent bacterial wound infections, especially 
Staphylococcus aureus wound infections. 
Many orthopedic surgeons consider that humans with prosthetic joints should 
be considered for antibiotic prophylaxis before dental treatment that 
could produce a bacteremia. Late deep infection is a serious complication 
sometimes leading to loss of the prosthetic joint and is accompanied by 
significant morbidity and mortality. It may therefore be possible to 
extend the use of the active agent as a replacement for prophylactic 
antibiotics in this situation. 
In addition to the therapy described above, the compositions of this 
invention may be used generally as a wound treatment agent to prevent 
adhesion of bacteria to matrix proteins exposed in wound tissue and for 
prophylactic use in dental treatment as an alternative to, or in 
conjunction with, antibiotic prophylaxis. 
Alternatively, the composition of the invention may be used to bathe an 
indwelling device immediately before insertion. The active agent will 
preferably be present at a concentration of 1 .mu.g/ml to 10 mg/ml for 
bathing of wounds or indwelling devices. 
A vaccine composition is conveniently in injectable form. Conventional 
adjuvants may be employed to enhance the immune response. A suitable unit 
dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is 
preferably administered 1-3 times and with an interval of 1-3 weeks. With 
the indicated dose range, no adverse toxicological effects will be 
observed with the compounds of the invention which would preclude their 
administration to suitable individuals. 
Sequence Databases, Sequences in a Tangible Medium, and Algorithms 
Polynucleotide and polypeptide sequences form a valuable information 
resource with which to determine their 2- and 3-dimensional structures as 
well as to identify further sequences of similar homology. These 
approaches are most easily facilitated by storing the sequence in a 
computer readable medium and then using the stored data in a known 
macromolecular structure program or to search a sequence database using 
well known searching tools, such as GCC. The polynucleotide and 
polypeptide sequences of the invention are particularly useful as 
components in databases useful for search analyses as well as in sequence 
analysis algorithms. As used in this section entitled "Sequence Databases, 
Sequences in a Tangible Medium, and Algorithms," and in claims related to 
this section, the terms "polynucleotide of the invention" and 
"polynucleotide sequence of the invention" mean any detectable chemical or 
physical characteristic of a polynucleotide of the invention that is or 
may be reduced to or stored in a tangible medium, preferably a computer 
readable form. For example, chromatographic scan data or peak data, 
photographic data or scan data therefrom, called bases, and mass 
spectrographic data. As used in this section entitled Databases and 
Algorithms and in claims related thereto, the terms "polypeptide of the 
invention" and "polypeptide sequence of the invention" mean any detectable 
chemical or physical characteristic of a polypeptide of the invention that 
is or may be reduced to or stored in a tangible medium, preferably a 
computer readable form. For example, chromatographic scan data or peak 
data, photographic data or scan data therefrom, and mass spectrographic 
data. 
The invention provides a computer readable medium having stored thereon 
polypeptide sequences of the invention and/or polynucleotide sequences of 
the invention. For example, a computer readable medium is provided 
comprising and having stored thereon a member selected from the group 
consisting of: a polynucleotide comprising the sequence of a 
polynucleotide of the invention; a polypeptide comprising the sequence of 
a polypeptide sequence of the invention; 
a set of polynucleotide sequences wherein at least one of the sequences 
comprises the sequence of a polynucleotide sequence of the invention; a 
set of polypeptide sequences wherein at least one of the sequences 
comprises the sequence of a polypeptide sequence of the invention; a data 
set representing a polynucleotide sequence comprising the sequence of 
polynucleotide sequence of the invention; a data set representing a 
polynucleotide sequence encoding a polypeptide sequence comprising the 
sequence of a polypeptide sequence of the invention; a polynucleotide 
comprising the sequence of a polynucleotide sequence of the invention; a 
polypeptide comprising the sequence of a polypeptide sequence of the 
invention; a set of polynucleotide sequences wherein at least one of the 
sequences comprises the sequence of a polynucleotide sequence of the 
invention; a set of polypeptide sequences wherein at least one of said 
sequences comprises the sequence of a polypeptide sequence of the 
invention; a data set representing a polynucleotide sequence comprising 
the sequence of a polynucleotide sequence of the invention; a data set 
representing a polynucleotide sequence encoding a polypeptide sequence 
comprising the sequence of a polypeptide sequence of the invention. The 
computer readable medium can be any composition of matter used to store 
information or data, including, for example, commercially available floppy 
disks, tapes, chips, hard drives, compact disks, and video disks. 
Also provided by the invention are methods for the analysis of character 
sequences or strings, particularly genetic sequences or encoded genetic 
sequences. Preferred methods of sequence analysis include, for example, 
methods of sequence homology analysis, such as identity and similarity 
analysis, RNA structure analysis, sequence assembly, cladistic analysis, 
sequence motif analysis, open reading frame determination, nucleic acid 
base calling, nucleic acid base trimming, and sequencing chromatogram peak 
analysis. 
A computer based method is provided for performing homology identification. 
This method comprises the steps of providing a polynucleotide sequence 
comprising the sequence a polynucleotide of the invention in a computer 
readable medium; and comparing said polynucleotide sequence to at least 
one polynucleotide or polypeptide sequence to identify homology. 
A computer based method is also provided for performing homology 
identification, said method comprising the steps of: providing a 
polypeptide sequence comprising the sequence of a polypeptide of the 
invention in a computer readable medium; and comparing said polypeptide 
sequence to at least one polynucleotide or polypeptide sequence to 
identify homology. 
A computer based method is still further provided for polynucleotide 
assembly, said method comprising the steps of: providing a first 
polynucleotide sequence comprising the sequence of a polynucleotide of the 
invention in a computer readable medium; and screening for at least one 
overlapping region between said first polynucleotide sequence and a second 
polynucleotide sequence. 
A further embodiment of the invention provides a computer based method for 
performing homology identification, said method comprising the steps of: 
providing a polynucleotide sequence comprising the sequence of a 
polynucleotide of the invention in a computer readable medium; and 
comparing said polynucleotide sequence to at least one polynucleotide or 
polypeptide sequence to identify homology. 
A further embodiment of the invention provides a computer based method for 
performing homology identification, said method comprising the steps of: 
providing a polypeptide sequence comprising the sequence of a polypeptide 
of the invention in a computer readable medium; and comparing said 
polypeptide sequence to at least one polynucleotide or polypeptide 
sequence to identify homology. 
A further embodiment of the invention provides a computer based method for 
polynucleotide assembly, said method comprising the steps of: providing a 
first polynucleotide sequence comprising the sequence of a polynucleotide 
of the invention in a computer readable medium; and screening for at least 
one overlapping region between said first polynucleotide sequence and a 
second polynucleotide sequence. 
In another preferred embodiment of the invention there is provided a 
computer readable medium having stored thereon a member selected from the 
group consisting of: a polynucleotide comprising the sequence of SEQ ID 
NO. 1 or 3; a polypeptide comprising the sequence of SEQ ID NO. 2 or 4; a 
set of polynucleotide sequences wherein at least one of said sequences 
comprises the sequence of SEQ ID NO. 1 or 3; a set of polypeptide 
sequences wherein at least one of said sequences comprises the sequence of 
SEQ ID NO. 2 or 4; a data set representing a polynucleotide sequence 
comprising the sequence of SEQ ID NO. 1 or 3; a data set representing a 
polynucleotide sequence encoding a polypeptide sequence comprising the 
sequence of SEQ ID NO. 2 or 4; a polynucleotide comprising the sequence of 
SEQ ID NO. 1 or 3; a polypeptide comprising the sequence of SEQ ID NO. 2 
or 4; a set of polynucleotide sequences wherein at least one of said 
sequences comprises the sequence of SEQ ID NO. 1 or 3; a set of 
polypeptide sequences wherein at least one of said sequences comprises the 
sequence of SEQ ID NO. 2 or 4; a data set representing a polynucleotide 
sequence comprising the sequence of SEQ ID NO. 1 or 3; 
a data set representing a polynucleotide sequence encoding a polypeptide 
sequence comprising the sequence of SEQ ID NO. 2 or 4. A further preferred 
embodiment of the invention provides a computer based method for 
performing homology identification, said method comprising the steps of 
providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 
1 or 3 in a computer readable medium; and comparing said polynucleotide 
sequence to at least one polynucleotide or polypeptide sequence to 
identify homology. 
A still further preferred embodiment of the invention provides a computer 
based method for performing homology identification, said method 
comprising the steps of: providing a polypeptide sequence comprising the 
sequence of SEQ ID NO. 2 or 4 in a computer readable medium; and comparing 
said polypeptide sequence to at least one polynucleotide or polypeptide 
sequence to identify homology. 
A further embodiment of the invention provides a computer based method for 
polynucleotide assembly, said method comprising the steps of: providing a 
first polynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3 
in a computer readable medium; and screening for at least one overlapping 
region between said first polynucleotide sequence and a second 
polynucleotide sequence. 
A further embodiment of the invention provides a computer based method for 
performing homology identification, said method comprising the steps of: 
providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 
1 or 3 in a computer readable medium; and comparing said polynucleotide 
sequence to at least one polynucleotide or polypeptide sequence to 
identify homology. 
A further embodiment of the invention provides a computer based method for 
performing homology identification, said method comprising the steps of: 
providing a polypeptide sequence comprising the sequence of SEQ ID NO. 2 
or 4 in a computer readable medium; and comparing said polypeptide 
sequence to at least one polynucleotide or polypeptide sequence to 
identify homology. 
A further embodiment of the invention provides a computer based method for 
polynucleotide assembly, said method comprising the steps of: providing a 
first polynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3 
in a computer readable medium; and screening for at least one overlapping 
region between said first polynucleotide sequence and a second 
polynucleotide sequence. 
All publications and references, including but not limited to patents and 
patent applications, cited in this specification are herein incorporated 
by reference in their entirety as if each individual publication or 
reference were specifically and individually indicated to be incorporated 
by reference herein as being fully set forth. Any patent application to 
which this application claims priority is also incorporated by reference 
herein in its entirety in the manner described above for publications and 
references. 
GLOSSARY 
The following definitions are provided to facilitate understanding of 
certain terms used frequently herein. 
"Antibody(ies)" as used herein includes polyclonal and monoclonal 
antibodies, chimeric, single chain, and humanized antibodies, as well as 
Fab fragments, including the products of an Fab or other immunoglobulin 
expression library. 
"Antigenically equivalent derivative(s)" as used herein encompasses a 
polypeptide, polynucleotide, or the equivalent of either which will be 
specifically recognized by certain antibodies which, when raised to the 
protein, polypeptide or polynucleotide according to the invention, 
interferes with the immediate physical interaction between pathogen and 
mammalian host. 
"Bispecific antibody(ies)" means an antibody comprising at least two 
antigen binding domains, each domain directed against a different epitope. 
"Bodily material(s) means any material derived from an individual or from 
an organism infecting, infesting or inhabiting an individual, including 
but not limited to, cells, tissues and waste, such as, bone, blood, serum, 
cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, 
urine, stool or autopsy materials. 
"Disease(s)" means any disease caused by or related to infection by a 
bacteria, including, for example, disease, such as, infections of the 
upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute 
epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung 
abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., 
secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., 
cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, 
endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), 
kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric 
absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, 
cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone 
and joint (e.g., septic arthritis, osteomyelitis). 
"Fusion protein(s)" refers to a protein encoded by two, often unrelated, 
fused genes or fragments thereof. In one example, EP-A-0464 discloses 
fusion proteins comprising various portions of constant region of 
immunoglobulin molecules together with another human protein or part 
thereof. In many cases, employing an immunoglobulin Fc region as a part of 
a fusion protein is advantageous for use in therapy and diagnosis 
resulting in, for example, improved pharmacokinetic properties [see, e.g., 
EP-A 0232262]. On the other hand, for some uses it would be desirable to 
be able to delete the Fc part after the fusion protein has been expressed, 
detected and purified. 
"Host cell(s)" is a cell which has been transformed or transfected, or is 
capable of transformation or transfection by an exogenous polynucleotide 
sequence. 
"Identity," as known in the art, is a relationship between two or more 
polypeptide sequences or two or more polynucleotide sequences, as the case 
may be, as determined by comparing the sequences. In the art, "identity" 
also means the degree of sequence relatedness between polypeptide or 
polynucleotide sequences, as the case may be, as determined by the match 
between strings of such sequences. "Identity" can be readily calculated by 
known methods, including but not limited to those described in 
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University 
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, 
Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of 
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana 
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von 
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, 
M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, 
H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to 
determine identity are designed to give the largest match between the 
sequences tested. Moreover, methods to determine identity are codified in 
publicly available computer programs. Computer program methods to 
determine identity between two sequences include, but are not limited to, 
the GCG program package (Devereux, J., et al., Nucleic Acids Research 
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. 
Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly 
available from NCBI and other sources (BLAST Manual, Altschul, S., et al., 
NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 
403-410 (1990). The well known Smith Waterman algorithm may also be used 
to determine identity. 
Parameters for polypeptide sequence comparison include the following: 
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) 
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. 
Acad. Sci. USA. 89:10915-10919 (1992) 
Gap Penalty: 12 
Gap Length Penalty: 4 
A program useful with these parameters is publicly available as the "gap" 
program from Genetics Computer Group, Madison Wis. The aforementioned 
parameters are the default parameters for peptide comparisons (along with 
no penalty for end gaps). 
Parameters for polynucleotide comparison include the following: 
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) 
Comparison matrix: matches=+10, mismatch=0 
Gap Penalty: 50 
Gap Length Penalty: 3 
Available as: The "gap" program from Genetics Computer Group, Madison Wis. 
These are the default parameters for nucleic acid comparisons. 
A preferred meaning for "identity" for polynucleotides and polypeptides, as 
the case may be, are provided in (1) and (2) below. 
(1) Polynucleotide embodiments further include an isolated polynucleotide 
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 
90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1, 
wherein said polynucleotide sequence may be identical to the reference 
sequence of SEQ ID NO: 1 or may include up to a certain integer number of 
nucleotide alterations as compared to the reference sequence, wherein said 
alterations are selected from the group consisting of at least one 
nucleotide deletion, substitution, including transition and transversion, 
or insertion, and wherein said alterations may occur at the 5' or 3' 
terminal positions of the reference nucleotide sequence or anywhere 
between those terminal positions, interspersed either individually among 
the nucleotides in the reference sequence or in one or more contiguous 
groups within the reference sequence, and wherein said number of 
nucleotide alterations is determined by multiplying the total number of 
nucleotides in SEQ ID NO: 1 by the integer defining the percent identity 
divided by 100 and then subtracting that product from said total number of 
nucleotides in SEQ ID NO: 1, or: 
EQU n.sub.n .ltoreq.x.sub.n -(x.sub.n .cndot.y), 
wherein n.sub.n is the number of nucleotide alterations, x.sub.n is the 
total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for 
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 
0.97 for 97% or 1.00 for 100%, and .cndot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.n 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.n. Alterations of a polynucleotide sequence encoding the polypeptide 
of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in 
this coding sequence and thereby alter the polypeptide encoded by the 
polynucleotide following such alterations. 
By way of example, a polynucleotide sequence of the present invention may 
be identical to the reference sequence of SEQ ID NO:1, that is it may be 
100% identical, or it may include up to a certain integer number of 
nucleic acid alterations as compared to the reference sequence such that 
the percent identity is less than 100% identity. Such alterations are 
selected from the group consisting of at least one nucleic acid deletion, 
substitution, including transition and transversion, or insertion, and 
wherein said alterations may occur at the 5' or 3' terminal positions of 
the reference polynucleotide sequence or anywhere between those terminal 
positions, interspersed either individually among the nucleic acids in the 
reference sequence or in one or more contiguous groups within the 
reference sequence. The number of nucleic acid alterations for a given 
percent identity is determined by multiplying the total number of nucleic 
acids in SEQ ID NO: 1 by the integer defining the percent identity divided 
by 100 and then subtracting that product from said total number of nucleic 
acids in SEQ ID NO: 1, or: 
EQU n.sub.n .ltoreq.x.sub.n -(x.sub.n .cndot.y), 
wherein n.sub.n is the number of nucleic acid alterations, x.sub.n is the 
total number of nucleic acids in SEQ ID NO: 1, y is, for instance 0.70 for 
70%, 0.80 for 80%, 0.85 for 85% etc., .cndot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.n 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.n. 
(2) Polypeptide embodiments further include an isolated polypeptide 
comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 
or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, 
wherein said polypeptide sequence may be identical to the reference 
sequence of SEQ ID NO: 2 or may include up to a certain integer number of 
amino acid alterations as compared to the reference sequence, wherein said 
alterations are selected from the group consisting of at least one amino 
acid deletion, substitution, including conservative and non-conservative 
substitution, or insertion, and wherein said alterations may occur at the 
amino- or carboxy-terminal positions of the reference polypeptide sequence 
or anywhere between those terminal positions, interspersed either 
individually among the amino acids in the reference sequence or in one or 
more contiguous groups within the reference sequence, and wherein said 
number of amino acid alterations is determined by multiplying the total 
number of amino acids in SEQ ID NO:2 by the integer defining the percent 
identity divided by 100 and then subtracting that product from said total 
number of amino acids in SEQ ID NO:2, or: 
EQU n.sub.a .ltoreq.x.sub.a -(x.sub.a .cndot.y), 
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the 
total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for 
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 
0.97 for 97% or 1.00 for 100%, and .cndot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.a 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.a. 
By way of example, a polypeptide sequence of the present invention may be 
identical to the reference sequence of SEQ ID NO:2, that is it may be 100% 
identical, or it may include up to a certain integer number of amino acid 
alterations as compared to the reference sequence such that the percent 
identity is less than 100% identity. Such alterations are selected from 
the group consisting of at least one amino acid deletion, substitution, 
including conservative and non-conservative substitution, or insertion, 
and wherein said alterations may occur at the amino- or carboxy-terminal 
positions of the reference polypeptide sequence or anywhere between those 
terminal positions, interspersed either individually among the amino acids 
in the reference sequence or in one or more contiguous groups within the 
reference sequence. The number of amino acid alterations for a given % 
identity is determined by multiplying the total number of amino acids in 
SEQ ID NO:2 by the integer defining the percent identity divided by 100 
and then subtracting that product from said total number of amino acids in 
SEQ ID NO:2, or: 
EQU n.sub.a .ltoreq.x.sub.a -(x.sub.a .cndot.y), 
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the 
total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 
70%, 0.80 for 80%, 0.85 for 85% etc., and .cndot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.a 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.a. 
"Immunologically equivalent derivative(s)" as used herein encompasses a 
polypeptide, polynucleotide, or the equivalent of either which when used 
in a suitable formulation to raise antibodies in a vertebrate, the 
antibodies act to interfere with the immediate physical interaction 
between pathogen and mammalian host. 
"Inmmunospecific" means that characteristic of an antibody whereby it 
possesses substantially greater affinity for the polypeptides of the 
invention or the polynucleotides of the invention than its affinity for 
other related polypeptides or polynucleotides respectively, particularly 
those polypeptides and polynucleotides in the prior art. 
"Individual(s)" means a multicellular eukaryote, including, but not limited 
to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a 
human. 
"Isolated" means altered "by the hand of man" from its natural state, i.e., 
if it occurs in nature, it has been changed or removed from its original 
environment, or both. For example, a polynucleotide or a polypeptide 
naturally present in a living organism is not "isolated," but the same 
polynucleotide or polypeptide separated from the coexisting materials of 
its natural state is "isolated", as the term is employed herein. Moreover, 
a polynucleotide or polypeptide that is introduced into an organism by 
transformation, genetic manipulation or by any other recombinant method is 
"isolated" even if it is still present in said organism, which organism 
may be living or non-living. 
"Organism(s)" means a (i) prokaryote, including but not limited to, a 
member of the genus Streptococcus, Staphylococcus, Bordetella, 
Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, 
Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, 
Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, 
Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, 
Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, 
Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, 
Shigella, Legionelia, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, 
Borrelia and Mycoplasma, and Aurther including, but not limited to, a 
member of the species or group, Group A Streptococcus, Group B 
Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G 
Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, 
Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, 
Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, 
Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium 
diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, 
Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, 
Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, 
Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, 
Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, 
Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella 
typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia 
pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia 
liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, 
Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, 
Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium 
tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii 
and Chlamydia trachomitis, (ii) an archaeon, including but not limited to 
Archaebacter, and (iii) a unicellular or filamentous eukaryote, including 
but not limited to, a protozoan, a fungus, a member of the genus 
Saccharomyces, Kluveromyces, or Candida, and a member of the species 
Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans. 
"Polynucleotide(s)" generally refers to any polyribonucleotide or 
polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified 
RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and 
double-stranded DNA, DNA that is a mixture of single- and double-stranded 
regions or single-, double- and triple-stranded regions, single- and 
double-stranded RNA, and RNA that is mixture of single- and 
double-stranded regions, hybrid molecules comprising DNA and RNA that may 
be single-stranded or, more typically, double-stranded, or triple-stranded 
regions, or a mixture of single- and double-stranded regions. In addition, 
"polynucleotide" as used herein refers to triple-stranded regions 
comprising RNA or DNA or both RNA and DNA. The strands in such regions may 
be from the same molecule or from different molecules. The regions may 
include all of one or more of the molecules, but more typically involve 
only a region of some of the molecules. One of the molecules of a 
triple-helical region often is an oligonucleotide. As used herein, the 
term "polynucleotide(s)" also includes DNAs or RNAs as described above 
that contain one or more modified bases. Thus, DNAs or RNAs with backbones 
modified for stability or for other reasons are "polynucleotide(s)" as 
that term is intended herein. Moreover, DNAs or RNAs comprising unusual 
bases, such as inosine, or modified bases, such as tritylated bases, to 
name just two examples, are polynucleotides as the term is used herein. It 
will be appreciated that a great variety of modifications have been made 
to DNA and RNA that serve many useful purposes known to those of skill in 
the art. The term "polynucleotide(s)" as it is employed herein embraces 
such chemically, enzymatically or metabolically modified forms of 
polynucleotides, as well as the chemical forms of DNA and RNA 
characteristic of viruses and cells, including, for example, simple and 
complex cells. "Polynucleotide(s)" also embraces short polynucleotides 
often referred to as oligonucleotide(s). 
"Polypeptide(s)" refers to any peptide or protein comprising two or more 
amino acids joined to each other by peptide bonds or modified peptide 
bonds. "Polypeptide(s)" refers to both short chains, commonly referred to 
as peptides, oligopeptides and oligomers and to longer chains generally 
referred to as proteins. Polypeptides may contain amino acids other than 
the 20 gene encoded amino acids. "Polypeptide(s)" include those modified 
either by natural processes, such as processing and other 
post-translational modifications, but also by chemical modification 
techniques. Such modifications are well described in basic texts and in 
more detailed monographs, as well as in a voluminous research literature, 
and they are well known to those of skill in the art. It will be 
appreciated that the same type of modification may be present in the same 
or varying degree at several sites in a given polypeptide. Also, a given 
polypeptide may contain many types of modifications. Modifications can 
occur anywhere in a polypeptide, including the peptide backbone, the amino 
acid side-chains, and the amino or carboxyl termini. Modifications 
include, for example, acetylation, acylation, ADP-ribosylation, amidation, 
covalent attachment of flavin, covalent attachment of a heme moiety, 
covalent attachment of a nucleotide or nucleotide derivative, covalent 
attachment of a lipid or lipid derivative, covalent attachment of 
phosphotidylinositol, cross-linking, cyclization, disulfide bond 
formation, demethylation, formation of covalent cross-links, formation of 
cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, 
GPI anchor formation, hydroxylation, iodination, methylation, 
myristoylation, oxidation, proteolytic processing, phosphorylation, 
prenylation, racemization, glycosylation, lipid attachment, sulfation, 
gamma-carboxylation of glutamic acid residues, hydroxylation and 
ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition 
of amino acids to proteins, such as arginylation, and ubiquitination. See, 
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. 
Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., 
Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 
1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. 
Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. 
Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: 
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 
(1992). Polypeptides may be branched or cyclic, with or without branching. 
Cyclic, branched and branched circular polypeptides may result from 
post-translational natural processes and may be made by entirely synthetic 
methods, as well. 
"Recombinant expression system(s)" refers to expression systems or portions 
thereof or polynucleotides of the invention introduced or transformed into 
a host cell or host cell lysate for the production of the polynucleotides 
and polypeptides of the invention. 
"Subtraction set" is one or more, but preferably less than 100, 
polynucleotides comprising at least one polynucleotide of the invention 
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide 
that differs from a reference polynucleotide or polypeptide respectively, 
but retains essential properties. A typical variant of a polynucleotide 
differs in nucleotide sequence from another, reference polynucleotide. 
Changes in the nucleotide sequence of the variant may or may not alter the 
amino acid sequence of a polypeptide encoded by the reference 
polynucleotide. Nucleotide changes may result in amino acid substitutions, 
additions, deletions, fusion proteins and truncations in the polypeptide 
encoded by the reference sequence, as discussed below. A typical variant 
of a polypeptide differs in amino acid sequence from another, reference 
polypeptide. Generally, differences are limited so that the sequences of 
the reference polypeptide and the variant are closely similar overall and, 
in many regions, identical. A variant and reference polypeptide may differ 
in amino acid sequence by one or more substitutions, additions, deletions 
in any combination. A substituted or inserted amino acid residue may or 
may not be one encoded by the genetic code. The present invention also 
includes include variants of each of the polypeptides of the invention, 
that is polypeptides that vary from the referents by conservative amino 
acid substitutions, whereby a residue is substituted by another with like 
characteristics. Typical such substitutions are among Ala, Val, Leu and 
Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn 
and Gln; and among the basic residues Lys and Arg; or aromatic residues 
Phe and Tyr. Particularly preferred are variants in which several, 5-10, 
1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any 
combination. A variant of a polynucleotide or polypeptide may be a 
naturally occurring such as an allelic variant, or it may be a variant 
that is not known to occur naturally. Non-naturally occurring variants of 
polynucleotides and polypeptides may be made by mutagenesis techniques, by 
direct synthesis, and by other recombinant methods known to skilled 
artisans.

EXAMPLES 
The examples below are carried out using standard techniques, which are 
well known and routine to those of skill in the art, except where 
otherwise described in detail. The examples are illustrative, but do not 
limit the invention. 
Example 1 
Strain selection, Library Production and Sequencing 
The polynucleotide having a DNA sequence given in Table l [SEQ ID NO:1 or 
3] was obtained from a library of clones of chromosomal DNA of 
Staphylococcus aureus in E. coli . The sequencing data from two or more 
clones containing overlapping Staphylococcus aureus DNAs was used to 
construct the contiguous DNA sequence in SEQ ID NO:I. Libraries may be 
prepared by routine methods, for example: 
Methods 1 and 2 below. 
Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according 
to standard procedures and size-fractionated by either of two methods. 
Method 1 
Total cellular DNA is mechanically sheared by passage through a needle in 
order to size-fractionate according to standard procedures. DNA fragments 
of up to 11 kbp in size are rendered blunt by treatment with exonuclease 
and DNA polymerase, and EcoRI linkers added. Fragments are ligated into 
the vector Lambda ZapII that has been cut with EcoRI, the library packaged 
by standard procedures and E.coli infected with the packaged library. The 
library is amplified by standard procedures. 
Method 2 
Total cellular DNA is partially hydrolyzed with a one or a combination of 
restriction enzymes appropriate to generate a series of fragments for 
cloning into library vectors (e.g., RsaI, PalI, AluI, Bsh1235I), and such 
fragments are size-fractionated according to standard procedures. EcoRI 
linkers are ligated to the DNA and the fragments then ligated into the 
vector Lambda ZapII that have been cut with EcoRI, the library packaged by 
standard procedures, and E. coli infected with the packaged library. The 
library is amplified by standard procedures. 
Example 2 
gidB Characterization 
The determination of expression during infection of a gene from 
Staphylococcus aureus 
Necrotic fatty tissue from a four day groin infection of Staphylococcus 
aureus WCUH29 in the mouse is efficiently disrupted and processed in the 
presence of chaotropic agents and RNAase inhibitor to provide a mixture of 
animal and bacterial RNA. The optimal conditions for disruption and 
processing to give stable preparations and high yields of bacterial RNA 
are followed by the use of hybridisation to a radiolabelled 
oligonucleotide specific to Staphylococcus aureus 16S RNA on Northern 
blots. The RNAase free, DNAase free, DNA and protein free preparations of 
RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using 
unique primer pairs designed from the sequence of each gene of 
Staphylococcus aureus WCUH29. 
a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from a 
mouse animal model of infection 
10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded with 
isolated, individual colonies of Staphylococcus aureus WCUH29 from an agar 
culture plate. The cultures are incubated aerobically (static culture) at 
37.degree. C. for 16-20 hours. 4 week old mice (female, 18 g-22 g, strain 
MF1) are each infected by subcutaneous injection of 0.5 ml. of this broth 
culture of Staphylococcus aureus WCUH29 (diluted in broth to approximately 
108 cfu/ml.) into the anterior, right lower quadrant (groin area). Mice 
should be monitored regularly during the first 24 hours after infection, 
then daily until termination of study. Animals with signs of systemic 
infection, i.e. lethargy, ruffled appearance, isolation from group, should 
be monitored closely and if signs progress to moribundancy, the animal 
should be culled immediately. 
Visible external signs of lesion development will be seen 24-48 h after 
infection. Examination of the abdomen of the animal will show the raised 
outline of the abscess beneath the skin. The localised lesion should 
remain in the right lower quadrant, but may occasionally spread to the 
left lower quadrant, and superiorly to the thorax. On occasions, the 
abscess may rupture through the overlying skin layers. In such cases the 
affected animal should be culled immediately and the tissues sampled if 
possible. Failure to cull the animal may result in the necrotic skin 
tissue overlying the abscess being sloughed off, exposing the abdominal 
muscle wall. 
Approximately 96 hours after infection, animals are killed using carbon 
dioxide asphyxiation. To minimise delay between death and tissue 
processing/storage, mice should be killed individually rather than in 
groups.The dead animal is placed onto its back and the fur swabbed 
liberally with 70% alcohol. An initial incision using scissors is made 
through the skin of the abdominal left lower quadrant, travelling 
superiorly up to, then across the thorax. The incision is completed by 
cutting inferiorly to the abdominal lower right quadrant. Care should be 
taken not to penetrate the abdominal wall. Holding the skin flap with 
forceps, the skin is gently pulled way from the abdomen. The exposed 
abscess, which covers the peritoneal wall but generally does not penetrate 
the muscle sheet completely, is excised, taking care not to puncture the 
viscera 
The abscess/muscle sheet and other infected tissue may require cutting in 
sections, prior to flash-freezing in liquid nitrogen, thereby allowing 
easier storage in plastic collecting vials. 
b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissue 
samples 
4-6 infected tissue samples(each approx 0.5-0.7 g) in 2 ml screw-cap tubes 
are removed from -80.degree. C. storage into a dry ice ethanol bath In a 
microbiological safety cabinet the samples are disrupted individually 
whilst the remaining samples are kept cold in the dry ice ethanol bath. To 
disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent 
(Gibco BRL, Life Technologies) is added followed by enough 0.1 mm 
zirconia/silica beads to almost fill the tube, the lid is replaced taking 
care not to get any beads into the screw thread so as to ensure a good 
seal and eliminate aerosol generation. The sample is then homogenised in a 
Mini-BeadBeater Type BX-4 (Biospec Products). Necrotic fatty tissue 
isstrain treated for 100 seconds at 5000 rpm in order to achieve bacterial 
lysis. In vivo grown bacteria require longer treatment than in vitro grown 
Staphylococcus aureus Staphylococcus which are disrupted by a 30 second 
bead-beat. 
After bead-beating the tubes are chilled on ice before opening in a 
fume-hood as heat generated during disruption may degrade the TRIzol and 
release cyanide. 
200 microliters of chloroform is then added and the tubes shaken by hand 
for 15 seconds to ensure complete mixing. After 2-3 minutes at room 
temperature the tubes are spun down at 12,000.times. g, 4.degree. C. for 
15 minutes and RNA extraction is then continued according to the method 
given by the manufacturers of TRIzol Reagent i.e.: The aqueous phase, 
approx 0.6 ml, is transferred to a sterile eppendorf tube and 0.5 ml of 
isopropanol is added. After 10 minutes at room temperature the samples are 
spun at 12,000.times. g, 4.degree. C. for 10 minutes. The supernatant is 
removed and discarded then the RNA pellet is washed with 1 ml 75% ethanol. 
A brief vortex is used to mix the sample before centrifuging at 
7,500.times. g, 4.degree. C. for 5 minutes. The ethanol is removed and the 
RNA pellet dried under vacuum for no more than 5 minutes. Samples are then 
resuspended by repeated pipetting in 100 microliters of DEPC treated 
water, followed by 5-10 minutes at 55.degree. C. Finally, after at least 1 
minute on ice, 200 units of Rnasin (Promega) is added. 
RNA preparations are stored at -80.degree. C. for up to one month. For 
longer term storage the RNA precipitate can be stored at the wash stage of 
the protocol in 75% ethanol for at least one year at -20.degree. C. 
Quality of the RNA isolated is assessed by running samples on 1% agarose 
gels. 1.times. TBE gels stained with ethidium bromide are used to 
visualise total RNA yields. To demonstrate the isolation of bacterial RNA 
from the infected tissue 1.times. MOPS, 2.2 M formaldehyde gels are run 
and vacuum blotted to Hybond-N (Amersham). The blot is then hybridised 
with a 32 P labelled oligonucletide probe specific to 16s rRNA of 
Staphylococcus aureus (K.Greisen, M. Loeffelholz, A. Purohit and D. Leong. 
J.Clin. (1994) Microbiol. 32 335-351 ). The oligonucleotide of the 
sequence is used as a probe. The size of the hybridising band is compared 
to that of control RNA isolated from in vitro grown Staphylococcus aureus 
WCUH29 in the Northern blot. Correct sized bacterial 16s rRNA bands can be 
detected in total RNA samples which show extensive degradation of the 
mammalian RNA when visualised on TBE gels. 
c) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA 
DNA was removed from 73 microliter samples of RNA by a 15 minute treatment 
on ice with 3 units of DNAaseI, amplification grade (Gibco BRL, Life 
Technologies) in the buffer supplied with the addition of 200 units of 
Rnasin (Promega) in a final volume of 90 microliters. 
The DNAase was inactivated and removed by treatment with TRIzol LS Reagent 
(Gibco BRL, Life Technologies) according to the manufacturers protocol. 
DNAase treated RNA was resuspended in 73 microliters of DEPC treated water 
with the addition of Rnasin as described in Method 1. 
d) The preparation of cDNA from RNA samples derived from infected tissue 10 
microliter samples of DNAase treated RNA are reverse transcribed using a 
SuperScript Preamplification System for First Strand cDNA Synthesis kit 
(Gibco BRL, Life Technologies) according to the manufacturers 
instructions. 1 nanogram of random hexamers is used to prime each 
reaction. Controls without the addition of SuperScriptII reverse 
transcriptase are also run. Both +/-RT samples are treated with RNaseH 
before proceeding to the PCR reaction 
e) The use of PCR to determine the presence of a bacterial cDNA species 
PCR reactions are set up on ice in 0.2 ml tubes by adding the following 
components: 45 microliters PCR SUPERMIX (Gibco BRL, Life Technologies); 1 
microliter 50 mM MgCl.sub.2, to adjust final concentration to 2.5 mM; 1 
microliter PCR primers(optimally 18-25 basepairs in length and designed to 
possess similar annealing temperatures), each primer at 10 mM initial 
concentration; and 2 microliters cDNA. 
PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 as follows: 
5 minutes at 95.degree. C., then 50 cycles of 30 seconds each at 
94.degree. C., 42.degree. C. and 72.degree. C. followed by 3 minutes at 
72.degree. C. and then a hold temperature of 4.degree. C. (the number of 
cycles is optimally 30-50 to determine the appearance or lack of a PCR 
product and optimally 8-30 cycles if an estimation of the starting 
quantity of cDNA from the RT reaction is to be made); 10 microliter 
aliquots are then run out on 1% 1.times. TBE gels stained with ethidium 
bromide with PCR product, if present, sizes estimated by comparison to a 
100 bp DNA Ladder (Gibco BRL, Life Technologies). Alternatively if the PCR 
products are conveniently labelled by the use of a labelled PCR primer 
(e.g. labelled at the 5' end with a dye) a suitable aliquot of the PCR 
product is run out on a polyacrylamide sequencing gel and its presence and 
quantity detected using a suitable gel scanning system (e.g. ABI PrismTM 
377 Sequencer using GeneScanTM software as supplied by Perkin Elmer). 
RT/PCR controls may include +/-reverse transcriptase reactions, 16s rRNA 
primers or DNA specific primer pairs designed to produce PCR products from 
non-transcribed Staphylococcus aureus WCUH29 genomic sequences. 
To test the efficiency of the primer pairs they are used in DNA PCR with 
Staphylococcus aureus WCUH29 total DNA. PCR reactions are set up and run 
as described above using approx. 1 microgram of DNA in place of the cDNA 
and 35 cycles of PCR. 
Primer pairs which fail to give the predicted sized product in either DNA 
PCR or RT/PCR are PCR failures and as such are uninformative. Of those 
which give the correct size product with DNA PCR two classes are 
distinguished in RT/PCR: 1.Genes which are not transcribed in vivo 
reproducibly fail to give a product in RT/PCR; and 2.Genes which are 
transcribed in vivo reproducibly give the correct size product in RT/PCR 
and show a stronger signal in the +RT samples than the signal (if at all 
present) in -RT controls. 
Two polynucleotide sequences of the invention, SEQ ID NOS:1 and 3, were 
identified in the above test as transcribed in vivo. SEQ ID NO:2 was 
deduced from the polynucleotide sequence given as SEQ ID NO: 1. SEQ ID 
NO:4 was deduced from the polynucleotide sequence given as SEQ ID NO:3. 
The pair of PCR primers used to identify the gene are given as SEQ ID 
NOS:5 and 6. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 6 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 810 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - TCTATATTAT TGATTTACTT AGAACAAGGT AAACTCCAAA GGGTGAGTGA CT - 
#AATGACTG 60 
- - TAGAATGGTT AGCAGAACAA TTAAAAGAAC ATAATATTGA ATTAACTGAG AC - 
#TCAAAAAC 120 
- - AACAGTTTCA AACATATTAT CGTTTACTTG TTGAATGGAA TGAAAAGATG AA - 
#TTTGACAA 180 
- - GTATTACAGA TGAACACGAT GTATATTTGA AACATTTTTA TGATTCCATT GC - 
#ACCTAGTT 240 
- - TTTATTTTGA TTTTAATCAG CCTATAAGTA TATGTGATGT AGGCGCTGGA GC - 
#TGGTTTTC 300 
- - CAAGTATTCC GTTAAAAATA ATGTTTCCGC AGTTAAAAGT GACGATTGTT GA - 
#TTCATTAA 360 
- - ATAAGCGTAT TCAATTTTTA AACCATTTAG CGTCAGAATT ACAATTACAG GA - 
#TGTCAGCT 420 
- - TTATACACGA TAGAGCAGAA ACATTTGGTA AGGGTGTCTA CAGGGAGTCT TA - 
#TGATGTTG 480 
- - TTACTGCAAG AGCAGTAGCT AGATTATCCG TGTTGAGTGA ATTGTGTTTA CC - 
#GCTAATTA 540 
- - AAAAAGGTGG ACAGTTTGTT GCATTAAAAT CTTCAAAAGG TGAAGAAGAA TT - 
#AGAAGAAG 600 
- - CAAAATTTGC AATTAGTGTG TTAGGTGGTA ACGTTACAGA AACACATACC TT - 
#TAAATTGC 660 
- - CAGAAGATGC TGGAGAGCGC CAGATGTTCA TTATTGATAA AAAAAGACAG AC - 
#GCCGAAAA 720 
- - AGTACCCAAG AAAACCAGGG ACGCCTAATA AGACTCCTTT ACTTGAAAAA TA - 
#ATGCATAA 780 
- - TCCTTTACAA CTAACATAAA AGGAGCGAAT - # - # 
810 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 239 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Thr Val Glu Trp Leu Ala Glu Gln Leu Ly - #s Glu His Asn Ile Glu 
1 5 - # 10 - # 15 
- - Leu Thr Glu Thr Gln Lys Gln Gln Phe Gln Th - #r Tyr Tyr Arg Leu Leu 
20 - # 25 - # 30 
- - Val Glu Trp Asn Glu Lys Met Asn Leu Thr Se - #r Ile Thr Asp Glu His 
35 - # 40 - # 45 
- - Asp Val Tyr Leu Lys His Phe Tyr Asp Ser Il - #e Ala Pro Ser Phe Tyr 
50 - # 55 - # 60 
- - Phe Asp Phe Asn Gln Pro Ile Ser Ile Cys As - #p Val Gly Ala Gly Ala 
65 - #70 - #75 - #80 
- - Gly Phe Pro Ser Ile Pro Leu Lys Ile Met Ph - #e Pro Gln Leu Lys Val 
85 - # 90 - # 95 
- - Thr Ile Val Asp Ser Leu Asn Lys Arg Ile Gl - #n Phe Leu Asn His Leu 
100 - # 105 - # 110 
- - Ala Ser Glu Leu Gln Leu Gln Asp Val Ser Ph - #e Ile His Asp Arg Ala 
115 - # 120 - # 125 
- - Glu Thr Phe Gly Lys Gly Val Tyr Arg Glu Se - #r Tyr Asp Val Val Thr 
130 - # 135 - # 140 
- - Ala Arg Ala Val Ala Arg Leu Ser Val Leu Se - #r Glu Leu Cys Leu Pro 
145 1 - #50 1 - #55 1 - 
#60 
- - Leu Ile Lys Lys Gly Gly Gln Phe Val Ala Le - #u Lys Ser Ser Lys 
Gly 
165 - # 170 - # 175 
- - Glu Glu Glu Leu Glu Glu Ala Lys Phe Ala Il - #e Ser Val Leu Gly Gly 
180 - # 185 - # 190 
- - Asn Val Thr Glu Thr His Thr Phe Lys Leu Pr - #o Glu Asp Ala Gly Glu 
195 - # 200 - # 205 
- - Arg Gln Met Phe Ile Ile Asp Lys Lys Arg Gl - #n Thr Pro Lys Lys Tyr 
210 - # 215 - # 220 
- - Pro Arg Lys Pro Gly Thr Pro Asn Lys Thr Pr - #o Leu Leu Glu Lys 
225 2 - #30 2 - #35 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 900 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - GTAAATCCAG CAGACATATC TATATTATTG ATTTACTTAG AACAAGGTAA AC - 
#TCCAAAGG 60 
- - GTGAGTGACT AATGACTGTA GAATGGTTAG CAGAACAATT AAAAGAACAT AA - 
#TATTGAAT 120 
- - TAACTGAGAC TCAAAAACAA CAGTTTCAAA CATATTATCG TTTACTTGTT GA - 
#ATGGAATG 180 
- - AAAAGATGAA TTTGACAAGT ATTACAGATG AACACGATGT ATATTTGAAA CA - 
#TTTTTATG 240 
- - ATTCCATTGC ACCTAGTTTT TATTTTGATT TTAATCAGCC TATAAGTATA TG - 
#TGATGTAG 300 
- - GCGCTGGAGC TGGTTTTCCA AGTATTCCGT TAAAAATAAT GTTTCCGCAG TT - 
#AAAAGTGA 360 
- - CGATTGTTGA TTCATTAAAT AAGCGTATTC AATTTTTAAA CCATTTAGCG TC - 
#AGAATTAC 420 
- - AATTACAGGA TGTCAGCTTT ATACACGATA GAGCAAAAAC ATNTGGTAAG GG - 
#TGTCTACA 480 
- - NGGAGTCTTA TGATGTTGTT ACTGCAAGAG CAGTANCTAA ATTATCCGTG TT - 
#GAGTGAAT 540 
- - TGTGTTTACC GCTAATTAAA AAAGGTGGAC AGTNTGTTGC ATTAAAATCT TC - 
#AAAAGGTG 600 
- - AAGAAAAATT ANAAAAAACA NAATTTGCAA TTAGTGTGTT AGGTGGTAAC GT - 
#TACAGAAA 660 
- - CACATACCTT TAAATTGCCA GAAGATGCTG GAGAGCGCCA GATGTTCATT AT - 
#TGATAAAA 720 
- - AAAGACAGAC GCCGAAAAAG TACCCAAGAA AACCAGGGAC GCCTAATAAG AC - 
#TCCTTTAC 780 
- - TTGAAAAATA ATGCATAATC CTTTACAACT AACATAAAAG GAGCGAATGG AT - 
#AATGAAAA 840 
- - AACCTTTTTC AAAATTATTT GGTTTGAAAA ACAAAGATGA CATCATTGGA CA - 
#TATTGAAG 900 
- - - - 2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 239 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Met Thr Val Glu Trp Leu Ala Glu Gln Leu Ly - #s Glu His Asn Ile 
Glu 
1 5 - # 10 - # 15 
- - Leu Thr Glu Thr Gln Lys Gln Gln Phe Gln Th - #r Tyr Tyr Arg Leu Leu 
20 - # 25 - # 30 
- - Val Glu Trp Asn Glu Lys Met Asn Leu Thr Se - #r Ile Thr Asp Glu His 
35 - # 40 - # 45 
- - Asp Val Tyr Leu Lys His Phe Tyr Asp Ser Il - #e Ala Pro Ser Phe Tyr 
50 - # 55 - # 60 
- - Phe Asp Phe Asn Gln Pro Ile Ser Ile Cys As - #p Val Gly Ala Gly Ala 
65 - #70 - #75 - #80 
- - Gly Phe Pro Ser Ile Pro Leu Lys Ile Met Ph - #e Pro Gln Leu Lys Val 
85 - # 90 - # 95 
- - Thr Ile Val Asp Ser Leu Asn Lys Arg Ile Gl - #n Phe Leu Asn His Leu 
100 - # 105 - # 110 
- - Ala Ser Glu Leu Gln Leu Gln Asp Val Ser Ph - #e Ile His Asp Arg Ala 
115 - # 120 - # 125 
- - Lys Thr Xaa Gly Lys Gly Val Tyr Xaa Glu Se - #r Tyr Asp Val Val Thr 
130 - # 135 - # 140 
- - Ala Arg Ala Val Xaa Lys Leu Ser Val Leu Se - #r Glu Leu Cys Leu Pro 
145 1 - #50 1 - #55 1 - 
#60 
- - Leu Ile Lys Lys Gly Gly Gln Xaa Val Ala Le - #u Lys Ser Ser Lys 
Gly 
165 - # 170 - # 175 
- - Glu Glu Lys Leu Xaa Lys Thr Xaa Phe Ala Il - #e Ser Val Leu Gly Gly 
180 - # 185 - # 190 
- - Asn Val Thr Glu Thr His Thr Phe Lys Leu Pr - #o Glu Asp Ala Gly Glu 
195 - # 200 - # 205 
- - Arg Gln Met Phe Ile Ile Asp Lys Lys Arg Gl - #n Thr Pro Lys Lys Tyr 
210 - # 215 - # 220 
- - Pro Arg Lys Pro Gly Thr Pro Asn Lys Thr Pr - #o Leu Leu Glu Lys 
225 2 - #30 2 - #35 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - ATGACTGTAG AATGGTTAGC - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - TTATTTTTCA AGTAAAGGAG - # - # 
- # 20 
__________________________________________________________________________