WO2001038345A1 - Regulatable fabh constructs - Google Patents

Abstract

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

Description

Regulatable FabH Constructs 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 novel polynucleotides and polypeptides of the Fab family, hereinafter referred to as "FabH". 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 a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.

The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, whilst the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeasts possess type I fatty acid synthases (FASs) in which all of the enzymatic activities are encoded on one or two polypeptide chains, respectively. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad spectrum antibacterial agents (Rock, C. & Cronan, J. 1996. Biochimica et Biophysica Acta 1302, 1-16; Jackowski, S. 1992. la Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadakou. Chapman & Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264, 7624-7629.)

The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. Prior to this, malonyl-ACP is synthesized from ACP and malonyl-CoA by FabD, malonyl CoA:ACP transacylase. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively). The second step in the elongation cycle is ketoester reduction by NADPH-depeiidcnt β-kctoacyl- ACP reductase (FabG). Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH- dependent enoyl-ACP reductase (Fabl). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, ( 1996), f.Biol.Chem. 271, 1833- 1836). Cerulenin and thiolactomycin are potent and selective inhibitors of bacterial fatty acid biosynthesis. Extensive work with these inhibitors in Gram-negative bacteria has proved that this biosynthetic pathway is essential for viability. Little work has been carried out in Gram- positive bacteria. No mutants totally lacking FabD activity have been described. No mammalian homologues to FabD have yet been identified. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics would be rendered inactive by known antibiotic resistance mechanisms. There is an unmet need for developing new classes of antibiotic compounds, such as those that target FabD.

Clearly, there exists a need for factors, such as the FabH embodiments of the invention, that have a present benefit of 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 amino acid sequence homology to a known FabH protein.

Clearly, there is a need for factors that may be used to screen compounds for antibiotic activity and which factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. There is a need, therefore, for identification and characterization of such factors which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.

The polypeptide of the present invention has amino acid sequence homology to known malonylCoA:ACP transacylase SUMMARY OF THE INVENTION

It is an object of the invention to provide a polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 expressibly linked to an isolated regulatable gene expression construct. It is a further object of the invention to provide a polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the FabH gene contained in the Staphylococcus aureus of the deposited strain expressibly linked to an isolated regulatable gene expression construct.

A still further embodiment of the invention is a polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 expressibly linked to an isolated regulatable gene expression construct.

A preferred polynucleotide of the invention comprises nucleotide 1 to the stop codon set forth in SEQ ID NO: 1. Another preferred polynucleotide of the invention comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

An embodiment of the invention provides a vector comprising a polynucleotide of the invention.

Another preferred embodiment of the invention provides a host cell comprising a vector of the invention.

The invention also provided a process for producing a polypeptide comprising: expressing from a host cell of the invention a polypeptide encoded by said DNA.

Another process of the invention provides producing a FabH polypeptide or fragment comprising culturing a host of the invention under conditions sufficient for the production of said polypeptide or fragment.

A method of the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprises the steps of: contacting a composition comprising the polypeptide with a compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.

Another method of the invention provides identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprising the steps of: contacting a cell comprising a polynucleotide of claim 1 with a compound to be screened; and detecting a phenotypic change is said cell.

A preferred embodiment of a method of the invention further comprises: (i) a step wherein said phenotypic change is lessened or does not occur after the level of expression of said polypeptide is raised, (ii) a step wherein said phenotypic change is increased or enhanced after the level of expression of said polypeptide is lowered, and/or (iii), a step wherein said phenotypic is altered after the level of expression of said polypeptide is altered.

A further method provided by the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprise the steps of: contacting a cell comprising a polynucleotide of the invention with a compound to be screened; and detecting a phenotypic change is said cell.

Another method of the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprises the steps of: contacting a cell comprising a polynucleotide of the invention with a compound to be screened; and detecting a phenotypic change is said cell.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 shows a determination of FabH expression levels by Western immunoblot

Fig 2 illustrates MIC testing of FabH compounds on S. aureus strain FabH2/pFF40

Fig 3 shows a schematic illustration a preferred construction of a regulatable fabH strain of S. aureus using Pspac promoter Fig 4 shows that strain FabHl has multiple integration of pSMUTery-fabH' Fig 5 shows a Southern hybridization confirms the genetic structure of FabHl and FabH2 Fig 6 shows a FabHl Growth Curve Fig 7 shows a FabHl/pFF40 Growth Curve Fig 8 shows a FabH2 Growth Curve

Fig 9 shows a FabH2/pFF40 Growth Curve Fig 10 illustrates growth dependence of Strain FabH l on IPTG Fig 1 1 illustrates growth dependence of Strain FabI 12 on IPTG

Fig 12 illustrates FabH cellular concentration and induction in regulatable fabH S. aureus strain FabH2

Fig 1 illustrates induction of FabH in strains FabH l and FabH2

DESCRIPTION OF THE INVENTION

The invention relates to novel FabH polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel FabH of Staphylococcus aureus, which is related by amino acid sequence homology to FabH polypeptide. The invention relates especially to FabH having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and to the FabH nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.

TABLE 1 FabH Polynucleotide and Polypeptide Sequences

(A) Sequences from Staphylococcus aureus FabH polynucleotide sequence [SEQ ID NO: l]. The start codon is shown in bold and underlined. The stop codon is underlined.

5 ' -ATGAACGTGGGTATTAAAGGTTTTGGTGCATATGCACCAGAAAAGA

TTATTGACAATGCCTATTTTGAGCAATTTTTAGATACATCTGATGAATGGATTTCTAAGATGACTGGAAT

TAAAGAAAGACATTGGGCAGATGACGATCAAGATACTTCAGATTTAGCATATGAAGCAAGTGTAAAAGCA ATCGCTGACGCTGGTATTCAGCCTGAAGATATAGATATGATAATTGTTGCCACAGCAACTGGAGATATGC CATTTCCAACTGTCGCAAATATGTTGCAAGAACGTTTAGGGACGGGCAAAGTTGCCTCTATGGATCAACT TGCAGCATGTTCTGGATTTATGTATTCAATGATTACAGCTAAACAATATGTTCAATCTGGAGATTATCAT AATATTTTAGTTGTCGGTGCAGATAAATTATCTAAAATAACAGATTTAACTGACCGTTCTACTGCAGTTC TATTTGGAGATGGTGCAGGTGCGGTTATCATCGGTGAAGTTTCAGAAGGCAGAGGTATTATAAGTTATGA AATGGGTTCTGATGGCACTGGTGGTAAACATTTATATTTAGATAAAGATACTGGTAAACTGAAAATGAAT GGTCGAGAAGTATTTAAATTTGCTGTTAGAATTATGGGTGATGCATCAACACGTGTAGTTGAAAAAGCGA ATTTAACATCAGATGATATAGATTTATTTATTCCTCATCAAGCTAATATTAGAATTATGGAATCAGCTAG AGAACGCTTAGGTATTTCAAAAGACAAAATGAGTGTTTCTGTAAATAAATATGGAAATACTTCAGCTGCG TCAATACCTTTAAGTATCGATCAAGAATTAAAAAATGGTAAACTCAAAGATGATGATACAATTGTTCTTG TCGGATTCGGTGGCGGCCTAACTTGGGGCGCAATGACAATAAAATGGGGAAAA AG-3 '

(B) Staphylococcus aureus FabH polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID NO:2].

NH--MNVGIKGFGAYAPEKIIDNAYFEQFLDTSDEWISKMTGIKERHWADDD

QDTSDLAYEASVKAIADAGIQPEDIDMIIVATATGDMPFPTVANMLQER GTGKVASMDQ AACSGF YS MITAKQYVQSGDYHNI WGADKLSKITDLTDRSTAVLFGDGAGAVI IGEVSEGRG I ISYEMGSDGTGGK HLYLDKDTGKLKMNGREVFKFAVRIMGDASTRWEKANLTSDDIDLFI PHQA IRIMESARERLGISKDK MSVSVNKYGNTSAASIPLSIDQELK GKLKDDDTIVLVGFGGGLTWGAMTIK GK-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 1RY. Scotland on 1 1 September 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 FabH gene. The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of the 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. §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. Polypeptides The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FabH, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO: 1 ]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% similarity (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO:2] 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 polypeptides of the formula:

X-(Rl)m-(R2)-(R3)n-Y wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Rj and R3 are any amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1. In the formula above R2 is oriented so that its amino terminal residue is at the left, bound to R_j and its carboxy terminal residue is at the right, bound to R3. Any stretch of amino 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.

A fragment is a variant polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides. As with FabH 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, 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 of variants thereof, such as a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention 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.

Also preferred are biologically active fragments which are those fragments that mediate activities of FabH, 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. Variants that are 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.

Figure 1 shows the results of a Western immunoblot to determine expression of FabH in S. aureus RN4220. Polynucleotides

An aspect of the invention is to provide a polynucleotide comprising a polynucleotide encoding a polypeptide comprising a FabH amino acid sequence, expressibly linked to an isolated regulatable gene expression construct. A regulatable gene expression construct includes, among other things, inducible and repressible promoters, temperature- sensitive promoters, pressure sensitive promoters, sigma factor driven promoters, operators, stem loops, DNA binding protein binding sites, and protein binding sites. A preferred embodiment of a regulatable gene expression construct is a construct that can be used to lower the level of FabH gene expression, for example, repressible promoters, an uniduced inducible promoter, an operator that can be repressed with a repressor, or a combination of these. An further aspect of the invention is a FabH construct comprising a 0.5 kb fragment of a fabH gene further comprising a 5' end of fabH gene together with its own ribosome binding site. This fragment was amplified by PCR from S. aureus strain RN4220. A preferred embodiment of this construct is an integration vector incorporating the DNA fragment, particularly constructed and used to transform S. aureus, especially S. aureus strain RN4220. fabH regulatable strains of the invention are a further preferred embodiment of the invention. In particular, constructs wherein FabH is regulatable, and bacteria comprising the same, are provided by this invention. These constructs may be singly or multiply present in a cell, and may be integrated into the genome or present as a non-integrated DNA. Regulation of the FabH gene may be achieved using a regulatable promoter, such as any inducible or repressible promoter known in the art or provided herein, as well as operator-repressor combinations known in the art. In a more preferred embodiment, cellular steady-state abundance of FabH expressed from the regulatable gene is about 0-500 molecules per cell, 500-1,000 molecules per cell, 1 ,000-3,000 molecules per cell, 3,000-10,000 molecules per cell, and greater than 10,000 molecules per cell. Another preferred embodiment is a regulatable FabH construct showing an induction ratio for FabH production of about 2-fold, 3-fold, 4-fold, 5-fold, or greater than 5-fold.

Polynucleotides of the invention are provided comprising FabH antisense constructs. These constructs are useful in methods wherein expression of antisense fragments down- regulate FabH gene expression. This approach has been demonstrated as useful to lowering gene expression (Kernodle et al., Infect. Immun., 1997, 65: 179-184). Moreover, the use of antisense can be a powerful tool to aid in understanding the function of FabH without necessarily completely eliminating its activity. In the case of an essential gene, like FabH, antisense technology allows the skilled artisan to very easily manipulate the expression of a gene in order to observe the consequences of a lethal mutation over time. Antisense constructs of the invention preferably further comprise an inducible promoter system to selectively induce expression of an antisense FabH fragment.

A promoter useful in the constructs and methods of the invention is the xylltet hybrid promoter (Geissendorfer et al., Appl. Microbiol. Biotechnol., 1990, 33:657-663), which allows tight regulation of downstream genes, with titratable induction. The xylltet hybrid promoter is functional in host cells, such as, S. aureus, as demonstrated by titratable activity that enabled monitoring of promoter activity over time. Inducible xylltet promoter constructs have also been shown to be functional in B. subtilis (Geissendorfer et al, Appl. Microbiol. Biotechnol, 1990, 33:657-663). This promoter incorporates elements of both the xylose- and tetracycline- inducible systems, resulting in a tightly-regulated, strong promoter that is induced with low concentrations of tetracycline. Certain preferred constructs are illustrated in Figures 3, and 4. Other useful promoters and expression modulations systems may be used, particularly in Staphylococcus aureus, such as those described in Zhang, et al. Gene 255: 297 (2000). This reference describes the Pspac, Pxyl and Pxyl/tet regulatable promoter systems in S. aureus. The constructs and host cells of the invention are useful in animal models, particularly in infection models in mice, hamsters, guinea pigs, gerbils, rats, chinchillas and rabbits. A preferred animal model in which host cells comprising the polynucleotiudes of the invention are useful is a murine model of hematogenous pyelonephritis. This model is particularly useful to illustrate certain embodiments of the invention, as it results in a localized kidney infection from which bacteria are readily recovered. For example, various regulatable promoter constructs can be induced or repressed using the appropriate inducer or repressor given orally to mice infected with bacteria comprising such promoter constructs. Using different concentrations of inducer or repressor to control the levels of antisense expressed, one can regulate expression to different degrees.

The inducible system that is an embodiment of the invention allows one to specifically increase, decrease or abolish expression of a particular gene. Therefore, the effects of the overexpression or absence of the gene product can be studied after synchronization of the cells by the addition of inducer. In addition, the titratability of this promoter systems provided herein makes it possible to observe the effects of different levels of up- or down-regulation of an FabH without completely inactivating the gene. This type of analysis can aid in the development of antimicrobial agents by, among other things, decreasing levels of a FabH gene product, thereby rendering cells more susceptible

Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the FabH polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof comprising a regulatable gene expression construct.

The DNA sequence set out in Table 1 [SEQ ID NO: 1 ] 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] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art. The polynucleotide of SEQ ID NO: 1 , between nucleotide number 1 and the stop codon which begins at the third nucleotide from the 3 -end of SEQ ID NO: 1 , encodes the polypeptide of SEQ ID NO:2.

The invention provides a polynucleotide comprising a sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO: l] expressibly linked to a regulatable gene expression construct. Also provided by the invention is the coding sequence for the mature polypeptide expressibly linked to a regulatable gene expression construct, or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence. The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode 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 tag (Wilson etal, Cell 37: 767 (1984). 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. The invention also includes polynucleotides of the formula:

X-(Rl)_m-(R₂)-(R₃)n-Y wherein, at the 5' end of the molecule, X is hydrogen, and at the 3^' end of the molecule, Y is hydrogen or a metal, R_j is any nucleic acid residue expressibly linked to a regulatable gene expression construct and R3 is any nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1. In the polynucleotide formula above R₂ is oriented so that its 5' end residue is at the left, bound to R_j and its 3' end residue is at the right, bound to R3. 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. In a preferred embodiment in and/or n is an integer between 1 and 1000.

It is most preferred that the polynucleotides of the inventions are derived from Staphylococcus aureus, however, they may preferably be obtained from organisms of the same taxonomic genus. They 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 FabH having an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) 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 for variants of the polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] expressibly linked to a regulatable gene expression construct. Variants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention. Further particularly preferred embodiments are polynucleotides encoding FabH variants, that have the amino acid sequence of FabH polypeptide of Table 1 [SEQ ID NO:2] expressibly linked to a regulatable gene expression construct , and in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of FabH.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding FabH polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2] and expressibly linked to a regulatable gene expression construct, 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 FabH polypeptide of the deposited strain 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 that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:l] expressibly linked to a regulatable gene expression construct.

The invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°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 1 1 therein. The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: l expressibly linked to a regulatable gene expression construct, under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO: l or a fragment thereof; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding FabH and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the FabH gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less. 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 disease, particularly human disease, as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived from the sequences of Table 1 [SEQ ID NOS: l or 2] expressibly linked to a regulatable gene expression construct, 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 may 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.

A precursor protein, having the 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 nucleic acid bases, the term "N" may also be used in describing certain polynucleotides of the invention. "N" means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a base 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

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.

A wide variety of vectors may be used in the present invention. For example, pCUl (Gram-negative to Gram-positive shuttle vector), pWM401 (Gram-negative to Gram-positive shuttle vector), pHV33 (Gram-negative to Gram-positive shuttle vector), pHV1431 (Gram- negative to Gram-positive shuttle vector), pNZ12 (Gram-negative to Gram-positive shuttle vector). Preferred vectors and related references are listed in Table 2 (below).

Table 2

Plasmid Description Related Reference

pMH109 contains multiple cloning sites Hudson et al., Gene, 1986, 48:93- upstream of the cat gene 100 pWH353 contains the tet regulatory Geissendorfer et al., Appl. elements including the tetR gene Microbiol. Biotechnol., 1990, and its promoter with a poly-A 33:657-663 block, and the xyl/tet promoter- operator fusion pYJ82 contains the cat gene cloned into Yanisch-Perron et al., Gene, 1985, the EcoRI and BamHl sites of 33: 103-119 pUC19 pYJ90 contains the origin of replication Horinouchi et al, J. BacterioL, from plasmids pE194 and pUC19, 1982, 150:804-814 which allows replication in Gram- positive and Gram-negative bacteria; contains Erm and Ap resistance markers and a multiple cloning site pYJIOl contains the tet regulatory element inserted into the Cla 1 and Hindlll sites of pBluescript II KS (Stratagene, La Jolla, CA) pYJ103 contains the cat gene cloned into EcoRl and Pstl sites of pYJ IOl pYJ335 contains the tet regulatory element and the cat gene cloned into the Sail site of pYJ90 pYJ318-7 contains a 621 -bp hla fragment in the antisense orientation cloned into the Sinai site of pYJ335 pYJ318-16 contains the hla fragment in the sense orientation cloned into the Smal site of pYJ335

The methods of the invention may be used with any Gram+ plasmid made into a shuttle vector by ligation with pBluescript. The skilled artisan will be readily able to make such vectors based on the teachings herein and in the art.

Inducible promoters useful in the methods of the invention may be any inducible promoter, for example, a doxycycline inducible promoter (see Kistner et al., PNAS USA 93: 10933 (1996)), erythromycin resistance promoter (see Ross et al, Gene 183: 143 (1996)), a macrolide resistance promoter (see Shuwsei et al., Antimicrobial Agents and Chemotherapy 41(3): 530 (1997), or a tetracycline resistance promoter (see Geissendorfer et al, Appl Microbiol. Biotchnol. 33:651-663 (1990); Gossen et al, Science 268: 1766 (1995)); an IPTG inducible promoter, such as pSpac; or an in vivo induced promoter, such as acetyl-CoA- acyltransferase promoter, identified by in vitro expression work by RT-PCR (see Yanasura, et al. Proc. Natl. Acad. Sci. USA 81 : 439 (1984). Termination sequences useful in the invention include, for example, rho-dependent termination signal, 5". aureus and S. pneumoniae termination signals, rho-independent termination.

For recombinant production, 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., MOLECUUXR CLONING: A L\BORATORY 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 streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drυsoplύla S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and vims-derived vectors, e.g., 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 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).

For secretion of the 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, hyd oxylapatite 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.

Antagonists and agonists - assays and molecules Regulatable FabH constructs of the invention and bacteria comprising such constructs may also be used in methods to screen for compounds that agonize or antagonize the activity of FabH. Compounds useful in such methods comprise small molecule substrates and ligands of FabH, as well as cell extracts, 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 of the same. See, e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of FabH polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bacteriocidal. 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 FabH 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 FabH agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the FabH 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 FabH polypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate 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 FabH polynucleotide or polypeptide activity, and binding assays known in the art.

A method of the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprises the steps of: contacting a composition comprising the polypeptide with a compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide. An example of MIC testing performed to demonstrate FabH inhibition by test compounds is shown in Figure 2. Another method of the invention provides identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprising the steps of: contacting a cell comprising a polynucleotide of claim 1 with a compound to be screened; and detecting a phenotypic change is said cell. A preferred embodiment of a method of the invention further comprises: (i) a step wherein said phenotypic change is lessened or does not occur after the level of expression of said polypeptide is raised, (ii) a step wherein said phenotypic change is increased or enhanced after the level of expression of said polypeptide is lowered, and/or (iii), a step wherein said phenotypic is altered after the level of expression of said polypeptide is altered. A further method provided by the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprise the steps of: contacting a cell comprising a polynucleotide of the invention with a compound to be screened; and detecting a phenotypic change is said cell. Another method of the invention for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprises the steps of: contacting a cell comprising a polynucleotide of the invention with a compound to be screened; and detecting a phenotypic change is said cell. Another example of an assay for FabH antagonists is a competitive assay that combines

FabH and a potential antagonist with FabH-binding molecules, recombinant FabH binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. FabH can be labeled, such as by radioactivity or a colorimetric compound, such that the number of FabH 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 small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. 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 FabH-induced activities, thereby preventing the action of FabH by excluding FabH 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, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of FabH.

Each of the DNA 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 DNA 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 or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and 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 bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block FabH 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 mammalian extracellular matrix proteins and bacterial FabH proteins that mediate tissue damage and; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques. The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat diseases.

Compositions and Kits

The invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above. The 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.

It is preferred that such carriers are fabricated from a rigid material, such as, for example, plastic, metal or glass.

Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety. GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "Disease(s)" means and disease caused by or related to infection by a bacteria, including 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 diarrhea, splenic abscess, 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 abscess, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).

"Antisense polynucleotide" means a polynucleotide sequence that is capable of hybridizing to or is complementary to, in whole or in part, another polynucleotide sequence.

"Expressibly linked" means a first polynucleotide sequence joined to a second polynucleotide sequence, such as by ligation, so that they act together to express a gene product, such as a DNA, an RNA or a protein.

"Host cell" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence and includes a cell or cells of 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, Legionella, Pseudo onas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further 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 pyogencs, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neissei iu meiiingitidis, Staphylococcus aureu.s, Staphy lococcus epidei idis R 4220, Staphylococcus epideimidis WCUH29, Staphylococcus epiderntidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium lcerans, Mycobacterium leprae, Actinomyctcs israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchi septica, Escherichia colt, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii. Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia inarcessetis, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium peifringens, Clostridium tetaui, 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. Herein these cells are also referred to as "organisms."

"Inducer" means a composition of matter or electromagnetic radiation to which an inducible gene control region responds by altering the expression of an expressibly linked polynucleotide. Examples of inducers include those well known in the art, such as UV radiation, IPTG, pressure, temperature, sugars, those disclosed herein, among others.

"Inducible gene control region" means a polynucleotide sequence that responds to a composition of matter or electromagnetic radiation and alters the expression of an expressibly linked polynucleotide. Examples of such regions include inducible promoters or derepressible operator/promoters combinations, many of which are well known.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, 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" and "similarity" 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 Pi inter, G ibs ov, M. and De . ercux, J., cd._., λl Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 4S: 1073 (198S). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity 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 (Atschul, 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 Bcthesda, MD 20894; Altschul, S., et al, J. Mol. Biol 215: 403-410 ( 1990). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously , by a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

"Isolated" means altered "by the hand of man" from its natural slate, 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.

"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, 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, oligopcptidcs 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 posl-tran_>latiunal 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 Iipid 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, glycosylation, 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. "Regulatable gene expression construct(s)" means a polynucleotide sequence that regulates gene expression in response to a compound, electromagnetic radiation, temperature, pressure, or other environmental factor to which said polynucleotide sequence or a cell comprising the same, is exposed. An example of a regulatable gene expression construct is an inducible gene control region.

"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, fusions 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. 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 1 [SEQ ID NO: l ] 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: 1. 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 l lkbp in size arc rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapTI that has been cut with EcoRI, the library packaged by standard procedures and E.cυli 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., Rsal, Pall, Alul, Bshl235I), 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 Construction of fabH regulated expression strains in 5. aureus

A 0.5 kb fragment (fabH') including 5' end of fabH gene together with its own ribosome binding site was amplified by PCR from S. aureus strain RN4220.

An integration vector incorporating this DNA fragment was constructed and used to transform S. aureus RN4220 by electroporation. Two putative fabH regulatable strains were identified by diagnostic PCR after five independent transformation attempts and screening of 33 erythromycin-resistant clones. Southern analysis showed that one of the mutant strain FabHl contained multiple integrations of pSMUTery-fabH' at the fabH locus, whereas the second mutant strain FabH2 had the correct single integration (See Figures 4 and 5). Both fabH strains were transformed with a Lacl-expressing plasmid pFF40. Strain FabHl showed growth dependence on IPTG with or without plasmid pFF40 (See Figures 6, 7 and 10). Strain FabH2 showed growth dependence on IPTG only in the presence of plasmid pFF40 (See Figures 8, 9 and 1 1 ). Cellular abundance of FabH in S. aureus was estimated to be around 3,000 molecules per cell (18 μM) using quantitative Western immunoblotting. S. aureus FabH2 strain showed an induction ratio of 3-fold for FabH production (see Figures 12 and 13).

Claims

What is claimed is:

I . A polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 expressibly linked to an isolated regulatable gene expression constntct.

2. A polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the FabH gene contained in the Staphylococcus aureus of the deposited strain expressibly linked to an isolated regulatable gene expression construct.

3. A polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of

SEQ ID NO:2 expressibly linked to an isolated regulatable gene expression construct.

4. A polynucleotide that is complementary to the polynucleotide of claim 1.

5. The polynucleotide of Claim 1 wherein the polynucleotide is DNA or RNA.

6. The polynucleotide of Claim 1 comprising the nucleic acid sequence set forth in SEQ ID NO: l .

7. The polynucleotide of Claim 1 comprising nucleotide 1 to the stop codon set forth in SEQ ID NO: 1.

8. The polynucleotide of Claim 1 which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

9. A vector comprising the polynucleotide of Claim 1.

10. A host cell comprising the vector of Claim 9.

I I . A process for producing a polypeptide comprising: expressing from the host cell of Claim 10 a polypeptide encoded by said DNA.

12. A process for producing a FabH polypeptide or fragment comprising culturing a host of claim 10 under conditions sufficient for the production of said polypeptide or fragment.

13. A method for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprising the steps of: contacting a composition comprising the polypeptide with a compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound; and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.

14. A method for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 707c identical to the amino acid sequence of SEQ ID NO:2 comprising the steps of: contacting a cell comprising a polynucleotide of claim 1 with a compound to be screened; and detecting a phenotypic change is said cell.

15. The method of claim 14 further comprising a step wherein said phenotypic change is lessened or does not occur after the level of expression of said polypeptide is raised.

16. The method of claim 14 further comprising a step wherein said phenotypic change is increased or enhanced after the level of expression of said polypeptide is lowered.

17. The method of claim 14 further comprising a step wherein said phenotypic is altered after the level of expression of said polypeptide is altered.

18. A method for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2 comprising the steps of: contacting a cell comprising a polynucleotide of claim 2 with a compound to be screened; and detecting a phenotypic change is said cell.

19. The method of claim 18 further comprising a step wherein said phenotypic change is lessened or does not occur after the level of expression of said polypeptide is raised.

20. The method of claim 18 further comprising a step wherein said phenotypic change is increased or enhanced after the level of expression of said polypeptide is lowered.

21. The method of claim 18 further comprising a step wherein said phenotypic is altered after the level of expression of said polypeptide is altered.

22. A method for identifying a compound that inhibits or activates an activity of a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 comprising the steps of: contacting a cell comprising a polynucleotide of claim 3 with a compound to be screened; and detecting a phenotypic change is said cell.

23. The method of claim 22 further comprising a step wherein said phenotypic change is lessened or does not occur after the level of expression of said polypeptide is raised.

24. The method of claim 22 further comprising a step wherein said phenotypic change is increased or enhanced after the level of expression of said polypeptide is lowered.

25. The method of claim 22 further comprising a step wherein said phenotypic is altered after the level of expression of said polypeptide is altered.