WO2003104391A2

WO2003104391A2 - Antibacterial targets in alloiococcus otitidis

Info

Publication number: WO2003104391A2
Application number: PCT/US2002/036122
Authority: WO
Inventors: Ellen Murphy; Steven Jay Projan
Original assignee: Wyeth Holdings Corporation
Priority date: 2001-11-29
Filing date: 2002-11-25
Publication date: 2003-12-18
Also published as: WO2003104391A3; AU2002367968A8; AU2002367968A1

Abstract

The present invention relates to the identification of polynucleotide sequences encoding polypeptides of Alloiococcus otitidis that are essential for the growth and survival of the bacteria. In particular, the invention relates to polypeptides encoded by the Alloiococcus otitidis open reading frames (ORFs), and to their use in pharmaceutical compositions, therapeutics, diagnostics and the like. The present invention also relates to methods for identifying pharmaceutical compounds that inhibit the activity of the polypeptides that are essential for the growth ofAlloiococcus otitidis, to pharmaceutical compositions containing these compounds and to their use in treatment and amelioration of diseases caused by Alloiococcus otitidis

Description

ANTIBACTERIAL TARGETS IN ALLOIOCOCCUS OTITIDIS

FIELD OF THE INVENTION

The present invention relates to the genomic sequence of Alloiococcus otitidis and polynucleotide sequences encoding polypeptides of the Gram-positive bacterium, Alloiococcus otitidis. The invention also relates to polynucleotides and polynucleotides encoding polypeptides, preferably antigenic polypeptides, encoded by the Alloiococcus otitidis open reading frames and the uses thereof.

BACKGROUND OF THE INVENTION

Since the discovery of penicillin, the use of antibiotics to treat the ravages of bacterial infections has saved millions of lives. With the advent of these "miracle drugs," for a time it was popularly believed that humanity might, once and for all, be saved from the scourge of bacterial infections. In fact, during the 1980s and early 1990s, many large pharmaceutical companies cut back or eliminated antibiotics research and development. They believed that infectious disease caused by bacteria finally had been conquered and that markets for new drugs were limited. Unfortunately, this belief was overly optimistic. The tide is beginning to turn in favor of the bacteria, as reports of drug resistant bacteria become more frequent. The United States Centers for Disease Control and Prevention announced that one of the most powerful known antibiotics, vancomycin, was unable to treat an infection of the common bacterial pathogen, Staphylococcus aureus. This organism, commonly found in our environment, is responsible for many nosocomial infections. The import of this announcement becomes clear when one considers that vancomycin was used for years to treat infections caused by Staphylococcus species as well as other stubborn strains of bacteria. In short, bacteria are becoming resistant to our most powerful antibiotics. If this trend continues, it is conceivable that we will return to a time when what are presently considered minor bacterial infections are fatal diseases.

Over-prescription and improper prescription habits by some physicians have caused an indiscriminate increase in the availability of antibiotics to the public. The patients are also partly responsible, since they will often improperly use the drug, thereby generating yet another population of bacteria that is resistant, in whole or in part, to traditional antibiotics.

The bacterial pathogens that have haunted humanity remain, in spite of the development of modern scientific practices to deal with the diseases that they cause. Drug resistant bacteria are now an increasing threat to the health of humanity. A new generation of antibiotics is needed to once again deal with the pending health threats that bacteria present.

As more and more bacterial strains become resistant to the panel of available antibiotics, new antibiotics are required to treat infections. In the past, practitioners of pharmacology relied upon traditional methods of drug discovery to generate novel, safe and efficacious compounds for the treatment of disease. Traditional drug discovery methods involve blindly testing potential drug candidate- molecules, often selected at random, in the hope that one might prove to be an effective treatment for some disease. The process is painstaking and laborious, with no guarantee of success.

Newly emerging practices in drug discovery utilize a number of biochemical techniques to provide for directed approaches to creating new drugs, rather than discovering them at random. For example, gene sequences and proteins encoded thereby that are required for the proliferation of a cell or microorganism make excellent targets since exposure of bacteria to compounds active against these targets would result in the inactivation of the cell or microorganism. Once a target is identified, biochemical analysis of that target can be used to discover or to design molecules that interact with and alter the functions of the target. Use of physical and computational techniques to analyze structural and biochemical properties of targets in order to derive compounds that interact with such targets is called rational drug design and offers great potential. Thus, emerging drug discovery practices use molecular modeling techniques, combinatorial chemistry approaches, and other means to produce and screen and/or design large numbers of candidate compounds. Nevertheless, while this approach to drug discovery is clearly the way of the future, problems remain. For example, the initial step of identifying molecular targets for investigation can be an extremely time consuming task. It may also be difficult to design molecules that interact with the target by using computer modeling techniques. Furthermore, in cases where the function of the target is not known or is poorly understood, it may be difficult to design assays to detect molecules that interact with and alter the functions of the target. To improve the rate of novel drug discovery and development, methods of identifying important molecular targets in pathogenic cells or microorganisms and methods for identifying molecules that interact with and alter the functions of such molecular targets are urgently required. The present invention is directed to identifying important molecular targets in a recently identified bacteria, Alloiococcus otitidis, which has been implicated in otitis media with effusion (OME). Otitis media, an inflammatory disease of the middle ear, is the most frequent cause of visits to pediatricians' offices in the United States (Schappert, 1991 ). Approximately 80% of all children experience at least one episode of otitis media by the age of three (Klein, 1994). There are three main types of otitis media: Acute otitis media (AOM), otorrhea, and otitis media with effusion (OME). Alloiococcus otitidis has only been associated with otitis media with effusion (OME), but this may be due to the difficulty of its detection by standard bacterial culturing methods. Its detection in the effusions is likely due to the fact that the effusions are normally sterile and few or no competing bacterial species are isolated from them. Without the interference of faster growing nasophryngeal species, the culture plates can be incubated for the longer duration needed to detect Alloiococcus otitidis colonies. Three other bacterial species are commonly isolated from middle ear effusions. These are nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. One or more of these species have been found in one study to be associated with about 77% of all cases of OME using a PCR detection method (Post, 2000). This study did not include assaying for Alloiococcus otitidis, so a portion of the unaccounted cases may be due to this organism.

The bacterium Alloiococcus otitidis was first isolated from the middle ear fluids of 10 children in the Buffalo, NY area with persistent OME and characterized as a large catalase negative, Gram-positive cocci that tend to occur in clumps, often in tetrads. It is slow growing and requires 2 to 5 days at 37°C before colonies can be seen on sheep blood agar plates. The bacterium was named Alloiococcus otitis by Aguirre and Collins (1992), who showed that it was different from other known Gram- positive species based on its 16S rRNA sequence. The bacterium's name has been changed from Alloiococcus otitis to Alloiococcus otitidis. (Hendolin, et al., (1999), and Hendolin et al., (2000)).

Several studies of the epidemiology Alloiococcus otitidis indicate it is associated with otitis media with effusion. These are summarized in Table 1. These studies have been done using both culture and PCR techniques. The number of cases detected by culture, as might be expected from the fastidious growth requirements of the bacterium, was less than the number detected by PCR. Assuming that the bacterium is detected more accurately by the PCR method, the bacterium is detected in between 10 and 50% of patients with OME. This frequency suggests that this organism represents a significant public health problem. Consequently, there is a need for identifying gene targets in Alloiococcus otitidis iox the development of anti-infectives. There is also a need for compositions for diagnosing Alloiococcus otitidis infection.

Number of persons in study.

SUMMARY OF INVENTION

The present invention broadly relates to Alloiococcus otitidis genomic sequence. Particularly, the invention relates to newly identified polynucleotide open reading frames (ORFs) comprised within the genomic nucleotide sequence of Alloiococcus otitidis, and to polypeptides encoded by the ORFs. More particularly, the ORFs encode polypeptides that are essential for the growth and survivablity of Alloiococcus otitidis.

Thus, in certain aspects, the invention relates to Alloiococcus otitidis ORFs that encode Alloiococcus otitidis polypeptides that function as enzymes in various biosynthetic pathways in the bacterium. In one embodiment, the invention relates to a purified or isolated Alloiococcus otitidis nucleic acid sequence comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, wherein expression of said nucleic acid is essential for the proliferation of a cell. In a preferred embodiment the ORF selected from one of the odd numbered sequence listings set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105 encodes an essential gene. The essential gene and the polypeptide encoded by them include ACPS (holo-(acyl carrier protein) synthase), murF (UDP-N- acetylmuramoylalanyl-D-glutamyl-2,6-diamino pimelate-D-alanyl-D-alanyl ligase) murA-2 (UDP-N-acetylglucosamine 1-carboxyvinyltransf erase), RpoE (DNA-directed RNA polymerase, delta subunit), rpoA (DNA-directed RNA polymerase alpha subunit), rpoC (RNA polymerase beta' subunit), rpoB (DNA-dependent RNA polymerase subunit beta), dnaB/C (DNA polymerase III delta prime subunit), gyrA (DNA gyrase A subunit), gyrB (DNA gyrase B subunit), dnaN (DNA polymerase III beta chain, folC-2 (folyl-polyglutamate synthetase), murE (UDP-N-acetylmuramoyl-L- alanyl-D-glutamyl-L-lysine Ligase), srtA (sortase), folC-1 (folyl-polyglutamate synthetase), folB (dihydroneopterin aldolase), folK (7,8-dihydro-6- hydroxymethylpterin-pyrophosphokinase), mvaS (hydroxymethylglutaryl-CoA synthase), mvaA (3-hydroxy-3-methylglutaryl-coenzyme a reductase), murB (UDP-N- acetylglucosaminyl-3-enolpyruvate reductase), mvaK2 (phosphomevalonate kinase), mvaD (mevalonate diphosphate decarboxylase), mvaK1 (mevalonate kinase), coaA (pantothenate kinase), nadE (NAD+ synthase), murl, Glutamate racemase), folP (Dihydropteroate synthase), folA (dihydrofolate reductase), grlB (topoisomerase IV B subunit), grlA (topoisomerase IV A subunit), rpoD (transcription initiation factor sigma), dnaG (DNA primase), era (GTP-binding protein), norA (drug-export protein), polC (DNA polymerase III, alpha subunit), obg (GTP-binding protein), yphC (similar to Escherichia coli GTP-binding protein Era), dnaE (DNA polymerase III, alpha subunit), coaBC (phosphopantothenoylcysteine synthetase/decarboxylase), holA (DNA polymerase III delta subunit), coaD (phosphopantetheine adenylyltransferase) ftsZ (Cell division protein ftsZ), ftsA (Cell division protein ftsA), murG (phospho-N- acetylmuramoyl-pentapeptide-transferase), murD (UDP-N-acetylmuramoylalanine D- glutamate ligase), nadD (nicotinic acid mononucleotide adenylyltransferase), coaE (dephospho-CoA kinase), murC (UDP-N-acetyl muramate-alanine ligase), fmhB FemX (factor essential for methicillin resistance), pcrA (ATP-dependent DNA helicase), murA-1 (UDP-N-acetylglucosamine 1-carboxyvinyltransf erase), holB (DNA polymerase III delta' subunit) and dnaX (DNA polymerase III -gamma and tau subunits). In another embodiment, the invention relates to purified or isolated nucleic acid of Alloiococcus otitidis comprising a fragment of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, wherein said fragment is selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50 and more than 50 consecutive nucleotides of one of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105. In yet another embodiment, the invention relates to a purified or isolated antisense nucleic acid comprising a nucleotide sequence complementary to at least a portion of an intragenic sequence, intergenic sequence, sequences spanning at least a portion of two or more genes, 5' noncoding region, or 3' noneoding region within an operon comprising a proliferation-required gene of Alloiococcus otitidis whose activity or expression is inhibited by an antisense nucleic acid and selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

In a nother embodiment, the invention relates to a purified or isolated nucleic acid comprising a nucleotide sequence having at least 70% identity to a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, fragments comprising at least 25 consecutive nucleotides selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, the nucleotide sequences complementary to one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, and the sequences complementary to fragments comprising at least 25 consecutive nucleotides of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

In another embodiment, the invention relates to a vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence of any one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

In another embodiment, the invention relates to purified or isolated polypeptide of Alloiococcus otitidis comprising a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a fragment selected from the group consisting of fragments comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of one of the said polypeptides. In yet another embodiment, the invention relates to purified or isolated

Alloiococcus otitidis polypeptide comprising a amino acid sequence having at least 25% amino acid identity to a polypeptide whose expression is inhibited by a nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or at least 25% amino acid identity to a fragment comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of a polypeptide whose expression is inhibited by a nucleic acid comprising a nucleotide sequence selected from the group consisting of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105. In one embodiment, the invention relates to a purified or isolated Alloiococcus otitidis polypeptide comprising selected from one of the even numbered sequences set forth in Seq. ID Nos: 2 to Seq. ID Nos: 106, wherein the polypeptide is essential for the proliferation of a cell..

In yet another embodiment, the invention relates to a method of producing an Alloiococcus otitidis polypeptide comprising introducing into a cell a vector comprising a promoter operably linked to a nucleic acid comprising a nucleotide sequence encoding a polypeptide whose expression is essential for the proliferation and viability of Alloiococcus otitidis, and which is inhibited by an antisense nucleic acid, and which is selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

In yet another embodiment, the invention relates to a method of inhibiting the proliferation of Alloiococcus otitidis in an individual comprising inhibiting the activity or reducing the amount of a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105 or inhibiting the activity or reducing the amount of a nucleic acid encoding said gene product.

In a preferred embodiment, the invention relates to method for identifying a compound which influences the activity of an Alloiococcus otitidis gene product , which is required for proliferation, said gene product comprising a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, said method comprising: (a) contacting said gene product with a candidate compound; and (b) determining whether said compound influences the activity of said gene product.

In a preferred embodiment, the invention relates to method for identifying a compound or an antisense nucleic acid having the ability to reduce activity or level of a Alloiococcus otitidis gene product, which is required for proliferation, said gene product comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, said method comprising the steps of: (a) contacting a target gene or RNA encoding said gene product with a candidate compound or antisense nucleic acid; and(b) measuring the activity of said target.

In yet another preferred embodiment, the invention relates to method for inhibiting cellular proliferation of Alloiococcus otitidis comprising introducing an effective amount of a compound with activity against a gene whose activity or expression is essential for cellular proliferation, and which is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a compound with activity against the product of said gene into a population of Alloiococcus otitidis cells expressing said gene. In a preferred embodiment, the invention relates to a composition comprising an effective concentration of an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a proliferation-inhibiting portion thereof in a pharmaceutically acceptable carrier.

In a preferred embodiment, the invention relates to method for identifying a compound having the ability to inhibit proliferation of Alloiococcus otitidis cell comprising: (a) identifying a homologue of a gene or gene product whose activity or level is inhibited by a nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, in a test cell, wherein said test cell is not Alloiococcus otitidis; (b) identifying an inhibitory nucleic acid sequence which inhibits the activity of said homologue in said test cell; (c) contacting said test cell with a sublethal level of said inhibitory nucleic acid, thus sensitizing said cell; (d) contacting the sensitized cell of step (c) with a compound; and (e) determining the degree to which said compound inhibits proliferation of said sensitized cell relative to a cell which does not contain said inhibitory nucleic acid.

In a preferred embodiment, the invention relates to a method for identifying a compound having activity against a biological pathway required for proliferation comprising: (a) sensitizing a cell by providing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding a gene product required for proliferation, wherein the activity or expression of said gene product is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, in said cell to reduce the activity or amount of said gene product; (b) contacting the sensitized cell with a compound; and (c) determining the degree to which said compound inhibits the growth of said sensitized cell relative to a cell which does not contain said antisense nucleic acid.

In a preferred embodiment, the invention relates to a method for identifying a compound having the ability to inhibit one of the Alloiococcus otitidis polypeptides encoded by a polynucleotide selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, and which is essential for cellular proliferation comprising: (a) contacting a cell which expresses the polypeptide with the compound; and (b) determining whether said compound reduces proliferation of said contacted cell by acting on said gene product.

In a preferred embodiment, the invention relates to a method for identifying a compound having the ability to inhibit one of the purified and isolated Alloiococcus otitidis polypeptides selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq. ID No.: 106, and which is essential for cellular proliferation comprising: (a) contacting the purified and isolated polypeptide with the compound in vitro in the presence or absence of a substrate, which is essential for the activity of the polypeptide; and (b) determining the effect of the compound on the polypeptide by measuring the effect of the polypeptide on the substrate.

In a preferred embodiment, the invention relates to a compound which interacts with an Alloiococcus otitidis polypeptide selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq. ID No.: 106 and inhibits its activity. In a preferred embodiment, the invention relates to a method for manufacturing an antimicrobial compound comprising the steps of screening one or more candidate compounds to identify a compound that reduces the activity or level of an Alloiococcus otitidis polypeptide selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq. ID No.: 106, said polypeptide comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105; and manufacturing the compound so identified.

In a preferred embodiment, the invention relates to a compound which inhibits proliferation of Alloiococcus otitidis by interacting with a gene encoding a polypeptide that is required for proliferation or with a polypeptide required for proliferation, wherein said polypeptide is selected from the group consisting of a gene product having at least 70% nucleotide sequence identity from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105, polypeptide encoded by a nucleic acid having at least 70% nucleotide sequence identity to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105, a polypeptide having at least 25% amino acid identity to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105, a polypeptide encoded by a nucleic acid comprising a nucleotide sequence which hybridizes to a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105 under stringent conditions, a gene product encoded by a nucleic acid comprising a nucleotide sequence which hybridizes to a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105 under moderate conditions, and a gene product whose activity may be complemented by the gene product whose activity is inhibited by a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions:

By "biological pathway" is meant any discrete cell function or process that is carried out by a gene product or a subset of gene products. Biological pathways include anabolic, catabolic, enzymatic, biochemical and metabolic pathways as well as pathways involved in the production of cellular structures such as cell walls. Biological pathways that are usually required for proliferation of cells or microorganisms include, but are not limited to, cell division, DNA synthesis and replication, RNA synthesis (transcription), protein synthesis (translation), protein processing, protein transport, fatty acid biosynthesis, electron transport chains, cell wall synthesis, cell membrane production, synthesis and maintenance, and the like. By "inhibit activity of a gene or gene product" is meant having the ability to interfere with the function of a gene or gene product in such a way as to decrease expression of the gene, in such a way as to reduce the level or activity of a product of the gene or in such a way as to inhibit the interaction of the gene or gene product with other biological molecules required for its activity.

Agents which inhibit the activity of a gene include agents that inhibit transcription of the gene, agents that inhibit processing of the transcript of the gene, agents that reduce the stability of the transcript of the gene, and agents that inhibit translation of the mRNA transcribed from the gene. In microorganisms, agents which inhibit the activity of a gene can act to decrease expression of the operon in which the gene resides or alter the folding or processing of operon RNA so as to reduce the level or activity of the gene product. The gene product can be a non- translated RNA such as ribosomal RNA, a translated RNA (mRNA) or the protein product resulting from translation of the gene mRNA. Of particular utility to the present invention are antisense RNAs that have activities against the operons or genes to which they specifically hybridze.

By "activity against a gene product" is meant having the ability to inhibit the function or to reduce the level or activity of the gene product in a cell. This includes, but is not limited to, inhibiting the enzymatic activity of the gene product or the ability of the gene product to interact with other biological molecules required for its activity, including inhibiting the gene product's assembly into a multimeric structure.

By "activity against a protein" is meant having the ability to inhibit the function or to reduce the level or activity of the protein in a cell. This includes, but is not limited to, inhibiting the enzymatic activity of the protein or the ability of the protein to interact with other biological molecules required for its activity, including inhibiting the protein's assembly into a multimeric structure.

By "activity against a nucleic acid" is meant having the ability to inhibit the function or to reduce the level or activity of the nucleic acid in a cell. This includes, but is not limited to, inhibiting the ability of the nucleic acid interact with other biological molecules required for its activity, including inhibiting the nucleic acid's assembly into a multimeric structure.

By "activity against a gene" is meant having the ability to inhibit the function or expression of the gene in a cell. This includes, but is not limited to, inhibiting the ability of the gene to interact with other biological molecules required for its activity. By "activity against an operon" is meant having the ability to inhibit the function or reduce the level of one or more products of the operon in a cell. This includes, but is not limited to, inhibiting the enzymatic activity of one or more products of the operon or the ability of one or more products of the operon to interact with other biological molecules required for its activity.

By "antibiotic" is meant an agent which inhibits the proliferation of a cell or microorganism. By "homologous coding nucleic acid" is meant a nucleic acid homologous to a nucleic acid encoding a gene product whose activity or level is inhibited by a nucleic acid selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 or a portion thereof. In some embodiments, the homologous coding nucleic acid may have at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and fragments comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides thereof. In other embodiments the homologous coding nucleic acids may have at least 97%, at least 5 95%, at least 90%, at least 85%, at least 80%, or at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequences complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and fragments comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides thereof. Identity may be measured using BLASTN version 2.0 with the default parameters or tBLASTX with the default parameters. (Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs, Nucleic Acid Res. 25: 3389- 3402 (1997)) Alternatively a "homologuous coding nucleic acid" could be identified by membership of the gene of interest to a functional orthologue cluster. All other members of that orthologue cluster would be considered homologues. Such a library of functional orthologue clusters can be found at hltp://www.nebi.nlm.nib.gov/COG. A gene can be classified into a cluster of orthologous groups or COG by using the COGNITOR program available at the above web site, or by direct BLASTP comparison of the gene of interest to the members of the COGs and analysis of these results as described by Tatusov, R.L., Galperin, M.Y., Natale, D. A. and

Koonin, EN. (2000) The COG database: a tool for genome- scale analysis of protein functions and evolution. Nucleic Acids Research v. 2 8 n. 1 , pp3 3 -3 6.

The term "homologous coding nucleic acid" also includes nucleic acids comprising nucleotide sequences which encode polypeptides having at least 99%, 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25% amino acid identity or similarity to a polypeptide comprising the amino acid sequence of one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 or to a polypeptide whose expression is inhibited by a nucleic acid comprising a nucleotide sequence of one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 or fragments comprising at least 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as determined using the FASTA version 3.O.78 algorithm with the default parameters. Alternatively, protein identity or similarity may be identified using BLASTP with the default parameters, BLASTX with the default parameters, TBLASTN with the default parameters, or tBLASTX with the default parameters. (Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs, Nucleic Acid Res. 25: 3389-3402 (1997)).

The term "homologous coding nucleic acid" also includes coding nucleic acids which hybridize under stringent conditions to a nucleic acid selected from the group consisting of the nucleotide sequences complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and coding nucleic acids comprising nucleotide sequences which hybridize under stringent conditions to a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides of the sequences complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105.

As used herein, "stringent conditions" means hybridization to filter-bound nucleic acid in 6xSSC at about 45'C followed by one or more washes in 0. lxSSC/0.2/ SDS at about 680C. Other exemplary stringent conditions may refer, e.g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37C, 48'C, 55'C, and 60'C as appropriate for the 5 particular probe being used.

The term "homologous coding nucleic acid" also includes coding nucleic acids comprising nucleotide sequences which hybridize under moderate conditions to a nucleotide sequence selected from the group consisting of the sequences complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and coding nucleic acids comprising nucleotide sequences which hybridize under moderate conditions to a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200,300,400, or 500 consecutive nucleotides of the sequences complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105. As used herein, "moderate conditions" means hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45'C followed by one or more washes in 0.2xSSC/0. 1 % SDS at about 42- 65'C.

The term "homologous coding nucleic acids" also includes nucleic acids comprising nucleotide sequences which encode a gene product whose activity may be complemented by a gene encoding a gene product whose activity is inhibited by a nucleic acid comprising a nucleotide sequence selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105. In some embodiments, the homologous coding nucleic acids may encode a gene product whose activity is complemented by the gene product encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting Seq ID Nos.: 1 to Seq. ID Nos.: 105. In other embodiments, the homologous coding nucleic acids may comprise a nucleotide sequence encodes a gene product whose activity is complemented by one of the polypeptides of Seq ID Nos.: 1 to Seq. ID Nos.: 105 . The term "homologous antisense nucleic acid" includes nucleic acids comprising a nucleotide sequence having at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of one of the sequences of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and fragments comprising at least 10, 15, 20, 25, 30,35,40, 50, 75, 100, 150, 200,300,400, or 500 consecutive nucleotides thereof. Homologous antisense nucleic acids may also comprising nucleotide sequences which have at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 70% nucleotide sequence identity to a nucleotide sequence selected from the group consisting of the sequences complementary to one of sequences of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and fragments comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides thereof.

Nucleic acid identity may be determined as described above. The term "homologous antisense nucleic acid" also includes antisense nucleic acids comprising nucleotide sequences which hybridize under stringent conditions to a nucleotide sequence complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and antisense nucleic acids comprising nucleotide sequences which hybridize under stringent conditions to a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive nucleotides of the sequence complementary to one Seq ID Nos.: 1 to Seq. ID Nos.: 105. Homologous antisense nucleic acids also include antisense nucleic acids comprising nucleotide sequences which hybridize under stringent conditions to a nucleotide sequence selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105, and antisense nucleic acids comprising nucleotide sequences which hybridize under stringent conditions to a fragment comprising at least 10, 15, 20,25, 30, 35, 40, 50, 75, 100,150,200,300,400, or 500 consecutive nucleotides of one of Seq ID Nos.: 1 to Seq. ID Nos.: 105.

The term "homologous antisense nucleic acid" also includes antisense nucleic acids comprising nucleotide sequences which hybridize under moderate conditions to a nucleotide sequence complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and antisense nucleic acids comprising nucleotide sequences which hybridize under moderate conditions to a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides of the sequence complementary to one of Seq ID Nos.: 1 to Seq. ID Nos.: 105.

Homologous antisense nucleic acids also include antisense nucleic acids comprising nucleotide sequences which hybridize under moderate conditions to a nucleotide sequence selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 and antisense nucleic acids which comprising nucleotide sequences hybridize under moderate conditions to a fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive nucleotides of one of Seq ID Nos.: 1 to Seq. ID Nos.: 105.

By "homologous polypeptide" is meant a polypeptide homologous to a polypeptide whose activity or level is inhibited by a nucleic acid comprising a nucleotide sequence selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 by a homologous antisense nucleic acid. The term "homologous polypeptide" includes polypeptides having at least 99%, 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25% amino acid identity or similarity to a polypeptide whose activity or level is inhibited by a nucleic acid selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 or by a homologous antisense nucleic acid, or polypeptides having at least 99%, 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25% amino acid identity or similarity to a polypeptide to a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids of a polypeptide whose activity or level is inhibited by a nucleic acid selected from the group consisting of Seq ID Nos.: 1 to Seq. ID Nos.: 105 or by a homologous antisense nucleic acid. Identity or similarity may be determined using the FASTA version 3. Ot78 algorithm with the default parameters. Alternatively, protein identity or similarity may be identified using BLASTP with the default parameters, BLASTX with the default parameters, or TBLASTN with the default parameters. (Altschul, S.F. et al. Gapped BLAST and PSI- BLAST: A New Generation of Protein Database Search Programs, Nucleic Acid Res. 25: 3389-3402 (1997).

The term homologous polypeptide also includes polypeptides having at least 99%, 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25% amino acid identity or similarity to a polypeptide selected from the group consisting of Seq ID Nos.: 2 to Seq. ID Nos.: 106 and polypeptides having at least 99%, 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25% amino acid identity or similarity to a fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 5 0, 75, 100, or 150 consecutive amino acids of a polypeptide selected from the group consisting of Seq ID Nos.: 2 to Seq. ID Nos.: 106. The invention also includes polynucleotides, preferably DNA molecules, that hybridize to one of the nucleic acids of Seq ID Nos.: 2 to Seq. ID Nos.: 106 or the complements of any of the preceding nucleic acids. Such hybridization may be under stringent or moderate conditions as defined above or under other conditions which permit specific hybridization. The nucleic acid molecules of the invention that hybridize to these DNA sequences include oligodeoxynucleotides ("oligos") which hybridize to the target gene under highly stringent or stringent conditions. In general, for oligos between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula:

Tm ff) = 81.5 + 16.6(log[monovalent cations (molar)] + 0.41 (% G+Q - (500N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation:

Tm('C) = 81.5 + 16.6(log[monovalent cations (niolar)] + 0.4 1 (% G+C) - (0.6 1) (% formamide) - (500N) where N is the length of the probe. In general, hybridization is carried out at about 20-25 degrees below Tin (for DNA-DNA hybrids) or about 10- 15 degrees below Tin (for RNA-DNA hybrids).

Other hybridization conditions are apparent to those of skill in the art (see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. 1 , Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3.

By "identifying a compound" is meant to screen one or more compounds in a collection of compounds such as a combinatorial chemical library or other library of chemical compounds or to characterize a single compound by testing the compound in a given assay and determining whether it exhibits the desired activity.

By "inducer" is meant an agent or solution which, when placed in contact with a cell or microorganism, increases transcription, or inhibitor and/or promoter clearance/fidelity, from a desired promoter. As used herein, "nucleic acid" means DNA, RNA, or modified nucleic acids.

Thus, the terminology "the nucleic acid of SEQ ID NO: V or "the nucleic acid comprising the nucleotide sequence" includes both the DNA sequence of SEQ ID NO: X and an RNA sequence in which the thymidines in the DNA sequence have been substituted with uridines in the RNA sequence and in which the deoxyribose backbone Of the DNA sequence has been substituted with a ribose backbone in the RNA sequence. Modified nucleic acids are nucleic acids having nucleotides or structures which do not occur in nature, such as nucleic acids in which the intemucleotide phosphate residues with methylphosphonates, phosphorothioates, phosphoramidates, and phosphate esters. Nonphosphate intemucleotide analogs such as siloxane bridges, carbonate bridges, thioester bridges, as well as many others known in the art may also be used in modified nucleic acids. Modified nucleic acids may also comprise, (x-anomeric nucleotide units and modified micleotides such as 1 2 dideoxy-d-ribofuranose, 1 ,2-dideoxy- 1 -phenylribof uranose, and N4, N4- ethano-5 -methyl-cytosine are contemplated for use in the present invention. Modified nucleic acids may also be peptide nucleic acids in which the entire deoxyribose-phosphate backbone has been exchanged with a chemically completely different, but structurally homologous, polyamide (peptide) backbone containing 2- aminoethyl glycogen units.

As used herein, "sub-lethal" means a concentration of an agent below the concentration required to inhibit all cell growth.

A proliferation-required gene or gene family is one where, in the absence or substantial reduction of a gene transcript and/or gene product, growth or viability of the cell or microorganism is reduced or eliminated. Thus, as used herein, the terminology "proliferation- required" or "required for proliferation" encompasses instances where the absence or substantial reduction of a gene transcript and/or gene product completely eliminates cell growth as well as instances where the absence of a gene transcript and/or gene product merely reduces cell growth. These proliferation-required genes can be used as potential targets for the generation of new antimicrobial agents. To achieve that goal, the present invention also encompasses assays for analyzing proliferation- required genes and for identifying compounds which interact with the gene and/or gene products of the proliferation- required genes. In addition, the present invention contemplates the expression of genes and the purification of the proteins encoded by the nucleic acid sequences identified as required proliferation genes and reported herein. The purified proteins can be used to generate reagents and screen small molecule libraries or other candidate compound libraries for compounds that can be further developed to yield novel antimicrobial compounds. The invention described herein addresses the need for identifying

Alloiococcus otitidis proliferation-required gene or gene family that may be used to identify compounds, which are effective in preventing or treating most or all of the disease caused by Alloiococcus otitidis. The invention further addresses the need for methods of diagnosing Alloiococcus otitidis infection using the genes and the polypeptides identified herein. The inventors have identified novel Alloiococcus otitidis open reading frames (Ors), which encode proteins/polypeptides that are essential for the growth and proliferation of the bacteria. More particularly, the newly identified Ors encode polypeptides that are essential for proliferation of Alloiococcus otitidis, and thus serve as potential targets for antimicrobial compounds. Thus, in certain embodiments, the invention comprises Alloiococcus otitidis Ors encoding polypeptides that are essential for cellular proliferation, transcription gene products of Alloiococcus otitidis Ors, including, but not limited to mRNA, antisense RNA, antisense oligonucleotides, and ribozyme molecules, which can be used to inhibit or control growth of the microorganism. The invention relates also to methods of detecting Alloiococcus otitidis nucleic acids or polypeptides and kits for diagnosing Alloiococcus otitidis infection. The invention also relates to pharmaceutical compositions, in particular antimicrobial compounds in pharmaceutical compositions, for the prevention and/or treatment of bacterial infection, in particular infection caused by or exacerbated by Alloiococcus otitidis.

B. ALLOIOCOCCUS OTITIDIS ORF POLYNUCLEOTIDES ENCODING POLYPEPTIDES ESSENTIAL FOR PROLIFERATION

Isolated and purified Alloiococcus otitidis ORF polynucleotides of the present invention are contemplated for use in the production of Alloiococcus otitidis polypeptides. More specifically, in certain embodiments, the ORFs encode

Alloiococcus otitidis polypeptides that are essential for cell proliferation. Thus, in one aspect, the present invention provides isolated and purified polynucleotides (ORFs) that encode Alloiococcus otitidis essential for cell proliferation. In particular embodiments, a polynucleotide of the present invention is a DNA molecule, wherein the DNA may be genomic DNA, plasmid DNA or cDNA. In a preferred embodiment, a polynucleotide of the present invention is a recombinant polynucleotide, which encodes an Alloiococcus otitidis polypeptide comprising an amino acid sequence that has at least 25% identity to an amino acid sequence of one of even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106 or a fragment thereof. In another embodiment, an isolated and purified ORF polynucleotide comprises a nucleotide sequence that has at least 70% identity to one of the ORF polynucleotide nucleotide sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105, a degenerate variant thereof, or a complement thereof. In yet another embodiment, an ORF polynucleotide of one of SEQ ID NO: 1 through SEQ ID NO: 105 is comprised in a plasmid vector and expressed in a host cell. In a preferred embodiment, the host cell is a prokaryotic host cell. As used herein, the term "polynucleotide" means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5' to the 3' direction. A polynucleotide of the present invention can comprise from about 10 to about several hundred thousand base pairs. Preferably, a polynucleotide comprises from about 10 to about 3,000 base pairs. Preferred lengths of particular polynucleotide are set forth hereinafter.

A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single- stranded or double-stranded, but preferably is double-stranded DNA. Where a polynucleotide is a DNA molecule, that molecule can be a gene, a cDNA molecule or a genomic DNA molecule. Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T) and cytosine (C).

"Isolated" means altered "by the hand of man" from the natural state. An "isolated" composition or substance is one that has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal 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. Preferably, an "isolated" polynucleotide is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated Alloiococcus otitidis nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0. 5 kb or 0. 1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. However, the Alloiococcus otitidis nucleic acid molecule can also be fused to heterologous protein encoding or regulatory sequences and still be considered isolated.

ORF polynucleotides of the present invention may also be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA. Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries (e.g., an Alloiococcus otitidis library) or can be synthesized using well-known and commercially available techniques. As contemplated in the present invention, ORF polynucleotides are obtained using Alloiococcus otitidis chromosomal DNA as the template.

The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences set forth in the odd numbered sequences listed in ID NO: 1 through SEQ ID NO: 105 (and fragments thereof) due to degeneracy of the genetic code, and thus encode the same Alloiococcus otitidis polypeptides as those encoded by the amino acid sequences shown in even numbered sequences set forth in SEQ ID NO:2 through SEQ ID NO: 106

Orthologs and allelic variants of the Alloiococcus otitidis polynucleotides are readily identified using methods well known in the art. An allelic variant or an orthologue of the polynucleotides comprises a nucleotide sequence that is typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more homologous to the nucleotide sequence shown in one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105, or a fragment of these nucleotide sequences. Such nucleic acid molecules are readily identified as being able to hybridize, preferably under stringent conditions, to the nucleotide sequence shown in one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105, or a fragment of these nucleotide sequences.

Moreover, the polynucleotides of the invention can comprise only a fragment of the coding region of an Alloiococcus otitidis polynucleotide or gene, such as a fragment of one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105.

When the ORF polynucleotides of the invention are used for the recombinant production of Alloiococcus otitidis polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be linked to the coding sequence (see Gentz et al., 1989, incorporated herein by reference). Thus, contemplated in the present invention is the preparation of polynucleotides encoding fusion polypeptides permitting His-tag purification of expression products. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals. Thus, a polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Alloiococcus otitidis, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105 or a fragment thereof; and isolating full-length cDNA and genomic clones containing the polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5" end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during the first- strand cDNA synthesis.

Thus, in certain embodiments, the polynucleotide sequence information provided by the present invention allows for the preparation of relatively short DNA (or RNA) oligonucleotide sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. The term "oligonucleotide" as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. Thus, in particular embodiments of the invention, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected nucleotide sequence, e.g., a sequence such as that shown in one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105. The ability of such nucleic acid probes to specifically hybridize to a polynucleotide encoding an Alloiococcus otitidis polypeptide lends them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotide primers. These primers are generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of an ORF polynucleotide that encodes an Alloiococcus otitidis polypeptide from prokaryotic cells using polymerase chain reaction (PCR) technology. In certain embodiments, it is advantageous to employ a polynucleotide of the present invention in combination with an appropriate label for detecting hybrid formation. A wide variety of appropriate labels are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO: 105, or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than Alloiococcus otitidis) that have a high sequence similarity to polynucleotide sequences set forth in one of the odd numbered sequences set forth in SEQ ID NO:1 through SEQ ID NO:105, or a fragment thereof. Typically these nucleotide sequences are from at least 70% identical to at least about 95% identical to that of the reference polynucleotide sequence. The probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, Frohman et al., 1988). Recent modifications of the technique, exemplified by the Marathon™ technology [Promega, Madison, Wl], for example, have significantly simplified the search for longer cDNAs. In the Marathon™ 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 cDNA 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 are then analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA 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.

To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes probe molecules that are complementary to at least a 10 to about 70 nucleotides long stretch of a polynucleotide that encodes an Alloiococcus otitidis polypeptide, such as that shown in one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106. A size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. It is generally preferable to design nucleic acid molecules with gene-complementary stretches of 25 to 40 nucleotides, 55 to 70 nucleotides, or even longer where desired. For example, such fragments are readily prepared by directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology (U.S. Patent 4,683,202, incorporated herein by reference), or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction enzyme sites.

In another aspect, the present invention contemplates an isolated and purified polynucleotide comprising a nucleotide sequence that is identical or complementary to a segment of at least 10 contiguous bases of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105, wherein the polynucleotide hybridizes to a polynucleotide that encodes an Alloiococcus otitidis polypeptide.

Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105. For example, the polynucleotide of the invention can comprise a segment of bases identical or complementary to from 40 to 55 contiguous bases of the disclosed nucleotide sequences.

Accordingly, a polynucleotide probe molecule of the invention can be used for its ability to selectively form duplex molecules with complementary stretches of the gene. Depending on the application envisioned, varying conditions of hybridization are employed to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, relatively stringent conditions are employed to form the hybrids. Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate an Alloiococcus otitidis homologous polypeptide coding sequence from other cells, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex (see Table 2). Cross-hybridizing species are thereby readily identified as positively hybridizing signals with respect to control hybridizations. Thus, hybridization conditions are readily manipulated, and thus will generally be a method of choice depending on the desired results.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate a homologous polypeptide coding sequence from other cells, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex. Cross-hybridizing species are thereby readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions are readily manipulated, and thus are generally a method of choice depending on the desired results.

The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. TABLE 2 STRINGENCY CONDITIONS

Stringency Polynucleotide Hybrid Length Hybridization Wash Temperature and Condition Hybrid (bp)¹ Temperature and BufferH

Buffer"

A DNA.DNA >50 65°C;1xSSC-or- 65 °C; 0.3xSSC

42°C;1xSSC,50% formamide

B DNA.DNA <50 T_B; 1XSSC T_B; 1XSSC

C DNA.RNA >50 67°C;1xSSC-or- 67 °C; 0.3xSSC

45°C;1xSSC, 50% formamide

D DNA:RNA <50 T_D; 1XSSC T_D; 1xSSC

E RNA: RNA >50 70°C;1xSSC-or- 70°C;0.3xSSC

50°C; 1xSSC, 50% formamide

F RNA:RNA <50 T_F; 1XSSC T_F; 1XSSC

G DNA.DNA >50 65°C;4xSSC-or- 65°C;1xSSC

42°C;4xSSC, 50% formamide

H DNA:DNA <50 T_H; 4XSSC T_H; 4XSSC

1 DNA.RNA >50 67°C;4xSSC-or- 67°C;1xSSC

45°C;4xSSC, 50% formamide

J DNA.RNA <50 TJ; 4XSSC TJ; 4XSSC

K RNA:RNA >50 70°C;4xSSC-or- 67°C;1xSSC

50EC; 4xSSC, 50% formamide

RNA:RNA <50 T_L; 2XSSC T_L; 2XSSC

M DNA:DNA >50 50°C;4xSSC-or- 50°C;2xSSC

40°C;6xSSC, 50% formamide

N DNA:DNA <50 T_N; 6XSSC TN; 6XSSC

O DNA:RNA >50 55°C;4xSSC-or- 55°C;2xSSC

42°C;6xSSC, 50% formamide

DNA:RNA <50 T_P; δxSSC T_P; δxSSC

Q RNA:RNA >50 60 °C; 4xSSC -or- 60°C;2xSSC

45°C;6xSSC, 50% formamide

R RNA:RNA <50 T_R; 4XSSC T_R; 4XSSC

(bp)': The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.

Buffer^H: SSPE (IxSSPE is 0.15M NaCl, 10mM NaH₂P0_4l and 1.25mM EDTA, pH 7.4), can be substituted for SSC (1xSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. T_B through T_R: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10EC less than the melting temperature (T_m) of the hybrid, where T_m is determined according to the following equations. For hybrids less than 18 base pairs in length, T_m(EC) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, T_m(EC) = 81.5 + 16.6(log₁₀[Na⁺]) + 0.41 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na⁺] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for 1xSSC = 0.165 M).

Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 , and Ausubel et al., 1995, Current Protocols in Molecular Biology, Eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

In addition to the nucleic acid molecules encoding Alloiococcus otitidis polypeptides described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire Alloiococcus otitidis coding strand, or to only a fragment thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an Alloiococcus otitidis polypeptide. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues, e.g., the entire coding region of each of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding an Alloiococcus otitidis polypeptide. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequence encoding the Alloiococcus otitidis polypeptides disclosed herein antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of Alloiococcus otitidis mRNA, but more preferably is an oligonucleotide which is antisense to only a fragment of the coding or noncoding region of Alloiococcus otitidis mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Alloiococcus otitidis mRNA.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, l-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- diaminopurine.

Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an Alloiococcus otitidis polypeptide to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site. Alternatively, an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically. For example, for systemic administration, an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual γ-units, the strands run parallel to each other (Gaultier et al., 1987). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleot.de (Inoue et al., 1987) or a chimeric RNA-DNA analogue (Inoue et al., 1987). In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes described in Haselhoff and Gerlach, 1988) can be used to catalytically cleave Alloiococcus otitidis mRNA transcripts to thereby inhibit translation of Alloiococcus otitidis mRNA. A ribozyme having specificity for an Alloiococcus otitidis-encod^'mg nucleic acid can be designed based upon the nucleotide sequence of an Alloiococcus otitidis cDNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an Alloiococcus otitidis-encodmg mRNA. See, e.g., Cech et al. U.S. 4,987,071 and Cech et al. U.S. 5,116,742 both incorporated herein in their entirety by reference. Alternatively, Alloiococcus otitidis mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak, 1993.

Alternatively Alloiococcus otitidis gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the Alloiococcus otitidis gene (e.g., the Alloiococcus otitidis gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the

Alloiococcus otitidis gene in target cells. See generally, Helene, 1991 ; Helene et al., 1992; and Maher, 1992.

Alloiococcus otitidis gene expression can also be inhibited using RNA interference (RNAi). This is a technique for post-transcriptional gene silencing (PTGS), in which target gene activity is specifically abolished with cognate double- stranded RNA (dsRNA). RNAi resembles in many aspects PTGS in plants and has been detected in many invertebrates including trypanosome, hydra, planaria, nematode and fruit fly (Drosophila melangnoster). It may be involved in the modulation of transposable element mobilization and antiviral state formation. RNAi in mammalian systems is disclosed in WO 00/63364, which is incorporated by reference herein in its entirety. Basically, dsRNA of at least about 600 nucleotides, homologous to the target is introduced into the cell and a sequence specific reduction in gene activity is observed. C. ALLOIOCOCCUS OTITIDIS POLYPEPTIDES

In particular embodiments, the present invention provides isolated and purified Alloiococcus otitidis polypeptides. Preferably, an Alloiococcus otitidis polypeptide of the invention is a recombinant polypeptide. In certain embodiments, an Alloiococcus otitidis polypeptide of the present invention comprises the amino acid sequence that has at least 25% identity to the amino acid sequence of one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106, a biological equivalent thereof, or a fragment thereof.

An Alloiococcus otitidis polypeptide according to the present invention encompasses a polypeptide that comprises: 1 ) the amino acid sequence shown in one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106) functional and non-functional naturally occurring variants or biological equivalents of Alloiococcus otitidis polypeptides of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106 and recombinantly produced variants or biological equivalents of Alloiococcus otitidis polypeptides set out in SEQ ID NO: 2 through SEQ ID NO: 106) polypeptides isolated from organisms other than Alloiococcus otitidis (orthologs of Alloiococcus otitidis polypeptides.) A biological equivalent or variant of an Alloiococcus otitidis polypeptide according to the present invention encompasses 1 ) a polypeptide isolated from Alloiococcus otitidis; and 2) a polypeptide that contains substantial homology to an Alloiococcus otitidis polypeptide.

Biological equivalents or variants of Alloiococcus otitidis include both functional and non-functional Alloiococcus otitidis polypeptides. Functional biological equivalents or variants are naturally occurring amino acid sequence variants of an Alloiococcus otitidis polypeptide that maintain the ability to elicit an immunological or antigenic response in a subject. Functional variants will typically contain only conservative substitutions of one or more amino acids in any one of even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106 or substitution, deletion or insertion of non-critical residues in non-critical regions of the polypeptide.

The present invention further provides non-/4//o/ococc-vs otitidis orthologues of Alloiococcus otitidis polypeptides. Orthologues of Alloiococcus otitidis polypeptides are polypeptides that are isolated from non- A//o/ococcus otitidis organisms and possess antigenic capabilities of the Alloiococcus otitidis polypeptide. Orthologues of an Alloiococcus otitidis polypeptide can readily be identified as comprising an amino acid sequence that is substantially homologous to one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106.

Modifications and changes can be made in the structure of a polypeptide of the present invention and still obtain a molecule having Alloiococcus otitidis antigenicity. For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of antigenicity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art (Kyte & Doolittle, 1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid residue determines the secondary and tertiary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within +/-2 is preferred, those within +/-1 are particularly preferred, and those within +/-0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101 , incorporated herein by reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the polypeptide. As detailed in U.S. Pat. No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +1); glutamate (+3.0 ±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine (See Table 3, below). The present invention thus contemplates functional or biological equivalents of an Alloiococcus otitidis polypeptide as set forth above. TABLE 3: AMINO ACID SUBSTITUTIONS

Biological or functional equivalents of a polypeptide are also prepared using site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of second generation polypeptides, or biologically functional equivalent polypeptides or peptides, derived from the sequences thereof, through specific mutagenesis of the underlying DNA. As noted above, such changes can be desirable where amino acid substitutions are desirable. The technique further provides a capacity to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the site of the alteration of the sequence.

In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a phage vector, that can exist in both a single stranded and double stranded form. Typically, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes all or a portion of the Alloiococcus otitidis polypeptide sequence selected. An oligonucleotide primer bearing the desired mutated sequence is prepared (e.g., synthetically). This primer is then annealed to the singled-stranded vector, and extended by the use of enzymes such as Escherichia coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as Escherichia coli cells and clones are selected which include recombinant vectors bearing the mutation. Commercially available kits come with all the reagents necessary, except the oligonucleotide primers. An Alloiococcus otitidis polypeptide or polypeptide antigen of the present invention is understood to be any Alloiococcus otitidis polypeptide comprising substantial sequence similarity, structural similarity and/or functional similarity to an Alloiococcus otitidis polypeptide comprising the amino acid sequence of one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106. In addition, an Alloiococcus otitidis polypeptide or polypeptide antigen of the invention is not limited to a particular source. Thus, the invention provides for the general detection and isolation of the polypeptides from a variety of sources.

It is contemplated in the present invention, that an Alloiococcus otitidis polypeptide may advantageously be cleaved into fragments for use in further structural or functional analysis, or in the generation of reagents such as Alloiococcus otitidis-re\ated polypeptides and Alloiococcus otitidis-specWic antibodies. This can be accomplished by treating purified or unpurified Alloiococcus otitidis polypeptides with a peptidase such as endoproteinase glu-C (Boehringer, Indianapolis, IN). Treatment with CNBr is another method by which peptide fragments may be produced from natural Alloiococcus otitidis polypeptides. Recombinant techniques also can be used to produce specific fragments of an Alloiococcus otitidis polypeptide.

In addition, the inventors also contemplate that compounds sterically similar to a particular Alloiococcus otitidis polypeptide antigen, called peptidomimetics, may be formulated to mimic the key portions of the peptide structure. Peptidemimetics are peptide-containing molecules that mimic elements of protein secondary structure. (See, for example, Johnson et al., 1993.) The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of receptor and ligand.

Successful applications of the peptide mimetic concept have thus far focused on mimetics of β-turns within proteins. Likely β-turn structures, within Alloiococcus otitidis, can be predicted by computer-based algorithms as discussed above. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains, as discussed in Johnson et al., 1993.

Fragments of the Alloiococcus otitidis polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as a part, but not all, of the amino acid sequence. The fragment can comprise, for example, at least 7 or more (e.g., 8, 10 12, 14, 16, 18, 20 or more) contiguous amino acids of an one of amino acid sequence selected from one of the even numbered sequences set forth in SEQ ID NO.: 2 through SEQ ID NO.: 106. Fragments may be "freestanding" or comprised within a larger polypeptide of which they form a part or region, most preferably as a single, continuous region. In one embodiment, the fragments include at least one epitope of the mature polypeptide sequence.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. For example, fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof have been described. 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 0232 2621). 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. D. ALLOIOCOCCUS OTITIDIS POLYNUCLEOTIDE AND POLYPEPTIDE VARIANTS

"Variant" 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 and 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 variant 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 or by direct synthesis.

"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" 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). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al 1984), BLASTP, BLASTN, and FASTA (Altschul, S. F., ef al., 1990. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., 1990). The well known Smith-Waterman algorithm may also be used to determine identity.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of one of SEQ ID NO:1 through SEQ ID NO: 105, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such 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. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105 by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105.

For example, the alterations in an isolated Alloiococcus otitidis polynucleotide comprise a polynucleotide sequence that has at least 70% identity to the nucleic acid sequence of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105; a degenerate variant thereof or a fragment thereof, wherein the polynucleotide sequence may include up to n_π nucleic acid alterations over the entire polynucleotide region of the nucleic acid sequence of any on of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105, wherein n_n is the maximum number of alterations and is calculated by the formula: n_n < x_n-(x_n ^#y), in which x_π is the total number of nucleic acids of one of SEQ ID NO:1 through SEQ ID NO:105 and y has a value of 0.70, wherein any non-integer product of x„ and y is rounded down to the nearest integer prior to subtracting such product from x_π. Of course, y may also have a value of 0.80 for 80%, 0.85 for 85%, 0.90 for 90% 0.95 for 95%, etc.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of any one of even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106, that is 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 percentage identity is less than 100%. 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 one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106 by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106, or: n_a < x_a-(x_a ^»y), wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in one of SEQ ID NO: 2 through SEQ ID NO: 106, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., 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_a.

E. VECTORS, HOST CELLS AND RECOMBINANT ALLOIOCOCCUS OTITIDIS POLYPEPTIDES

In a preferred embodiment, the present invention provides expression vectors comprising ORF polynucleotides that encode Alloiococcus otitidis polypeptides. Preferably, the expression vectors of the present invention comprise ORF polynucleotides that encode Alloiococcus otitidis polypeptides comprising the amino acid residue sequence of one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106. More preferably, the expression vectors of the present invention comprise a polynucleotide comprising the nucleotide base sequence of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105. Even more preferably, the expression vectors of the invention comprise a polynucleotide operatively linked to promoter. Still more preferably, the expression vectors of the invention comprise a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, the expression vectors of the present invention comprise a polynucleotide operatively linked to an enhancer-promoter, that is, an eukaryotic promoter. The expression vectors further comprise a polyadenylation signal that is positioned 3' of the carboxy-terminal amino acid and within a transcriptional unit of the encoded polypeptide.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988), pMAL (New England Biolabs, Beverly; MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the Alloiococcus otitidis polynucleotide is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-_>4//o ococct/s otitidis polypeptide. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant Alloiococcus otitidis polypeptide unfused to GST can be recovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion Escherichia coli expression vectors include pTrc (Amann et al., 1988) and pET 1 1 d (Studier ef al., 1990). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 1 d vector relies on transcription from a T7 gn1 0-lac fusion promoter mediated by a coexpressed viral RNA polymerase T7 gnl. This viral polymerase is supplied by host strains BL21 (DE3) or HMS I 74(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in Escherichia coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in Escherichia coli. Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA mutagenesis or synthesis techniques.

In another embodiment, the Alloiococcus otitidis polynucleotide expression vector is a yeast expression vector. Examples of vectors for expression in a yeast such as S. cerevisiae include pYepSec I (Baldari, et al., 1987), pMFa (Kurjan and Herskowitz, 1982), pJRY88 (Schultz et al., 1987), and pYES2 (invitrogen Corporation, San Diego, CA).

Alternatively, an Alloiococcus otitidis polynucleotide is expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 or Sf 21 cells) include the pAc series (Smith et al., 1983) and the pVL series (Lucklow and Summers, 1989).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987) and pMT2PC (Kaufman ef al., 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. As used herein, a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes. As used herein, the term "promoter" includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA Polymerase II transcription unit.

Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer can function when located at variable distances from transcription start sites so long as a promoter is present. As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase "operatively linked" means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art. As is also well known in the art, the precise orientation and location relative to a coding sequence whose transcription is controlled, is dependent inter alia upon the specific nature of the enhancer-promoter. Thus, a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site. In contrast, an enhancer can be located downstream from the initiation site and can be at a considerable distance from that site. An enhancer-promoter used in a vector construct of the present invention can be any enhancer-promoter that drives expression in a cell to be transfected. By employing an enhancer-promoter with well-known properties, the level and pattern of gene product expression can be optimized. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus (CMV) and Simian Virus 40 (SV40). For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., "Molecular Cloning: A Laboratory Manual" 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, incorporated herein by reference.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue- specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987), lymphoid-specific promoters (Calame and Eaton, 1988), in particular promoters of T cell receptors (Winoto and Baltimore, 1989) and immunoglobulins (Banerji et al., 1983), Queen and Baltimore (1983), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989), pancreas-specific promoters (Edlund et al., 1985), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. 4, 873,316 and EP 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss, 1990) and the α-fetoprotein promoter (Campes and Tilghman, 1989). The invention further provides a recombinant expression vector comprising a

DNA molecule encoding an Alloiococcus otitidis polypeptide cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to Alloiococcus otitidis mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, an Alloiococcus otitidis polypeptide can be expressed in bacterial cells such as Escherichia coli, insect cells, yeast or mammalian cells

(such as Chinese hamster ovary cells (CHO), NIH3T3, PER C6, NSO, VERO or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA is can be introduced into prokaryotic or eukaryotic cells via conventional transformation, infection or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran- mediated transfection, lipofection, protoplast fusion, direct microinfection. Another recognized technique for introducing DNA into a host cell is "infection", such as by adenovirus infection or electroporation. Suitable methods for transforming, infecting or transfecting host cells can be found in Sambrook, ef al. ("Molecular Cloning: A Laboratory Manual" 2nd ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains unclear, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells can be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for experiments that require transient expression of the foreign DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome. In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of copies of plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacteria are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus. Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome. The application of brief, high-voltage electric pulses (electroporation) to a variety of mammalian and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA. Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear, but transfection efficiencies can be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well known in the art. Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet, etal. 1992).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, is used to produce (i.e., express) an Alloiococcus otitidis polypeptide. Accordingly, the invention further provides methods for producing an Alloiococcus otitidis polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an Alloiococcus otitidis polypeptide has been introduced) in a suitable medium until the Alloiococcus otitidis polypeptide is produced. In another embodiment, the method further comprises isolating the Alloiococcus otitidis polypeptide from the medium or the host cell.

A coding sequence of an expression vector is operatively linked to a transcription-terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). Transcription-terminating regions are well known in the art. A preferred transcription-terminating region used in an adenovirus vector construct of the present invention comprises a polyadenylation signal of SV40 or the protamine gene.

An expression vector comprises a polynucleotide that encodes an Alloiococcus otitidis polypeptide. Such a polypeptide is meant to include a sequence of nucleotide bases encoding an Alloiococcus otitidis polypeptide sufficient in length to distinguish the segment from a polynucleotide segment encoding a non-

Alloiococcus otitidis polypeptide. A polypeptide of the invention can also encode biologically functional polypeptides or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score of the amino acids being exchanged. These variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.

Preferably, an expression vector of the present invention comprises a polynucleotide that encodes a polypeptide comprising the amino acid residue sequence of one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO:.4036 An expression vector can include an Alloiococcus otitidis polypeptide coding region itself of any of the Alloiococcus otitidis polypeptides noted above or it can contain coding regions bearing selected alterations or modifications in the basic coding region of such an Alloiococcus otitidis polypeptide. Alternatively, such vectors or fragments can also encode larger polypeptides or polypeptides which nevertheless include the basic coding region. In any event, it should be appreciated that due to codon redundancy as well as biological functional equivalence, this aspect of the invention is not limited to the particular DNA molecules corresponding to the polypeptide sequences noted above.

Exemplary vectors include the mammalian expression vectors of the pCMV family including pCMV6b and pCMV6c (Chiron Corp., Emeryville CA.). In certain cases, and specifically in the case of these individual mammalian expression vectors, the resulting constructs can require co-transfection with a vector containing a selectable marker such as pSV2neo. Via co-transfection into a dihydrofolate reductase-deficient Chinese hamster ovary cell line, such as DG44, clones expressing Alloiococcus otitidis polypeptides by virtue of DNA incorporated into such expression vectors can be detected.

A DNA molecule of the present invention can be incorporated into a vector by a number of techniques that are well known in the art. For instance, the vector pUC18 has been demonstrated to be of particular value in cloning and expression of genes. Likewise, the related vectors M13mp18 and M13mp19 can also be used in certain embodiments of the invention, in particular, in performing dideoxy sequencing. An expression vector of the present invention is useful both as a means for preparing quantities of the Alloiococcus otitidis polypeptide-encoding DNA itself, and as a means for preparing the encoded polypeptide and peptides. It is contemplated that where Alloiococcus otitidis polypeptides of the invention are made by recombinant means, one can employ either prokaryotic or eukaryotic expression vectors as shuttle systems. In another aspect, the recombinant host cells of the present invention are prokaryotic host cells. Preferably, the recombinant host cells of the invention are bacterial cells of the DH5α strain of Escherichia coli. In general, prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention. For example, Escherichia coli K12 strains can be particularly useful. Other microbial strains that can be used include Escherichia coli B, Escherichia co//W3110 (ATCC No. 273325) and Escherichia. co/χ1976 (ATCC No. 31537). Bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or other Salmonella species or Serratia marcesans, and various pseudomonas species can be used. These examples are, of course, intended to be illustrative rather than limiting.

In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, Escherichia coli can be transformed using pBR322, a plasmid derived from an Escherichia coli species (Bolivar, ef al. 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage, must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own polypeptides.

Those promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems (Chang, ef al. 1978; Itakura., ef al. 1977, Goeddel, et al. 1979; Goeddel, ef al. 1980) and a tryptophan (TRP) promoter system (EP 0036776; Siebwenlist et al. 1980). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to introduce functional promoters into plasmid vectors (Siebwenlist, et al. 1980).

In addition to prokaryotes, eukaryotic microbes such as yeast can also be used. Saccharomyces cerevisiase or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb, ef al. 1979; Kingsman, ef al. 1979;

Tschemper, et al. 1980). This plasmid already contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoter sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (PGK) (Hitzeman, et al. 1980) or other glycolytic enzymes (Hess, ef al. 1968; Holland, ef al. 1978) such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also introduced into the expression vector downstream from the sequences to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3- phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms can also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are AtT-20, VERO, HeLa, NSO, PER C6, Chinese hamster ovary (CHO) cell lines, W138, BHK, COSM6, COS-7, 293 , VERO and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. Where expression of recombinant Alloiococcus otitidis polypeptides is desired and a eukaryotic host is contemplated, it is most desirable to employ a vector, such as a plasmid, that incorporates a eukaryotic origin of replication. Additionally, for the purposes of expression in eukaryotic systems, one desires to position the Alloiococcus otitidis encoding sequence adjacent to and under the control of an effective eukaryotic promoter such as promoters used in combination with Chinese hamster ovary cells (CHO). To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5' end of the translation initiation side of the proper translational reading frame of the polypeptide between about 1 and about 50 nucleotides 3' of or downstream with respect to the promoter chosen. Furthermore, where eukaryotic expression is anticipated, one would typically desire to incorporate an appropriate polyadenylation site into the transcriptional unit that includes the Alloiococcus otitidis polypeptide. A transfected cell can be prokaryotic or eukaryotic. Preferably, the host cells of the invention are prokaryotic host cells. Where it is of interest to produce an Alloiococcus otitidis polypeptide, cultured prokaryotic host cells are of particular interest.

In yet another embodiment, the present invention contemplates a process or method of preparing Alloiococcus otitidis polypeptides comprising transfecting, transforming or infecting cells with a polynucleotide that encodes an Alloiococcus otitidis polypeptide to produce transformed host cells; and maintaining the transformed host cells under biological conditions sufficient for expression of the polypeptide. Preferably, the transformed host cells are prokaryotic cells. Alternatively, the host cells are eukaryotic cells. More preferably, the prokaryotic cells are bacterial cells of the DH5α strain of Escherichia coli. Even more preferably, the polynucleotide transfected into the transformed cells comprises the nucleic acid sequence of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105. Additionally, transfection is accomplished using an expression vector disclosed above. A host cell used in the process is capable of expressing a functional, recombinant Alloiococcus otitidis polypeptide.

Following transfection, the cell is maintained under culture conditions for a period of time sufficient for expression of an Alloiococcus otitidis polypeptide. Culture conditions are well known in the art and include ionic composition and concentration, temperature, pH and the like. Typically, transfected cells are maintained under culture conditions in a culture medium. Suitable media for various cell types are well known in the art. In a preferred embodiment, temperature is from about 20°C to about 50°C, more preferably from about 30°C to about 40°C and, even more preferably about 37°C. The pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8 and, most preferably about 7.4. Osmolality is preferably from about 200 milliosmols per liter (mosm/L) to about 400 mosm/l and, more preferably from about 290 mosm/L to about 310 mosm/L. Other biological conditions needed for transfection and expression of an encoded protein are well known in the art.

Transfected cells are maintained for a period of time sufficient for expression of an Alloiococcus otitidis polypeptide. A suitable time depends inter alia upon the cell type used and is readily determinable by a skilled artisan. Typically, maintenance time is from about 2 to about 14 days.

Recombinant Alloiococcus otitidis polypeptide is recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises isolating and purifying the Alloiococcus otitidis polypeptide. Isolation and purification techniques for polypeptides are well known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like.

F. ANTIBODIES IMMUNOREACTIVE WITH ALLOIOCOCCUS OTITIDIS POLYPEPTIDES In still another embodiment, the present invention provides antibodies immunoreactive with Alloiococcus otitidis polypeptides. Preferably, the antibodies of the invention are monoclonal antibodies. Additionally, the Alloiococcus otitidis polypeptides comprise the amino acid residue sequence of one of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies "A Laboratory Manual", E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988). Polyclonal antisera is obtained by bleeding an immunized animal into a glass or plastic container, incubating the blood at 25°C for one hour, followed by incubating at 4°C for 2-18 hours. The serum is then recovered by centrifugation. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide or polynucleotide of the present invention, and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given polypeptide or polynucleotide may vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or polynucleotide) of the present invention with a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.

Means for conjugating a polypeptide or a polynucleotide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As is also well known in the art, immunogencity to a particular immunogen can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvants, cholera toxin (e.g. mutant cholera toxin E29H; see published International Patent Application WO 00/18434), and aluminum hydroxide adjuvant.

The amount of immunogen used for the production of polyclonal antibodies depends upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen

(subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies is monitored by sampling blood from the immunized animal at various points following immunization. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored.

In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with an Alloiococcus otitidis polypeptide comprising the steps of (a) transfecting recombinant host cells with a polynucleotide that encodes an Alloiococcus otitidis polypeptide; (b) culturing the host cells under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptides; and (d) preparing the antibodies to the polypeptides. Preferably, the host cell is transfected with the polynucleotide of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 4035. Even more preferably, the present invention provides antibodies prepared according to the process described above. A monoclonal antibody of the present invention can be readily prepared through use of well-known techniques such as those exemplified in U.S. Pat. No. 4,196,265, herein incorporated by reference. Typically, a technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the present invention) in a manner sufficient to provide an immune response. Rodents such as mice and rats are preferred animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, e.g., by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides. Where azaserine is used, the media is supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptide. The selected clones can then be propagated indefinitely to provide the monoclonal antibody.

By way of specific example, to produce an antibody of the present invention, mice are injected intraperitoneally with between about 1 -200 μg of an antigen comprising a polypeptide of the present invention. B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete

Freund's adjuvant (CFA; a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis). At some time (e.g., at least two weeks) after the first injection, mice are boosted by injection with a second dose of the antigen mixed with incomplete Freund's adjuvant (I FA; lacks the killed mycobacterium of CFA). A few weeks after the second injection, mice are tail bled and the sera titered by immunoprecipitation against radiolabeled antigen. Preferably, the process of boosting and titering is repeated until a suitable titer is achieved. The spleen of the mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5x10⁷ to 2x10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they can be propagated indefinitely in tissue culture, and are thus denominated immortal. Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established. Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen of the mouse or rat injected with the antigen/polypeptide of the present invention. Fusion conditions include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma cells grow indefinitely in culture. Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as HAT media (hypoxanthine, aminopterin, thymidine). Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) can grow in the selection media.

Each of the surviving hybridoma cells produces a single antibody. These cells are then screened for the production of the specific antibody immunoreactive with an antigen/polypeptide of the present invention. Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence of the monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody of the present invention in convenient quantity. By use of a monoclonal antibody of the present invention, specific polypeptides and polynucleotide of the invention are identified as antigens. Once identified, those polypeptides and polynucleotide are isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified. Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. 5,223,409; WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809, which are incorporated herein in their entirety by reference. Additionally, recombinant anti->4-7o/Ococcι/s otitidis antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non- human fragments, which are made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies are produced by recombinant DNA techniques known in the art, for example using methods described in PCT/US86/02269; EP 184, 187; EP 171 ,496; EP 173,494; WO 86/01533; U.S. 4,816,567; and EP 125,023.

An anti-/4//o/ococct/s otitidis antibody (e.g., monoclonal antibody) is used to isolate Alloiococcus otitidis polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. An antM/fo/ococcus otitidis antibody facilitates the purification of a natural Alloiococcus otitidis polypeptide from cells and recombinantly produced Alloiococcus otitidis polypeptides expressed in host cells. Moreover, an anti-y4//o/ococc_-s otitidis antibody is used to detect Alloiococcus otitidis polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance of the Alloiococcus otitidis polypeptide. The detection of circulating fragments of an Alloiococcus otitidis polypeptide is used to identify Alloiococcus otitidis polypeptide turnover in a subject. Anti-yA//o/ococct/s otitidis antibodies are used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection is facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylarnine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and acquorin, and examples of suitable radioactive material include ¹²⁵l, ¹³¹l, ¹⁵S or ³H.

G. PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the present invention provides pharmaceutical compositions comprising compounds that inhibit the activities of Alloiococcus otitidis polypeptides, and physiologically acceptable carriers. Compounds that inhibit the activities of Alloiococcus otitidis polypeptides polypeptides, which are essential for the proliferation of the bacteria, are identified using one or more assay systems set forth in Examples 5-38. More preferably, the pharmaceutical compositions comprise one or more compounds that inhibit the activities of Alloiococcus otitidis polypeptides comprising the amino acid residue sequence of one or more of the even numbered sequences set forth in SEQ ID NO: 2 through SEQ ID NO: 106. In other embodiments, the pharmaceutical compositions of the invention comprise antisense polynucleotides of polynucleotides selected from one of the odd numbered sequences set forth in Seq. ID NO. 1 to Seq. ID No. 105, and physiologically acceptable carriers.

Various tests are to be used to assess the in vitro and in vivo efficacy of anitmicrobial and pharmaceutical compounds that inhibit the activities of Alloiococcus otitidis polypeptides, and these are set forth in detail in Examples 5 through 38. For example, an in vitro activity of the compounds may be assayed by incubating together a mixture of Alloiococcus otitidis or other heterologous bacterial cells such as E. coli cells expressing Alloiococcus otitidis polypeptides set forth in one of the even numbered sequences from Seq. ID No. 2 to Seq. ID No. 106, and then measuring the activity of the polypeptide using one or more of the assay systems detailed in Example 5 through 38.

The Alloiococcus otitidis polynucleotides, polypeptides, compounds that modulate the activity of an Alloiococcus otitidis polypeptides, and anti-/4//o/ococct/s otitidis antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration to a host or subject, e.g., a human. Such compositions typically comprise the nucleic acid molecule, protein, antimicrobial compound, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, (e.g., intravenous, intradermal, subcutaneous, intraperitoneal), transmucosal (e.g., oral, rectal, intranasal, vaginal, respiratory), and transdermal (topical). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an Alloiococcus otitidis polypeptide inhibitory compound or anti- Alloiococcus otitidis antisense polynucleotide or antibody directed against an Alloiococcus otitidis polypeptide) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods

H. DIAGNOSTIC ASSAYS

The invention also provides methods for detecting the presence of an Alloiococcus otitidis polypeptide or Alloiococcus otitidis polynucleotide, or fragment thereof, in a biological sample. The method involves contacting the biological sample with a compound or an agent capable of detecting an Alloiococcus otitidis polypeptide or mRNA such that the presence of the Alloiococcus otitidis polypeptide/encoding nucleic acid molecule is detected in the biological sample. A preferred agent for detecting Alloiococcus otitidis mRNA or DNA is a labeled or labelable oligonucleotide probe capable of hybridizing to Alloiococcus otitidis mRNA or DNA. The nucleic acid probe can be, for example, a full-length Alloiococcus otitidis polynucleotide of one of the odd numbered sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 105, a complement thereof, or a fragment thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to Alloiococcus otitidis mRNA or DNA. Alternatively, the sample can be contacted with an oligonucleotide primer of an Alloiococcus otitidis polynucleotide of SEQ ID NO: 1 through SEQ ID :105, a complement thereof, or a fragment thereof, in the presence of nucleotides and a polymerase, under conditions permitting primer extension. A preferred agent for detecting Alloiococcus otitidis polypeptide is a labeled or labelable antibody capable of binding to an Alloiococcus otitidis polypeptide. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled or labelable," with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect Alloiococcus otitidis mRNA, DNA or protein in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of Alloiococcus otitidis mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of Alloiococcus otitidis polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Alternatively, Alloiococcus otitidis polypeptides can be detected in vivo in a subject by introducing into the subject a labeled anti-/4//o/ococcus otitidis antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. The polynucleotides according to the invention may also be used in analytical

DNA chips, which allow sequencing, the study of mutations and of the expression of genes, and which are currently of interest given their very small size and their high capacity in terms of number of analyses.

The principle of the operation of these chips is based on molecular probes, most often oligonucleotides, which are attached onto a miniaturized surface, generally of the order of a few square centimeters. During an analysis, a sample containing fragments of a target nucleic acid to be analyzed, for example DNA or RNA labeled, for example, after amplification, is deposited onto the DNA chip in which the support has been coated beforehand with probes. Bringing the labeled target sequences into contact with the probes leads to the formation, through hybridization, of a duplex according to the rule of pairing defined by J.D. Watson and F. Crick. After a washing step, analysis of the surface of the chip allows the effective hybridizations to be located by means of the signals emitted by the labels tagging the target. A hybridization fingerprint results from this analysis which, by appropriate computer processing, will make it possible to determine information such as the presence of specific fragments in the sample, the determination of sequences and the presence of mutations.

The chip consists of a multitude of molecular probes, precisely organized or arrayed on a solid support whose surface is miniaturized. It is at the center of a system where other elements (imaging system, microcomputer) allow the acquisition and interpretation of a hybridization fingerprint.

The hybridization supports are provided in the form of flat or porous surfaces (pierced with wells) composed of various materials. The choice of a support is determined by its physicochemical properties, or more precisely, by the relationship between the latter and the conditions under which the support will be placed during the synthesis or the attachment of the probes or during the use of the chip. It is therefore necessary, before considering the use of a particular support, to consider characteristics such as its stability to pH, its physical strength, its reactivity and its chemical stability as well as its capacity to nonspecifically bind nucleic acids. Materials such as glass, silicon and polymers are commonly used. Their surface is, in a first step, called "functionalization", made reactive towards the groups which it is desired to attach thereon. After the functionalization, so-called spacer molecules are grafted onto the activated surface. Used as intermediates between the surface and the probe, these molecules of variable size render unimportant the surface properties of the supports, which often prove to be problematic for the synthesis or the attachment of the probes and for the hybridization.

Among the hybridization supports, there may be mentioned glass which is used, for example, in the method of in situ synthesis of oligonucleotides by photochemical addressing developed by the company Affymetrix (E.L. Sheldon, 1993), the glass surface being activated by silane. Genosensor Consortium (P. Merel, 1994) also uses glass slides carrying wells 3 mm apart, this support being activated with epoxysilane. The probes according to the invention may be synthesized directly in situ on the supports of the DNA chips. This in situ synthesis may be carried out by photochemical addressing (developed by the company Affymax (Amsterdam, Holland) and exploited industrially by its subsidiary Affymetrix (United States)) or based on the VLSI PS (very large scale immobilized polymer synthesis) technology (S.P.A. Fodor ef al., 1991 ) which is based on a method of photochemically directed combinatory synthesis and the principle of which combines solid-phase chemistry, the use of photolabile protecting groups and photolithography.

The probes according to the invention may be attached to the DNA chips in various ways such as electrochemical addressing, automated addressing or the use of probe printers (T. Livache et al., 1994; G. Yershov et al., 1996; J. Derisi et al., 1996, and S. Borman, 1996).

The revealing of the hybridization between the probes of the invention, deposited or synthesized in situ on the supports of the DNA chips, and the sample to be analyzed, may be determined, for example, by measurement of fluorescent signals, by radioactive counting or by electronic detection.

The use of fluorescent molecules such as fluorescein constitutes the most common method of labeling the samples. It allows direct or indirect revealing of the hybridization and allows the use of various fluorochromes. Affymetrix currently provides an apparatus or a scanner designed to read its Gene Chip™ chips. It makes it possible to detect the hybridizations by scanning the surface of the chip in confocal microscopy (R.J. Lipshutz ef al., 1995).

The nucleotide sequences according to the invention are also used in DNA chips to carry out the analysis of the expression of the Alloiococcus otitidis genes. This analysis of the expression of Alloiococcus otitidis genes is based on the use of chips where probes of the invention, chosen for their specificity to characterize a given gene, are present (D.J. Lockhart et al., 1996; D.D. Shoemaker et al., 1996). For the methods of analysis of gene expression using the DNA chips, reference may, for example, be made to the methods described by D.J. Lockhart ef al. (1996) and Sosnowsky ef al. (1997) for the synthesis of probes in situ or for the addressing and the attachment of previously synthesized probes. The target sequences to be analyzed are labeled and in general fragmented into sequences of about 50 to 100 nucleotides before being hybridized onto the chip. After washing as described, for example, by D.J. Lockhart ef al. (1996) and application of different electric fields (Sosnowsky et al., 1997), the labeled compounds are detected and quantified, the hybridizations being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, determine the differential expression of RNA or of DNA derived from the sample.

The nucleotide sequences according to the invention are, in addition, used in DNA chips where other nucleotide probes specific for other microorganisms are also present, and allow the carrying out of a serial test allowing rapid identification of the presence of a microorganism in a sample. Accordingly, the subject of the invention is also the nucleotide sequences according to the invention, characterized in that they are immobilized on a support of a DNA chip.

The DNA chips, characterized in that they contain at least one nucleotide sequence according to the invention, immobilized on the support of the said chip, also form part of the invention.

The chips preferably contain several probes or nucleotide sequences of the invention of different length and/or corresponding to different genes so as to identify, with greater certainty, the specificity of the target sequences or the desired mutation in the sample to be analyzed.

Accordingly, the analyses carried out by means of primers and/or probes according to the invention, immobilized on supports such as DNA chips, make it possible, for example, to identify, in samples, mutations linked to variations such as intraspecies variations. These variations may be correlated or associated with pathologies specific to the variant identified and make it possible to select the appropriate treatment.

The invention thus comprises a DNA chip according to the invention, characterized in that it contains, in addition, at least one nucleotide sequence of a microorganism different from Alloiococcus otitidis, immobilized on the support of the said chip; preferably, the different microorganism is chosen from an associated microorganism, a bacterium of the Streptococcus family, and a variant of the species Alloiococcus otitidis.

The principle of the DNA chip as explained above, is also used to produce protein "chips" on which the support has been coated with a polypeptide or an antibody according to the invention, or arrays thereof, in place of the DNA. These protein "chips" make it possible, for example, to analyze the biomolecular interactions (BIA) induced by the affinity capture of target analytes onto a support coated, for example, with proteins, by surface plasma resonance (SPR). Reference may be made, for example, to the techniques for coupling proteins onto a solid support which are described in EP 524 800 or to the methods describing the use of biosensor-type protein chips such as the BIAcore-type technique (Pharmacia) (Arlinghaus etal., 1997, Krone et al., 1997, Chatelier et al., 1995). These polypeptides or antibodies according to the invention, capable of specifically binding antibodies or polypeptides derived from the sample to be analyzed, are thus used in protein chips for the detection and/or the identification of proteins in samples. The said protein chips may in particular be used for infectious diagnosis and preferably contain, per chip, several polypeptides and/or antibodies of the invention of different specificity, and/or polypeptides and/or antibodies capable of recognizing microorganisms different from Alloiococcus otitidis.

Accordingly, the subject of the present invention is also the polypeptides and the antibodies according to the invention, characterized in that they are immobilized on a support, in particular, on a protein chip. The protein chips, characterized in that they contain at least one polypeptide or one antibody according to the invention immobilized on the support of the said chip, also form part of the invention.

The invention comprises, in addition, a protein chip according to the invention, characterized in that it contains, in addition, at least one polypeptide of a microorganism different from Alloiococcus otitidis or at least one antibody directed against a compound of a microorganism different from Alloiococcus otitidis, immobilized on the support of the chip.

The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Alloiococcus otitidis or to an associated microorganism, or for the detection and/or the identification of a microorganism characterized in that it comprises a protein chip according to the invention.

The present invention also provides a method for the detection and/or the identification of bacteria belonging to the species Alloiococcus otitidis or to an associated microorganism in a biological sample, characterized in that it uses a nucleotide sequence according to the invention.

The invention also encompasses kits for detecting the presence of an Alloiococcus otitidis polypeptide in a biological sample. For example, the kit comprises reagents such as a labeled or labelable compound or agent capable of detecting Alloiococcus otitidis polypeptide or mRNA in a biological sample; means for determining the amount of Alloiococcus otitidis polypeptide in the sample; and means for comparing the amount of Alloiococcus otitidis polypeptide in the sample with a standard. The compound or agent are packaged in a suitable container. The kit further comprises instructions for using the kit to detect Alloiococcus otitidis mRNA or protein.

In certain embodiments, detection involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. 4,683,195 and U.S. 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR). This method includes the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an Alloiococcus otitidis polynucleotide under conditions such that hybridization and amplification of the Alloiococcus of/^'f/αfe-polynucleotide (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.

I. TRANSGENIC ANIMALS

It is contemplated that in some instances the genome of a transgenic animal of the present invention will have been altered through the stable introduction of one or more of the Alloiococcus otitidis polynucleotide compositions described herein, either native, synthetically modified or mutated. As described herein, a "transgenic animal" refers to any animal, preferably a non-human mammal (e.g. mouse, rat, rabbit, squirrel, hamster, rabbits, guinea pigs, pigs, micro-pigs, baboons, squirrel monkeys and chimpanzees, etc), bird or an amphibian, in which one or more cells contain a heterologous nucleic acid sequence introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly, by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

The host cells of the invention are also used to produce non-human transgenic animals. The non-human transgenic animals are used in screening assays designed to identify infections or compounds, e.g., drugs, pharmaceuticals, efc, which are capable of ameliorating Alloiococcus otitidis symptoms or infections. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which an Alloiococcus otitidis polypeptide-coding sequence has been introduced. Such host cells are then used to create non-human transgenic animals in which exogenous Alloiococcus otitidis gene sequences have been introduced into their genome or homologous recombinant animals in which endogenous Alloiococcus otitidis gene sequences have been altered. Such animals are useful for studying the effects of an Alloiococcus otitidis polypeptide and for identifying and/or evaluating modulators of Alloiococcus otitidis polypeptide infectivity.

A transgenic animal of the invention is created by introducing an Alloiococcus otitidis polypeptide-encoding nucleic acid sequence into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human Alloiococcus otitidis cDNA sequence of one or more of SEQ ID NO:1 through SEQ ID NO: 4035 can be introduced as a transgene into the genome of a non-human animal.

Moreover, a non-y4//o/ococcts otitidis homologue of the Alloiococcus otitidis gene can be isolated based on hybridization to the Alloiococcus otitidis cDNA

(described above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the Alloiococcus otitidis transgene to direct expression of an Alloiococcus otitidis polypeptide to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. 4,736,866 and 4,870, 009, U.S. 4,873,191 and in Hogan, 1986. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the Alloiococcus otitidis transgene in its genome and/or expression of Alloiococcus otitidis mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an Alloiococcus otitidis polypeptide can further be bred to other transgenic animals carrying other transgenes.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage Pλ. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al., 1992. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gon-nan et al., 1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al., 1997, and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

All patents and publications cited herein are hereby incorporated by reference.

The following examples 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 following examples are presented for illustrative purposes, and should not be construed in any way limiting the scope of this invention.

EXAMPLE 1 CONFIRMATION OF THE IDENTITY OF THE ALLOIOCOCCUS OTITIDIS 1104-92 ISOLATE

The Alloiococcus otitidis isolate 1104-92 was obtained from Dr. Richard Facklam of the Centers for Disease Control in Atlanta. It was isolated from the middle ear fluid of a child in the Atlanta, Georgia area. It was confirmed to be A. otitidis by comparing it to the type strain, ATCC51267, obtained from the American Type Culture Collection [Aguirre, 1992 #1 ]. Both the 1104-92 and type strain are characterized as Gram positive cocci. Both grow on Columbia agar supplemented with 5% yeast extract, 0.5% polysorbate 80 (Tween 80), and 0.7% phospatidyl choline when incubated at 37°C. On this medium, both strains form slow growing small white colonies that require nearly two days to be easily observed with the naked eye. Both are sensitive to lysis by hen egg white lysozyme and Streptococcus globisporus mutanolysin. Both grow in the presence of 2% sodium azide. Both are killed by incubation at 55°C for 30 minutes. Finally, to further confirm that the 1104- 92 was a strain of A. otitidis, it was subject to polymerase chain reaction (PCR) identification based on its 16s rRNA gene. This was done using two of the primers specified by Aguirre and Collins [Aguirre, 1992 #2]. The antisense primer used was 5'-ATCTTCCTGCTTGCAGGAAGAGG-3' and the sense primer was 3'-CGCTTCATCTCTGAAGCTAGC-5'. Thus by multiple criteria, the 1104-92 strain was confirmed to be an isolate of A. otitidis.

EXAMPLE 2 STORAGE, GROWTH, AND HARVEST OF ALLOIOCOCCUS OTITIDIS 1104-92 FOR ISOLATION

OF DNA

The A. otitidis isolate 1104-92 was stored at -70°C in Todd-Hewlett broth containing 40% glycerol. A small portion of the frozen stock was streaked onto the agar medium described in Example 1 and incubated at 37°C for two days. The growth from the plate was swabbed into a 17 x 100 cm tube containing 6 ml of a serum-free broth medium. This broth medium was prepared with 30 g Todd-Hewlett medium, 5 g yeast extract, 10 ml polysorbate 80 (Tween 80), and 1 liter distilled water. This medium was sterilized by autoclaving for 35 minutes. The bacteria were incubated aerobically without shaking in an aerobic incubator at 37°C for two days. The tube containing the growing bacteria was then shaken to resuspend the bacteria and added to a liter of the same medium in a Fembach flask. This flask, in turn, was incubated aerobically for three days without shaking. The bacteria were harvested by first swirling the flask to suspend the bacteria and then low speed centrifugation at about 5,000 x g for 30 minutes. The pellet of bacteria was washed by resuspending it in 10 to 15 mL of phosphate buffered saline (PBS), and centrifuging the suspension at about 8,000 x g for 20 minutes. The pellet of bacteria was retained and stored frozen at -20°C. The yield of wet bacterial pellet was typically about 1 g per liter of broth. EXAMPLE 3 PREPARATION OF ALLOIOCOCCUS OTITIDIS GENOMIC DNA

To prepare genomic DNA, 0.95 g frozen pellet of bacteria was defrosted and suspended in 10 mL of PBS containing 1 mM MgCI₂. The bacteria were killed by incubating the suspension at 55°C for 20 minutes. The suspension was allowed to cool before adding 25 μl of a 10 mg/mL stock of hen egg white lysozyme and 50 μl of a 25,000 unit/mL stock of Streptococcus globisporus mutanolysin to the suspension. It was then incubated for one hour at 37°C. Then 50 μl of a 10 mg/mL stock of RNase was added and the suspension incubated an additional hour at 37°C. After these incubations, sodium dodecylsulfate (SDS) was added to a final concentration of 0.3% (0.3 mL of a 10% stock). This was followed by the addition of 0.3 mL of a 1 mg/mL stock of proteinase K. The suspension was then incubated for two hours at 37°C. After this time, an equal volume of water saturated phenol/chloroform/isopropyl (25:24:1 ) was added to the digested suspension and gently mixed. The upper aqueous layer was retained after a low speed centrifugation and 2.5 volumes of ethanol were added and the tube gently inverted to mix. The DNA was then spooled out on a glass rod and allowed to air dry.

The DNA at this stage still contained obvious impurities and needed further purification. The DNA dried on the glass rod was soaked in 70% ethanol to remove excess phenol and air-dried once again. It was then suspended in 2 ml of Tris-EDTA buffer to which 2 μl of RNase cocktail was added and incubated at room temperature for 75 minutes. Then 100 μl of protease, 100 μl SDS and 40 μl of 100 mM CaCI₂ were added and the suspension incubated for 3.5 hours. An equal volume of chloroform was added, gently mixed, then centrifuged at a low speed. The aqueous layer was collected and re-extracted with the phenol, chloroform, isopropyl alcohol reagent. In turn, the aqueous layer was extracted with chloroform. At this point, 3 M sodium acetate was added to the aqueous phase collected form the last extraction and then 3.75 ml of ethanol was added and gently mixed. The DNA was spooled out, soaked in 70% ethanol and allowed to air-dry. The DNA was finally suspended in 2 ml of Tris-EDTA buffer. Based on absorption at 260 nm, the final yield of DNA was 482 μg of DNA. The DNA was confirmed to be that of A. otitidis by the PCR method described in example 1. This DNA was submitted for sequencing. EXAMPLE 4 CLONING AND SEQUENCING ALLOIOCOCCUS OTITIDIS GENOME

This invention provides nucleotide sequences of the genome of Alloiococcus otitidis which thus comprises a DNA sequence library of Alloiococcus otitidis genomic DNA. The detailed description that follows provides nucleotide sequences of Alloiococcus otitidis, and also describes how the sequences were obtained and how ORFs (Open Reading Frames) and protein-coding sequences can be identified. To construct a library, genomic DNA was hydrodynamically sheared in an

HPLC and then separated on a standard 1% agarose gel. A fraction corresponding to 3000-3500 bp in length was excised from the gel and purified by the GeneClean procedure (BIO101 , Inc.).

The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The blunt-ended DNA was then ligated to unique BstXI -linker adapters. These linkers are complimentary to the pGTC vector, while the overhang is not self- complimentary. Therefore, the linkers will not concatermerize nor will the cut-vector religate itself easily. The liner-adapted inserts were separated from the unincorporated linkers on a 1% agarose gel and again purified using GeneClean. The linker-adapted inserts were then ligated to BstXI -cut vector to construct "shotgun" subclone libraries.

Only major modifications to the protocols are highlighted. Briefly, the library was transformed into DH10B competent cells (Gibco/BRL, DH5a transformation protocol). Transformed cells were detected by plating onto antibiotic plates containing ampicillin. The plates were incubated overnight at 37° C. Transformant clones were then selected for sequencing. The cultures were grown overnight at 37°C. DNA was purified using a silica bead DNA preparation (Egelstein, 1996) method. In this manner, 25 mg of DNA was obtained per clone.

These purified DNA samples were then sequenced using ABI dye-terminator chemistry. All subsequent steps were based on sequencing by automated DNA sequencing methods. The ABI dye terminator sequence reads were run on MegaBace™ 10000 (Amersham) machines and the data transferred to UNIX based computers. Base calls and quality scores were determined using the PHRED software program (Ewing et al., 1998, Genome Res. 8: 175-185; Ewing and Green, 1998, Genome Res. 8:685-734). Reads were assembled using PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, Jan. 1996, p 157) with default program parameters and quality scores. To identify Alloiococcus otitidis genome encoded polypeptides, the complete genomic sequence of Alloiococcus otitidis was analyzed essentially as follows: First, all possible stop-to-stop open reading frames (ORFs) > 222 nucleotides in all three reading frames were translated into amino acid sequences.

Second, the identified ORFs were analyzed for homology to known protein sequences. Third, the coding potential of non-homologous sequences were evaluated with the GENEMARKTM software program (Borodovsky and Mclninch, 1993, Comp. Chem. 17:123). The results of these analysis are set forth in tables 2- 16.

EXAMPLE 5

IDENTIFICATION OF SPECIFIC GENES IN ALLOIOCOCCUS OTITIDIS

Alloiococcus otitidis homologs of the genes listed in Table 4 were identified as follows: Protein sequences of interest ("query sequences", Table 4) were extracted from Genbank from one or more species; query species included but were not limited to Staphylococcus aureus, Streptococcus pnuemoniae, Streptococcus pyogenes, Lactococcus lactis, Escherichia coli, and Bacillus subtilis. These queries were compared to the Alloiococcus otitidis sequence by several methods in order to determine which Alloiococcus sequence was the ortholog for the query gene.

First, the query sequences were compared to the translated Alloiococcus otitidis ORF set using BLASTP. The ORF set was generated as described in Vaccines patent, except that for each ORF that had multiple potential start codons, only the longest ORF was used. The top 10 Alloiococcus otitidis hits for each query were saved, without regard to score.

These Alloiococcus otitidis hits were then compared to NR, the nonredundant Genpept database, using BLASTP. An Alloiococcus otitidis ORF was considered the ortholog of a query sequence if the genes were reciprocal best hits in Alloiococcus otitidis and the query genome. This analysis is also sumarized in Table 4 (excel file AOT_PATENT_FILE.xls, Sheet TopHitsAndClustalKey). Specific numerical cutoffs were not used; however all top hits had Expect values of less than 3 x 10^~28.

Several query sequences had more than one high-scoring hit in Alloiococcus otitidis. In most cases, however, only the first, best hit to the original query sequence had that query sequence as its reciprocal best hit. For example, the Streptococcus pyogenes query sequence GyrA (alpha subunit of DNA gyrase) has two high-scoring hits in Alloiococcus otitidis. These were distinguished by the reciprocal blast analysis; the first, ORF_505 (60% identity, Expect = 0) is the GyrA homolog and the second, ORF_1907 (38% identity, Expect = 1 x 10 ^~154) is the homolog of the query sequence GrlA or ParC (topoisomerase IV, A subunit). Other examples of closely related proteins include the B subunits of DNA gyrase (GyrB) and Topoisomerase IV (GrlB or ParE); and YphC and Era, both of which are putative GTP binding proteins of unknown function. These Alloiococcus otitidis ORFS were assigned based on their top hit in Genpept.

In two cases the multiple high-scoring hits in Alloiococcus otitidis were the result of gene duplication. In the case of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) two separate Alloiococcus otitidis ORFS were determined to be the desired orthologs, because both had MurA (or MurZ, alternate notation) as their best hit in Genpept. Likewise, there are two FolC (folylpolyglutamate synthase) homologs in Alloiococcus otitidis. It is known that other bacteria, particularly Gram- positive bacteria, may carry two homologs of each of these genes.

As a further step in verification of gene assignments, the Alloiococcus otitidis ORFS identified as orthologs of the query genes by the analysis above were then compared to an internal copy of the COGS database (Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS, Kiryutin B, Galperin MY, Fedorova ND, Koonin EV, 2001 , Nucleic Acids Res 2001 Jan 1 ;29(1):22-8. The COG database: new developments in phylogenetic classification of proteins from complete genomes) using BLASTP. The COGS database is a curated set of proteins from a set of finished bacterial genomes, which have been grouped into specific protein families on the basis of protein similarity. In all cases, the Alloiococcus otitidis ORF was most closely related to the COGS family of the initial query protein, if that protein had been assigned to a COGS family. Examples of proteins for which there is no COGS family defined (in our local version of the database) include SrtA (sortase) and MvaK1 (phosphomevalonate kinase).

As a final confirmation, all query proteins were compared to the complete Alloiococcus otitidis nucleotide sequence using TBLASTN, in order to determine if there were additional and/or better hits that had not been predicted as ORFS. In all cases, the same sequence was identified as the best hit by TBLASTN and by BLASTP.

For one query sequence, sortase, the Alloiococcus otitidis ORF that was the top hit (Expect = 0.42) by the initial BLASTP or TBLASTN using the Staphylococcus aureus sortase sequence as query was found by additional analysis (reciprocal blast) to be a putative ABC-transport protein. The true sortase homolog in Alloiococcus otitidis was identified by construction of a Hidden Markov Model based on a multiple alignment of 72 known and putative sortase proteins that had been identified previously using similar computational methods. The model was constructed using "hmmbuild" and the Alloiococcus otitidis ORF set was searched using "hmmsearch", both of the hmmer package (S.R. Eddy. Profile hidden Markov models. Bioinformatics 14:755-763, 1998). The assignment of ORF_876 as sortase was then confirmed by reciprocal blast as described above and in Table 2. ORF_876 was also found to be the top hit in Alloiococcus otitidis when the Bacillus subtilis putative sortase (YhcS) was used as the query sequence in a BLASTP search. The Bacillus halodurans BH3596 Bacillus subtilis YhcS and proteins that are the top hits for RF_876 have recently been placed into a COGS group of sortases, further confirming the identity of ORF_876 as the Alloiococcus otitidis sortase.

TABLE 4

Table 4

EXAMPLE 6

IDENTIFICATION OF THE GENE ENCODING COENZYME A (COA) IN ALLOIOCOCCUS

OTITIDIS

Pantothenate kinase (PanK, CoaA) encoded by the coa-4 gene catalyzes the initial step in Coenzyme A (CoA) biosynthesis. CoA is an essential co-factor in a number of metabolic pathways in bacteria and mammals. Short-chain thioesters such as acetyl-CoA and succinyl-CoA are essential intermediates in carbon metabolism. CoA-thioesters of long chain fatty acids feed into β-oxidation and are also the source of fatty acids for phospholipids. In addition, CoA and its thioesters play important roles in the regulation of several enzymes in intermediary metabolism, including pyruvate dehydrogenase and phosphoenolpyruvate carboxylase. Finally, synthesis of holo acyl carrier protein (ACP) is dependent on CoA for the 4'- phosphopantetheine moiety linked to ACP. ACP is essential for fatty acid biosynthesis. The two major acyl-carrier groups in cells: CoA and ACP, are derived from pantothenate. Pantothenate can be obtained exogenously through uptake via a permease, the product of the panF gene. Alternately, pantothenate is the product of condensation of pantoate and β-alanine via pantothenate synthetase, the product of the panC gene. The initial step in CoA biosynthesis is the phosphorylation of pantothenate by pantothenate kinase (PanK, CoaA).

The coaA gene was originally identified by Dunn and Snell in S. typhimurium as a temperature sensitive allele. Similarly, a temperature sensitive allele of coa-4 was reported for E. coli in 1987. CoaA was found to be essential in E. coli in a recent genetic footprinting analysis. In the temperature sensitive strains, accumulation of phosphorylated CoA intermediates rapidly ceased following shift to the non-permissive temperature. CoaA was shown to be a homo-dimer of 35 kDa subunits that bound ATP cooperatively. ATP is bound first in a sequential mechanism of action; CoA has been shown to be a potent inhibitor of the reaction and competitively competes for binding with ATP. Therefore CoaA is under feedback regulation and is the major regulatory step in CoA biosynthesis.

Lysine 101 in bacterial pantothenate kinase (CoaA) was found to be essential for both ATP and CoA binding. This supports kinetic data that CoA is a competitive inhibitor of ATP binding to CoaA and that both substrates bind to the same site.

Homologues of E. coli CoaA have been identified in B. subtilis, S. pyogenes, M. tuberculosis, H. influenzae and V. cholerae. Homologues have not been identified in either the S. cerevisiae genome or in a mammalian expressed sequence tag database. Calder ef al. identified a homologue, through functional complementation of an E. coli coaA ts mutant, in A. nidulans. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 47. The protein encoded by the gene is set forth in Seq. ID No. 48.

The A. nidulans gene was then used to identify a yeast homologue. The bacterial and Aspergillus enzymes were found to be 16% identical and 32% similar. Although this level of similarity is quite weak the essential lysine residue involved in nucleotide binding appears to be conserved; however, the sequence surrounding the lysine residue were not conserved and further study will be required to validate this finding. The most striking difference between the eukaryotic and prokaryotic enzymes is found in the sensitivity of each to competitive inhibition by CoA and acetyl-CoA. The yeast enzyme was most sensitive to acetyl-CoA and less sensitive to CoA, whereas the converse was true for the bacterial enzyme. Later studies demonstrated that mammalian pantothenate kinase is activated by CoA and inhibited by acetyl-CoA.

Nucleotide binding

Binding of ATP to CoaA is directly demonstrated by equilibrium dialysis employing the non-hydrolyzable ATP analogue ATPyS. The K_d measured for ATP binding is reported to be 2.1 μM.

CoA binding

Binding of CoA to CoaA is directyl demonstrated by equilibrium dialysis and the K_d is reported to be 6.7 μM.

Pantothenate kinase activity

Specific kinase activity of CoaA is demonstrated using D-[1 - ⁴CJpantothenate and capturing 4'-phospho[1 -¹⁴C]pantothenate on DE81 filters. Using this assay the following kinetic values were derived: specific activity - 470+/- 200 nmol/min/mg; pantothenate K_m - 36 μM; K_m ATP - 136 μM.

Suitability of target for anti-infective development

Coenzyme A biosynthesis is essential for bacterial viability. CoaA catalyzes the first step of biosynthesis of CoA and appears to be the point of regulation for the pathway. The essentiality of CoaA is demonstrated through the construction of temperature sensitive alleles in coaA. Although the yeast enzyme is found to functionally complement the bacterial temperature sensitive allele, sequence and kinetic differences suggest the possibility of identifying inhibitors of the bacterial enzyme with high selectivity. As CoaA is essential and conserved in gram-negative and gram-positive pathogens, such inhibitors will have broad-spectrum utility. Suitable assays for measuring CoaA function

CoaA is purified by standard methods using widely available molecular tags following expression at high level from E. coli. Pantothenate kinase activity is measured as follows: CoaA and D-[1-¹⁴C]pantothenate is incubated in a buffer consisting of 100 mM Tris (pH 7.4), 2.5 mM MgCI₂, 2.5 mM ATP for 5-60 minutes at 37^°C. Product, 4'-phospho[1 -¹⁴C] pantothenate, is monitored through retention of labeled material on DE81 filters. This assay is amenable to high-throughput screening using high-density well-filter plates.

EXAMPLE 7

IDENTIFICATION OF THE GENE ENCODING COABC (DFP) IN ALLOIOCOCCUS OTITIDIS

The E. coli dfp gene, which encodes the previously designated Dfp protein, was originally identified as encoding an enzyme required for CoA biosynthesis. The gene, coding for the protein of interest, was renamed coaBC to reflect the enzyme function in CoA biosynthesis. CoA is an essential co-factor in a number of metabolic pathways in bacteria and mammals. Short-chain thioesters such as acetyl-CoA and succinyl-CoA are essential intermediates in carbon metabolism.

CoaBC carries out the second and third steps of coenzyme A biosynthesis: the conjugation of 4'-phosphopantetheate with cysteine by the CoaB (PPCS : 4'phosphopantethenoyl cysteine synthase) activity followed by the conversion to 4'-phosphopantetheine by the CoaC (PPCDC: 4'phosphopantenoylcysteine decarboxylase) activity. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 77). The protein encoded by the gene is set forth in Seq. ID No. 78.

Enzyme activity of CoaBC (Dfp):

Initially it was demonstrated that Dfp enzyme catalyzing oxidative decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine (PPC) to form 4'- phosphopantetheine (PP) - the third step in CoA biosynthesis from pantothenate The _M for this reaction is 800 μM for PPC.

Subsequently, it was established that Dfp is a bifunctional enzyme, catalyzing the second step of CoA biosynthesis, coupling of 4'-phosphopantothenate with cysteine to form PPC, as well. This reaction is a two-step process and requires CTP for initial 4'-phosphopantothenate activation. Second step couples cysteine to the phosphopantothenate moiety with a release of CMP. Estimated ^" _M's are 300 μM for 4'-phosphopantothenate and CTP, and 250 μM for cysteine.

CoaBC as target for antibacterial development.

Coenzyme A (CoA) plays a vital role in the metabolism of living cells. According to a recent report, 4% of all enzymes in the cell require CoA, its thioesters or 4'-phosphopantetheine. Recent genetic footprinting experiments on E. coli and direct gene knockout have established that this coaBC is essential for bacterial growth. Homologs of coaBC have been identified in a number of gram-positive and gram-negative organisms, which suggested the possibility of developing a broad- spectrum antibacterials from coaBC inhibitors. Considering the bifunctional nature of CoaBC, it is feasible to identify inhibitors that will inhibit both enzymic functions, thus arresting two steps in the CoA pathway. Another important factor in favor of selecting CoaBC as a target for antibacterials is low homology of the bacterial enzyme to eukaryotic counterparts. In most of the higher organisms including humans, two separate enzymes carry out these functions. Moreover, mammalian (R)-4'-phospho-N-pantothenoylcysteine decarboxylase is a pyruvate-dependent enzyme, while CoaBC requires flavine mononucleotide for its function.

Assays for measuring CoaBC activity.

PPC synthetase activity is be monitored by detecting the released pyrophosphate. This is achieved by converting pyrophosphate to inorganic phospate with pyrophosphatase and detection by the Malachite Green assay, or by the MESG assay spectrophotometrically. CoaBC (2 μg) is incubated in the reaction buffer containing 10 mM DTT, 2 mM MgCI₂₎ 50 mM Tris-HCl, pH 8, 300 μM 4'- phosphopantothenate, 3.5 mM CTP, 5 μg pyrophosphatase. The reaction is started by addition of appropriate amount (10-500 μM final) of cysteine. The reaction is stopped at different time points by addition of equal volume of 5M H₂S0 . The amount of inorganic phosphate released will be determined according to the one of described techniques.

PPC synthetase activity is also monitored by detecting the release of carbon dioxide from ¹⁴C-labeled cysteine. CoaBC (2 μg) is incubated in the reaction buffer containing 10 mM DTT, 2 mM MgCI₂, 50 mM Tris-HCl, pH 8, 2.5 μM 4'- phosphopantothenate, 3.5 mM CTP. The reaction is started by addition of appropriate amount (30 mM, final concentration) of ¹⁴C-labeled cysteine. The reaction is stopped at different time points by addition of equal volume of 5M H₂S0₄. Amount of released ¹⁴C-labeled C0₂ is determined according to published technique.

Example 8

Identification of the gene encoding phosphopantetheine adenylyltransferase (CoaD) in Alloiococcus otitidis

Phosphopantetheine adenylyltransferase, (PPAT, CoaD, KdtB) catalyzes the penultimate step in Coenzyme A (CoA) biosynthesis. The fourth step in CoA biosynthesis is the addition of AMP to 4'-phosphopantetheine by phosphopantetheine adenylyltransferase (CoaD) to form 3' dephospho-CoA (dPCoA).

The coaD gene was first identified in E coli by Geerlof ef al. CoaD is essential for viability in E coli and S. aureus. The enzyme has a mass of 18 kDa and was determined to be a hexamer through cross-linking studies. Crystallography confirmed the oligomeric state of the enzyme. Moreover, co-crystallography of CoaD with dPCoA has also been carried out mapping the binding pocket for the major product of the reaction. Interestingly, in mammals PPAT has been shown to be in a complex with dephospho Coenzyme A kinase (dPCoA kinase, DPCK). This enzyme, purified from pig liver, is referred to as CoA Synthase. The yeast PPAT is associated with a protein complex that is in excess of 375 kDa and composed of six proteins. There is no detectable homology between the bacterial PPAT (CoaD) and the recently identified human PPAT, the activity of which is contained in a bifunctional PPAT/DPCK enzyme. Homologues of E. coli CoaD have been identified in P. aeruginosa, S. pneumoniae, S. aureus, H. influenzae, H. pylori, B. anthracis and M. tuberculosis. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 81). The protein encoded by the gene is set forth in Seq. ID No. 82.

Enzyme activity CoaD (PPAT) carries out the reversible transfer of AMP to 4'- phosphopantetheine, forming dephosphocoenzyme A and releasing PPi. The reverse reaction was demonstrated by Geerlof ef al. using a coupled assay to tie ATP production to NADP reduction, which is monitored at 340 nm. The following kinetic constants were calculated: k_cat = 3.3 +/- 0.1 /sec; K_m(d_Pco_A) = 7.0 +/- 1.4 uM; K_m(Ppi₎ = 0.22 +/- 0.04 mM.

CoaD as target for anti-infective development.

Coenzyme A biosynthesis is essential for bacterial viability. CoaD, phosphopantetheine adenylyltransferase, catalyzes the fourth step in the pathway and was shown to be essential in both E. coli and S. aureus. There is no measurable homology between CoaD and the human PPAT enzyme, so the liability of poorly selective compounds is quite low. As CoaD is essential and conserved in gram- negative and gram-positive pathogens, inhibitors developed against this target will have broad-spectrum utility.

Assays for measuring CoaD function

CoaD will be expressed and purified using standard methodologies for bacterial expression and affinity tag-based purification. Two assay formats can be used to monitor enzymatic activity: the forward reaction and the reverse reaction. The forward reaction assay was initially described for measuring the activity of the human PPAT activity in the PPAT/DPCK enzyme. The enzyme assay is carried out in 50 mM Tris (pH 8.0), 2 mM MgCI₂, 5 mM ATP, 5-500 uM 4'- phosphopantotheine, 7.5 mM NADH and enzyme (initially 0.1 - 1.0 μg/ml). The production of PPj is detected using the protocol of O'Brien in which PPi production is coupled to the oxidation of NADH to NAD. This system requires the addition of 4 enzymes (PP_rdependent phosphofructokinase, aldolase, triosephophate isomerase and glycerol-3-P dehydrogenase) to the basic reaction mix and presents the added issue of deconvolution, which limits the use of the assay as a primary screen. The reverse direction assay is carried out also as a coupled assay to tie ATP production to NADP reduction following the method described by Lamprecht & Trautschold. The assay is set up in reaction buffer containing the following: 50 mM Tris (pH 8.0), 1 mM DTT, 2 mM MgCI₂, 1 mM NADP, 5 mM glucose, 2 mM PP|, 0.1 mM dPCoA. Hexokinase (4 units) and glucose-6-phosphate dehydrogenase (1 unit) will be added to the assay as the coupling enzymes in addition to CoaD (initially 0.1 - 1 μg/ml). The assay is monitored at 340 nm. Deconvolution of hits is required with this assay, however with only 2 additional enzymes the task will be less cumbersome when compared to the forward assay described above.

Example 9

IDENTIFICATION OF THE GENE ENCODING DEPHOSPHOCOA KINASE (DPCK, YACE,

COAE) IN ALLOIOCOCCUS OTITIDIS

DephosphoCoA kinase (DPCK, YacE, CoaE) encoded by the coaE gene catalyzes the final step in Coenzyme A (CoA) biosynthesis. The final step in CoA biosynthesis is the phosphorylation of the 3'-hydroxyl group of dephospho-CoA to form CoA by dephosphocoenzyme A kinase (DPCK, YacE, CoaE).

The determination that the previously identified yacE gene encoded the dephosphocoenzyme A kinase activity was reported by Mishra ef al. These authors previously determined that separate enzymes encode the phosphopantetheine adenyltransferase (PPAT) and dephosphocoenzyme A kinase (DPCK) activity in Corynebacterium ammoniagenes in contrast to the eukaryotic enzymes in which the PPAT and DPCK activities are coupled. The E. coli gene, encoding a 25 kDa protein, was cloned based on the sequence of the C. ammoniagenes gene and found to be identical to the previously described yacE gene. The gene was designated coaE to follow existing nomenclature in E. coli. CoaE (YacE) was shown to be essential in E. coli through genetic footprinting. CoaE is widely distributed in bacteria. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 93). The protein encoded by the gene is set forth in Seq. ID No. 94. Assays for measuring CoaE function

CoaE carries out the phosphorylation of dephosphocoenzyme A at the 3' hydroxyl group, consuming ATP, to form CoA. Dephosphocoenzyme A kinase activity is measured in a coupled reaction in which NADH oxidation to NAD is tied to ADP production. In this assay, the standard pyruvate kinase/lactose dehydrogenase coupling system is used to generate NAD in a 1 :1 molar equivalent to the ADP produced by the test enzyme. NADH oxidation to NAD is monitored at 340 nm in a standard spectrophotometer. The following kinetic values were determined for CoaE: K_m (ATP) = 0.74 mM; K_{m (}de_P ospho-coA) = 0.14 mM (7). The formation of CoA is monitored using a coupled enzyme system in which acetyl-CoA is formed in proportion to the amount of CoA in the assay. Three enzymes (phosphate acetyl transferase, citrate synthase and malate dehydrogenase) are added to the reaction that results in the formation of NADH from NAD, which is monitored at 340 nm.

CoaE as a target for anti-infective development

Coenzyme A biosynthesis is essential for bacterial viability. CoaE, dephosphocoenzyme A kinase, catalyzes the final step in CoA synthesis and is shown to be essential by genetic footprinting in E coli. A degree of homology between CoaE and the human DPCK enzyme has been noted, such that selectivity assays is necessary to determine a high therapeutic index for CoaE inhibitory compounds. CoaE is conserved in gram-negative and gram-positive pathogens and should have broad-spectrum utility in the clinic.

CoaE is expressed and purified using standard methodologies for bacterial expression and affinity tag-based purification. DephosphocoA kinase activity is monitored using a coupled enzyme system to tie ADP production to oxidation of NADH to NAD. The decay of absorbance at 340 nm will be the assay readout. The assay will be setup in the following buffer: 50 mM Tris (pH 8.5), 20 mM KCl, 10 mM MgCI₂, 10 mM ATP, 0.3 mM NADH and 0.4 mM phosphoenolpyruvate. The coupling enzymes: pyruvate kinase (10 U) and lactate dehydrogenase (4 U) will be added along with dephosphocoenzyme A kinase (initially 0.1- 1.0 ug/ml). The assay will be started by the addition of 0.4 mM dephosphocoenzyme A. In this assay system, the release of ADP is tied to the oxidation of NADH to NAD, and is monitored at 340 nm. This assay is transferable to a high-density microtiter plate format and suitable for HTS.

EXAMPLE 10 IDENTIFICATION OF DNAB AND PCRA, GENES ENCODING HELICASES IN ALLOIOCOCCUS

OTITIDIS

Helicases unwind double-stranded DNA in a reaction that couple nucleotide binding and hydrolysis to strand unwinding. Their activity is required for a number of biological processes such as separation of the chromosome during replication, recombination and repair. Homologue of thse genes were identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 15 and 99). The protein encoded by the gene is set forth in Seq. ID No. 16 and 100.

Due to the essential roles modulated by these molecules they represent an important target for antibacterial therapy. Homologs of dnaB and pcr>4 genes encoding helicases were identified as described in Example 5. A primary assay, which detects helicase function in vitro, is used to identify inhibitors of each enzyme and is described below. Genes encoding DnaB and PcrA is obtained using polymerase chain reaction amplification of the genomic region encoding them. The genes is subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme is then over-expressed in Escherichia coli and purified using a standard tag system. Most helicases require a region of single-stranded DNA flanking the duplex region that it unwinds. As a result, providing a single stranded region to either the 3' or 5' end of a duplex allows for determination of the polarity of helicase unwinding. These types of experiments have demonstrated that PcrA and DnaB are 3'-5' and 5'- 3' helicases, respectively. None the less, a convenient filtration assay has previously been described that is formatted for high-through-put screening of inhibitors of either enzyme, regardless of polarity. Assays (90 ul) contained 15 pM single-stranded M13 DNA to which a radiolabeled oligonucleotide had been annealed as a substrate for unwinding. Reactions are carried out in 96-well GF/C unifilter hydrophobic plates (Polyfiltronics Inc.) in 70 ul helicase buffer [20 mM Hepes (pH 7.6), 4 mM MgCI₂ 4 mM ATP, 100 ug/ml BSA, 5% glycerol and 2 mM DTT] and 10 ul of DMSO or compound. Reactions are initiated by adding 10 ul of purified helicase protein and are incubated for 1 hr at room temperature. 100 ul of 2X capture buffer containing silica beads [25% methanol, 3 M Nal, 0.03% NP-40, and 10% GlassFog beads (BI0101 )] were added. The mixture was incubated for 30 min at room temperature. Plates are then washed 5X on a Bio-Teck instruments, Auto Washer EL403) with wash buffer (50% ethanol, 0.2% NP-40 and 50 mM NaCl). Scintillation fluid was added and plates are counted (Packard Topcount).

EXAMPLE 11

IDENTIFICATION OF DNAE. THE GENE ENCODING DNAE-POLYMERASE IN ALLOIOCOCCUS

OTITIDIS

DnaE is an enzyme that catalyzes the DNA template directed polymerization of deoxyribonucleotides into deoxyribonucleic acid. The enzyme has been reported to modulate lagging strand synthesis at gram-positive replication forks. Functions for DnaE have been defined biochemically, in Bacillus subtilis and Streptococcus pyogenes. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 75). The protein encoded by the gene is set forth in Seq. ID No. 76.

Because DnaE is an essential protein in gram-positive bacteria and has high homology to the gram-negative dnaE, which is an essential polymerase subunit of the DNA polymerase III holoenzyme, it serves as a good target for antibacterial drug discovery. A primary assay, which detects processive DnaE mediated DNA synthesis in vitro, is useful identify inhibitors of the enzyme and is described below. The gene encoding DnaE I in Alloiococcus otitidis was identified as described in Example 5. Purification of DnaE DNA polymerase from Alloiococcus. The gene encoding DnaE is obtained using polymerase chain reaction amplification of the dnaE gene. The gene is subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme is then over-expressed in Escherichia coli and purified using a standard tag system.

Because DnaE catalyzes the incorporation of single deoxyribonucleotides into DNA, the incorporation of radiolabelled deoxyribonucleotides into larger deoxyribonucleic acid molecules is monitored to measure activity of the enzyme. A filtration assay has been previously described for Streptococcus pyogenes DnaE that uses filterplates containing DE81 filters to capture polymerized DNA. This assay is amenable to high-through-put screening format for DnaE. Assays contained 70 ng of 30-mer primed M13mp18 single stranded DNA as a template for replication. The reaction contained 3.3-300 ng of DnaE in 23.5 μl of replication buffer [20 mM Tris- HCL (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCI₂, 40 μg/ml BSA] and 60 μM of both dGTP and dCTP. NaCl was added to the reaction mixture to a final concentration of 40 mM. DNA synthesis was initiated by the addition of 1.5 μl of 1.5 mM dATP and 0.5 mM [μ-³²P]dTTP. Reactions were incubated at 37°C for various lengths of time and were quenched by adding an equal volume of 1 % SDS and 40 mM EDTA. One-half of the terminated reaction was applied to DE81 filter paper and washed 3X with wash solution (0.3 M Ammonium formate and 0.01 M Sodium pyrophosphate). Filters were then placed in scintillation vials and 1 ml scintillation counting liquid was added. Radioactivity was counted using a scintillation counter.

EXAMPLE 12 IDENTIFICATION OF DNAG, THE GENE ENCODING PRIMASE IN ALLOIOCOCCUS OTITIDIS

DnaG is an enzyme that catalyzes the DNA template directed polymerization of ribonucleotides into ribonucleic acid de novo . Ribonucleic acid molecules that are synthesized by DnaG primase subsequently serve as primers for synthesis of the leading- and lagging-strands during chromosomal replication. Functions for DnaG have been defined biochemically, and the crystal structure of the RNA polymerase domain has been determined in Escherichia coli. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 63). The protein encoded by the gene is set forth in Seq. ID No. 64.

Because DnaG primase plays an essential role in both leading- and lagging- strand synthesis during chromosomal replication, and DnaG has homologs in all prokaryotes but not eukaryotes, it serves as a good target for antibacterial drug discovery. A primary assay, which detects DnaG mediated RNA synthesis in vitro, can be used to identify inhibitors of the enzyme and is described below. Assay for the activity of DNA polymerase and identification of compounds that inhibit DnaG

The gene encoding DnaG is obtained using polymerase chain reaction amplification of the dnaG gene. The gene is subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme is then over-expressed in Escherichia coli and purified using a standard tag system.

Because DnaG catalyzes the incorporation of single ribonucleotides into RNA, the incorporation of radiolabelled ribonucleotides into larger ribonucleic acid molecules is monitored to measure activity of the enzyme. A high-throughput scintillation proximity assay (SPA) assay, previously described for E. coli DnaG, is used to meadure activity of DnaG activity in a coupled reaction with DnaB helicase. The assay, which was shown to work with DnaG alone, is used to screen for compounds that inhibit DnaG function. Assays are run in 96-well Packard Optiplate plates. First, 1 μl DMSO or test compound was added, followed by 20 μl of DnaG (208 nM) and 3.3 nM M13mp18 single-stranded DNA. Reactions are initiated by adding 10 ul of primase assay buffer [50 mM Tris-HCl (pH 7.5), 4% sucrose, 8 mM DTT, 5 mM MgCI₂, 40 ug/ml BSA, 0.1 μg/ul Rifampicin, 25 U/ml RNA guard, 100 μM GTP, 100 μM UTP, 3 μM CTP, 1 mM ATP] and 0.4 μCi [³H]CTP. Reactions are incubated at 30°C for 30 min. Next, a suspension of 50 μl of 2.5 mg/ml PVT-PEI SPA beads (Amersham; prepared in 0.3 M NaCitrate, pH 3.0) were added. Plates were read after 1 hr on a Topcount instrument (Packard).

EXAMPLE 13

DNAN, DNAX, HOLA, HOLB, AND POLC, THE GENES ENCODING THE SUBUNITS OF ALLOIOCOCCUS OTITIDIS DNA POLYMERASE III HOLOENZYME: BETA (β), TAU (T), DELTA

(A), DELTA' (Δ') AND POLC.

DNA polymerase III holoenzyme is an enzyme complex comprised of multiple highly conserved subunits that catalyzes the DNA template directed polymerization of deoxyribonucleotides into deoxyribonucleic acid. In gram positive organisms the holoenzyme is composed of a polymerase subunit, PolC, and accessory proteins. The accessory proteins act in a coordinated manner to clamp the polymerase tightly to the DNA template allowing the polymerase to synthesize DNA with high speed and processivity. Homologue of these genes identified in Alloiococcus otitidis are described in Example 5 (Seq. ID Nos. 21 , 105, 79, 103, and 105 respectively). The protein encoded by the gene is set forth in Seq. ID No. 22, 106, 80, 104 and 106 respectively). Functions for the individual subunits have been defined biochemically and interactions between them have now been deduced structurally by crystallographic analysis of the enzyme from Escherichia coli. Tau interacts directly with both delta and delta' to form a clamp loader complex. Upon binding ATP the complex undergoes a conformational change altering an interaction between delta and delta', which allows delta to subsequently interact with the beta-clamp. The beta-clamp is a ring-shaped homomultimer assembly that can be opened by delta and placed onto a primed DNA template. ATP hydrolysis results in closing the clamp around DNA and dissociation of the clamp-loading complex. PolC then couples with the beta clamp to form a highly processive polymerase. Because DNA polymerase III holoenzyme is comprised of multiple subunits, the opportunity exists to inhibit its activity at a number of different sites. A primary assay, which detects processive DNA synthesis in vitro, can be used to identify inhibitors of the enzyme and is described below. Deconvolution of inhibitors, based on either activity of physical interaction, follow the primary assay.

Assay for the activity of DNA polymerase

Purification of DNA polymerase III holoenzyme subunits from Alloiococcus. Genes encoding the subunits of DNA polymerase is obtained using polymerase chain reaction (PCR) amplification of the genomic region encoding them. The genes are subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme is then over-expressed in Escherichia coli and purified using a standard tag system.

Because DNA polymerase III catalyzes the incorporation of single deoxyribonucleotides into DNA, the incorporation of radiolabeled deoxynucleotides into larger deoxyribonucleic acid molecules is monitored to measure activity of the enzyme. A filtration assay is previously described for Streptococcus pyogenes DNA polymerase III that uses filterplates containing DE81 filters to capture polymerized DNA (2). This assay is amenable to high-through-put screening format. Assays contained 70 ng of 30-mer primed M13mp18 single stranded DNA as a template for replication. The reaction contained 43 ng of β and 140 ng of PolC-Tflfl' complex in 23.5 μl of replication buffer (20 mM Tris-HCL (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCI₂, 40 ug/ml BSA, and 60 u,M of both dGTP and dCTP. DNA synthesis was initiated by the addition of 1.5 uj of dATP and [ι\\- ³²P]dTTP. Reactions were incubated at 37°C for various lengths of time and were quenched by adding an equal volume of 1 % SDS and 40 mM EDTA. One-half of the terminated reaction was applied to DE81 filter paper and washed 3X with wash solution (0.3 M Ammonium formate and 0.01 M Sodium pyrophosphate). Filters were then placed in scintillation vials and 1 ml scintillation counting liquid was added. Radioactivity was counted using a scintillation counter.

Compounds inhibiting PolC subunit is identified by modifying the above reaction to include only the PolC subunit and using 2.5 μg activated calf thymus DNA as a substrate, instead of singly-primed M13mp18 DNA, as previously described. Several techniques are utilized to determine the interaction of inhibitors with individual subunits. These have been described in the literature and include the following: (1 ) Nuclear magnetic resonance and capillary electrophoresis.

EXAMPLE 14

ERA GTPASE IN ALLOIOCOCCUS OTITIDIS

The era (E. coli Ras) gene was initially identified while sequencing around the rnc gene; era lies downstream of rnc. While a function for era has yet to be determined, conditional (temperature sensitive) mutants revealed that the product of the era gene, Era, is essential for E. co//viability. A hint as to an in vivo function for Era was uncovered when a suppressor of a dnaG (primase) allele was found to map in the era coding sequence and a second suppressor, which mapped upstream of the era open reading frame, affected expression of era. These data suggest that Era could play one or more roles in DNA replication, regulation of primase activity or otherwise effect cell cycle progression. More recent data has confirmed that the eral mutant causes a defect in cell growth at the two-cell stage and delays cell division Moreover, Britton et al demonstrated that cell division was coupled with the level of Era in the cell: division arrest, through reduction in Era levels, is reversed when Era levels return to threshold amount. A current model suggests that Era acts as a checkpoint regulator in the bacterial cell cycle. Era is a GTP-binding protein with GTPase activity, a threshold level of functional/activated Era may be required to initiate septation.

Era is associated with additional cellular functions, specifically translation, as Era specifically interacts with the translation machinery. E. coli Era binds both 16S rRNA and the 30S ribosomal subunit; whereas, the S. pneumoniae 16S rRNA co- purifies with Era. A putative RNA binding "KH motif" has been identified in the carboxyl-terminal domain. The RNA binding activity is critical to Era cellular function as mutation of the putative RNA binding region of the S. pneumoniae Era prevents complementation of an E. coli era mutant strain. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID No 65). The protein encoded by the gene is set forth in Seq. ID No. 66.

Nucleotide binding

Filter-binding assays are utilized to demonstrate nucleotide-binding specific to GTP and not UTP, CTP or ATP. Both GTP and GDP (unlabeled) were capable of inhibiting α³ P-GTP binding. The Kd for GTP and GDP binding were reported to be 5.5 and 1.0 μM, respectively.

A large number of GTP-binding proteins have been studied and all members of the family contain three regions of highly homologous amino acid residues that define a GTP-binding pocket. Era contains well-conserved regions defining the so- called G1 (G/AXXXXGKT/S: residues 15-22), G3 (DXXG: residues 62-65) and G4 (NKXD: residues 124-128) consensus sequences. The G2 domain (residues 33-38, see below), located between G1 and G3, is generally more variable.

GTPase activity

Purified Era showed a significant GTPase activity, which is inhibitable by GTP or GDP but not by UTP, CTP, ATP or ADP. The maximum hydrolysis rate is measured at 9.8 mmol GTP hydrolyzed/min/mol Era. The Km was found to be 9 μM.

It should be noted that Sullivan ef al demonstrated, using mant (Λ/-methyl-3'- O-anthraniloyl) labeled GTP and GDP, very rapid exchange kinetics for guanine nucleotide binding. Era exchanges guanine nucleotides 10-fold more rapidly than the GTP hydrolysis rate suggesting that guanine nucleotide binding and release should be considered as a regulatory point in addition to the more well-studied hydrolysis step.

Autophosphorylation

When γ³²P-GTP is used as a substrate for the GTPase activity , Era is phosphorylated. The autophosphorylation reaction is specific for GTP, as incubation with γ³²P-ATP did not result in phosphorylation of Era. Moreover, α³²P-GTP is not a suitable substrate for detection of Era autophosphorylation. Tryptic digestion and HPLC were utilized to resolve the sites(s) of phosphorylation. Using γ³²P-GTP as a substrate the major radioactive peak contained the tryptic peptide, ISITSR, corresponding to Era residues 33-38 and containing 3 potential phosphorylation sites. Mutagenesis of both Thr-36 and Ser-37 to alanine abolished enzymatic activity. However, individual alanine substitutions at either site had no effect on Era function. The autophosphorylation site is located in the so-called G2 domain of Era.

Suitability of target for anti-infective development

Era is an essential protein for bacterial viability. Knock-down mutations as well as conditional-lethal alleles revealed that Era function is required for cytokinesis. An additional phenotype of the Era-depleted strains is an aberrant response to temperature induced stress. This target is novel and may well lead to the identification of new classes of anti-infectives. The widespread distribution of Era homologues in both gram-negative and gram-positive pathogens suggests that broad-spectrum agents could result from an effort to define Era inhibitory compounds.

Assays for measuring Era function

NUCLEOTIDE BINDING ASSAYS

Era binding to nucleotide is monitored by a simple filter-binding assay. Era (1 -5 μg) is incubated with α³²P-GTP (0.2 μCi) in a buffer consisting of 100 mM Tris (pH 7.5), 10 mM MgCI₂, 0.2% NP-40, 0.2 mg/ml BSA for 30 minutes at 32°C. A portion of the reaction mix is spotted on nitrocellulose membrane, washed (50 mM Tris (pH 7.5), 5 mM MgCI₂, 1 mM DTT) and dried. The membrane is then exposed to X-ray film. Alternatively, the spots are excised and counted. This assay is directly amenable to HTS using filter plates.

GTPASE ACTIVITY ASSAY

The GTP hydrolytic activity of Era is monitored using thin-layer chromatography. Era and α³²P-GTP is incubated in 50 mM Tris (pH 7.5), 5 mM MgCI2, 0.1 % NP-40, 0.2 mg/ml BSA for 30 minutes at 37°C. An aliquot of the reaction is placed on PEI cellulose and the strip developed with 0.5 M KH₂P0₄, 1.0 M NaCl (pH 3.7). The spots conforming to GDP and GTP are identified by UV shadowing, excised and counted. This assay represents an acceptable secondary/confirmatory assay.

Alternatively, the hydrolysis of γ³²P-GTP is monitored by assaying for liberated P,. Obg and α³²P-GTP is incubated in 50 mM Tris (pH 8.5), 1.5 mM MgCI₂, 0.1 mM EDTA, 100 mM KCl, 10% glycerol for 30 minutes to 3 hours at 37°C. The reaction will be stopped by the addition of a slurry of charcoal in 1 mM Kpi (pH 7.5), which selectively binds the GTP and GDP. The liberated Pi in the supernatant is monitored by Cerenkov counting. Free Pi can also be monitored with the Malachite Green reagent.

AUTOPHOSPHORYLATION ASSAY

Era autophosphorylation is monitored by incubating Era with γ³²P-GTP in 50 mM morpholinopropane sulphate (pH 6.8), 5 mM MgCI2, 1 mM DTT at 37°C (14). Samples are analyzed following separation on SDS polyacrylamide gels, drying the gel and exposure to film. This assay represents an acceptable secondary/confirmatory assay for Era activity.

EXAMPLE 15 FMHB(FEMX) GENES IN ALLOIOCOCCUS OTITIDIS

The femA, femB, and fmhB(femX) genes have been shown to be essential for incorporation of glycine into the side chain of peptidoglycan precursors in Staphylococcus aureus,. The femAB locus was initially identified as a factor essential for methicillin resistance (fern) based on random insertional inactivation of chromosomal genes and a screen for reduced expression of resistance mediated by the penicillin binding protein 2A (PBP2A). Inactivation of femA or femB was subsequently reported to prevent incorporation of glycine residues at positions 2 to 5 or positions 4 to 5 of the penta-glycine cross bridge since muropeptides cross-linked by one or three glycine residues were detected in the corresponding mutants. Inactivation of fmhB, formerly femX, is lethal, but the construction of a mutant conditionally expressing fmhB under the control of a xylose-inducible promoter showed that the gene was essential for synthesis of branched peptidoglycan precursors . These studies show that the fern gene products were required for incorporation of glycine at positions 1 (FmhB), 2 and 3 (FemA), and 4 and 5 (FemB) of the cross bridge, although the catalytic activity of the proteins has not been directly assessed. Similarly, inactivation of two fmhB homologues in Streptococcus pneumoniae, designated murM (fibA) and murN (fibB), reduced addition of L-Ala or L- Ser to the -amino group of L-Lys and subsequent addition of a second L-Ala residue, respectively. Overall, disruption of the murMN operon reduced the proportion of branched peptide stems in the peptidoglycan from 89 to 33% . In contrast to what occurs in S. aureus, direct cross-linking of L-Lys to D-Ala occurs in S. pneumoniae, and the murMN operon was accordingly reported to be unessential.

BLAST analysis of Alloiococcus otitis genome revealed an ORF similar to femXoi Weissella viridescent , and f hB oi S. aureus. It suggests that in Alloiococcus otitis there is an enzyme with similar to FhmB function. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5 /Table 4 (Seq. ID No 97). The protein encoded by the gene is set forth in Seq. ID No. 98. Assays for measuring FmhB function

There are no in vitro biochemical assays to test enzymatic activity of S. aureus FmhB because the reaction occurs at the membrane-bound lipid II precursor GlcNAc-(β-1 ,4)-N- acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala)- pyrophosphoryl-undecaprenol.

Lipid II is a minor component of bacterial cell membrane which is detected by thin-layer chromatography separation of presolubilized membranes supplied with the cytoplasmic precursors, UDP-/V-acetylmuramyl-pentapeptide (UDP-MurNAc- pentapeptide) and [¹⁴C]UDP-Λ/-acetylglucosamine ([¹⁴C]UDP-GlcNAc). The in vitro biosynthesis of branched lipid II of S. aureus requires whole-cell membranes, cytoplasmic PG precursors, glycine (¹⁴C labeled for detection of reaction products), purified tRNA, and an intracellular fraction that contains tRNA-activating enzymes. Therefore, the in vitro assay of S. aureus FmhB is a tedious procedure. One way to facilitate this procedure is to use Weissella viridescens FemX or E. faecalis UDP-MurNac-pentapetide:L-alanine ligase. Recombinant Weissella viridescensFemX and E. faecalis UDP-MurNac-pentapetide:L-alanine ligase were purified, and their in vitro activity was demonstrated. The distinctive feature of these enzymes is that they catalyze the addition of a branching amino acid (Ala) to the cytoplasmic cell wall precursor UDP-MurNac-pentapetide. Other bacteria for which the biosynthesis of Gly-containing branched UDP-

MurNac-hexapeptide in cytoplasm was shown are Streptomyces lividans and Streptomyces hydroscopicus , although the enzymes were not isolated and their ligase activity remain to be demonstrated.

These new data open an opportunity to develop an assay to detect the activity of FmhB(FemX) by using cytoplasmic UDP-MurNac-pentapetide.

Products of the reaction are detected by HPLC. HPLC separation of precursors are performed by the method of Flouret et al. The precursors are separated by reverse- phase HPLC on aμBondapak C₁₈ column (3.9 by 300 mm; Waters) in 50 mM ammonium formate (pH 3.9) at a flow rate of 0.5 ml/min. The elution of precursors is monitored at a wavelength of 254 nm. EXAMPLE 16 FOLA- DIHYDROFOLATE REDUCTASE (DHFR)

The Alloiococcus ORF-1863 encodes a homolog of S. aureus dihydrofolate reductase that catalyzes the NADPH-dependent conversion of dihydrofolate to tetrahydrofolate, one of the steps in bacterial folate biosynthesis. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 55). The protein encoded by the gene is set forth in Seq. ID No. 56.

FOLA as a target for anti-infective development

Folate is an essential cofactor in many important metabolic processes in bacteria, such as purine, pyrimidine. amino acid and pantothenate biosynthesis. Unlike mammalian cells, bacteria are unable to utilize exogenous folate derivatives, and therefore must synthesize folate αfe novo. Bacterial folate biosynthesis occurs via two converging pathways, the non-essential para-amino-benzoate (PABA) synthesis pathway, and synthesis of the pterin precursor, to which pABA is subsequently attached to form the folate precursor. Bacterial DHFRs are essential for viability and well conserved across all bacterial species. Although bacterial DHFR shares similarity with human DHFR, selective inhibitors against bacterial DHFR have been identified in the past such as trimethoprim which specifically blocks the bacterial DHFR step. Thus DHFR still remains an attractive target for development of broad- spectrum antibacterial agents.

Assays for measuring DHFR activity

DHFR activity is monitored spectrophotometrically, recording the change of absorbance at 340 nm due to the equimolar consumption of NADPH in the course of dihydrofolate substrate reduction. DHFR (10 ng) is preincubated in reaction buffer containing 50 mM 2-(N-morpholino)ethanesulfonic acid, 25 mM Tris-HCl, 25 mM ethanolamine, and 100 mM NaCl at pH 7.5 for 3 minutes. The reaction is started by addition of 0.5-10 μM 7,8-dihydrofolate. The amount of processed substrate is calculated from the decrease of absorbance at 340 nm due to oxidation of NADPH (D=11800 M^{' 1}) to NADP÷. EXAMPLE 17 FOLB- DIHYPRONEOPTERIN ALDOLASE (DHNA)

The Alloiococcus otitidis ORF-959 encodes a homolog of S. aureus dihydroneopterin aldolase that catalyzes the conversion of 7,8-dihydroneopterin to 6- hydroxymethyl-7,8-dihydropterin, one of the early steps in bacterial folate biosynthesis. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 31). The protein encoded by the gene is set forth in Seq. ID No. 32.

FOLB as a target for anti-infective development

Folate is an essential cofactor in many important metabolic processes in bacteria, such as purine, pyrimidine, amino acid and pantothenate biosynthesis. Unlike mammalian cells, bacteria are unable to utilize exogenous folate derivatives, and therefore must synthesize folate de novo. Bacterial folate biosynthesis occurs via two converging pathways, the non-essential para-amino-benzoate (pABA) synthesis pathway, and synthesis of the pterin precursor, to which pABA is subsequently attached to form the folate precursor. Enzymes that catalyze steps in the folate biosynthesis pathway are essential and well conserved across all bacterial species, and those that act in early steps such as FolB have no direct homologs in mammals. Thus FolB becomes an attractive target for development of broad-spectrum antibacterial agents.

Assays for measuring FOLB activity

FolB (DHNA) 7,8-dihydroneopterin aldolase activity is monitored individually or in conjunction with downstream enzymes in folic acid biosynthesis pathway (FolK and Sul).

FolB activity is monitored directly by HPLC assay. FolB substrate (7,8- dihydro-D-neopterin) is commercially available from Schircks Laboratories

(Swizerland). FolB (0.5 μg) is preincubated in reaction buffer containing 50 mM Tris- HCl (pH 8.0), 50 mM KCl, 0.1 mg/ml BSA, 2.5 mM dithiothrietol for 5 min. Reaction is started by addition of stock solution of 7,8-dihydro-D-neopterin in DMSO (100 μM final concentration). Reaction is terminated by addition of 1/3 of reaction volume of 1 % l₂, 2% Kl in 1 M HCl with subsequent incubation at room temperature for 5 minutes. Quenched reaction will be applied directly to HPLC. Oxidized starting material and reaction products are efficiently separated on ODS (C18) column. Reaction components are detected and quantified by analysis of UV absorbance at 254 nm, or fluorescence (excitation at 365 nm; emission at 446 nm).

FolB activity are also monitored in the coupled assay with FolK (HPPK) and Sul (DHPS) enzymes. FolB activity is measured by detection of radioactive dihydropteroate formation as described in FolK and Sul assays, under conditions of excess of the later enzymes. FolB enzyme and substrate 7,8-dihydro-D-neopterin are added to the described assay to replace the 6-hydroxymethyl-7,8-dihydropterin (FolK substrate).

EXAMPLE 18 FOLC- DIHYDROFOLATE SYNTHASE (DHFS)

The Alloiococcus otitidis ORF-956 and ORF-528 both encode a homolog of B. subtilis dihydrofolate synthase that catalyzes the conversion of 7,8-dihydropteroate and glutamate to dihydrofolate, one of the steps in bacterial folate biosynthesis [. Homologue of this gene identified in Alloiococcus otitidis as described in Example 5 (Seq. ID Nos. 29 and 23). The protein encoded by the gene is set forth in Seq. ID Nos. 30 and 24.

Use of FOLC as a target for anti-infective development Folate is an essential cofactor in many important metabolic processes in bacteria, such as purine, pyrimidine, amino acid and pantothenate biosynthesis. Unlike mammalian cells, bacteria are unable to utilize exogenous folate derivatives, and therefore must synthesize folate de novo. Bacterial folate biosynthesis occurs via two converging pathways, the non-essential para-amino-benzoate (pABA) synthesis pathway, and synthesis of the pterin precursor, to which pABA is subsequently attached to form the folate precursor. Enzymes that catalyze steps in the folate biosynthesis pathway are essential, and are well conserved across all bacterial species. Bacterial FolC appears to be a bifunctional enzyme possessing both dihydrofolate synthase (DHFS) activity and folyl-polyglutamate synthetase (FPGS) activity, which are probably mediated through different sites of the protein. The bacterial DHFS activity but not the FPGS activity is essential for viability. Although bacterial FolC shares similarity with human FPGS, the human enzymes apparently lack DHFS activity and display a folate substrate specificity quite distinct from that of bacterial enzymes. Thus targeting bacterial FolC/DHFS activity selectively might lead to identification of broad-spectrum antibacterial agents.

Assays for measuring FOLC activity FolC (DHFS) 7,8-dihydrofolate synthase activity in the presence or absence of antimicrobial compounds or putative inhibitory compounds are monitored by several methods.

In one method, FolC activity is monitored directly by simple HPLC assay. FolC substrate (7,8-dihydropteroic acid) is commercially available form Schircks Laboratories (Switzerland). FolC (15 ng) is added to reaction mix, containing 10 mM glutamate, 5 mM ATP, 50 mM Tris-HCl (pH 8.0), 20 mM Mg₂CI, 50 mM KCl, 0.1 mg/ml BSA, 5 mM dithiothreitol. Reaction is started by addition of stock solution of 7,8-dihydropteroic acid in DMSO (10 μM final concentration). Reaction is terminated by addition of equal volume of 8M Guanidinium hydrochloride. Stopped reaction is applied directly to HPLC. Starting material and reaction products are efficiently separated on ODS (C18) column. Reaction components are detected and quantified by analysis of UV absorbance at 254 nm, or fluorescence (excitation at 280 nm; emission at 420 nm).

In another method, the FolC activity monitoring is by detection of ADP accumulation. ADP is released in the amount equimolar to the amount of the product formed. ADP detection is performed by coupling its conversion to ATP by pyruvate kinase in the presence of phospho(enol)pyruvate producing pyruvate. Lactate dehydrogenase reduces pyruvate to S-lactate in the presence of NADH. Course of reaction is monitored by decrease in absorbance at 340 nm due to oxidation of NADH (ε=6220 cm^"1M^"1) to NAD⁺. Reaction conditions are as following: 5 mM dithiothreitol, 5 mM ATP, 380 μM NADH, 10 mM glutamate, 2 mM phospho(enol)pyruvate, 50 mM KCl, 20 mM Mg₂CI, 50 mM Tris-HCl, 50 μg of pyruvate kinase, 50 μg of S-lactate dehydrogenase. Reaction is started by addition 7,8-dihydropteroic acid in DMSO (10 μM final concentration).

In yet another method, FolC activity is monitored through detection of inorganic phospate release. Amount of inorganic phosphate in solution is quantified by:

(i) its conversion by purinenucleoside phosphorylase leading to phosphorylation of MESG. Later assay kit is available from Molecular Probes as EnzCheck™ Phosphate Assay Kit; (ii) its reaction with Malachite Green reagent; and (iii) detecting the release of radioactive inorganic phosphate in reaction with γ-

³³P-labeled ATP following the absorption of unprocessed ATP by charcoal. First method is applied in rate-based assay format; the later two in end-point format. Reaction conditions are similar to the ones described in HPLC-based assay.

EXAMPLE 19 FOLK- 6-HYDROXYMETHYL-7, 8-DIHYDROPTERIN PYROPHOSPHOKINASE (HPPK)

The Alloiococcus otitidis OFR-961 (Seq. ID No. 33) encodes a homolog of S. aureus 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase that catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, one of the early steps in bacterial folate biosynthesis. The protein encoded by this ORF is set forth in Seq. ID No. 34. (see Example 5/Table 4).

Use of FolK as a target for anti-infective development

Folate is an essential cofactor in many important metabolic processes in bacteria, such as purine, pyrimidine, amino acid and pantothenate biosynthesis. Unlike mammalian cells, bacteria are unable to utilize exogenous folate derivatives, and therefore must synthesize folate de novo. Bacterial folate biosynthesis occurs via two converging pathways, the non-essential para-amino-benzoate (pABA) synthesis pathway, and synthesis of the pterin precursor, to which pABA is subsequently attached to form the folate precursor. Enzymes that catalyze steps in the folate biosynthesis pathway are essential and well conserved across all bacterial species, and those that act in early steps such as FolK have no direct homologs in mammals. Thus FolK is an attractive target for the development of broad-spectrum antibacterial agents.

Assays for measuring FolK activity

FolK (HPPK) 7,8-dihydroxymethylpterin-pyrophosphokinase activity is monitored individually or in conjunction with downstream enzyme in folic acid biosynthesis pathway.

FolK activity is monitored directly by HPLC assay. FolK substrate (7,8- dihydro-6-hydroxymethylpterin) is commercially available from Schircks Laboratories

(Swizerland). FolK is preincubated in reaction buffer containing 50 mM Tris-HCl (pH

8.0), 50 mM KCl, 20 mM MgCI₂, 5 mM ATP, 0.1 mg/ml BSA, 2.5 mM dithiothrietol.

Reaction is started by addition of stock solution of 7,8-dihydro-6-hydroxymethylpterin in DMSO (100 μM final concentration). Reaction is terminated by addition of equal volume of 8M Guanidinium hydrochloride and applied directly on HPLC. Starting material and reaction products are efficiently separated on ODS (C18) column.

Reaction components are detected and quantified by analysis of UV absorbance at

254 nm.

FolK activity is monitored by end-point assay coupled with excess of Sul enzyme. Activity is calculated from quantification of the radioactivity incorporated in final product (7,8-dihydropteroate).

EXAMPLE 20 ALLOIOCOCCUS OTITIDIS ENCODED FOLP (SUL)- DIHYDROPTEROATE SYNTHASE (DHPS)

The Alloiococcus otitidis ORF-1811 (Seq. ID No. 53) encodes a homolog of B. subtilis dihydropteroate synthase that catalyzes the condensation of pABA (para- aminobenzoic acid) with 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, one of the early steps in bacterial folate biosynthesis. The polypeptide encoded by this ORF is set forth in Seq. ID No. 54. (see Example 5/Table 4)

FOLP AS A TARGET FOR ANTI-INFECTIVE DEVELOPMENT

Folate is an essential cofactor in many important metabolic processes in bacteria, such as purine, pyrimidine, amino acid and pantothenate biosynthesis. Unlike mammalian cells, bacteria are unable to utilize exogenous folate derivatives, and therefore must synthesize folate de novo. Bacterial folate biosynthesis occurs via two converging pathways, the non-essential para-amino-benzoate (pABA) synthesis pathway, and synthesis of the pterin precursor, to which pABA is subsequently attached to form the folate precursor. Enzymes that catalyze steps in the folate biosynthesis pathway are essential and well conserved across all bacterial species, and those that act in early steps such as FolP (Sul) have no direct homologs in mammals. In fact, dihydropteroate synthase (FolP or Sul) is the target for known antibiotics sulfonamides which are competitive inhibitors of FolP/Sul as pABA analogues. Thus FolP (Sul) still remains an attractive target for development of broad-spectrum antibacterial agents.

Suitable assays for measuring FolP/Sul activity

Sul (DHPS) 6-hydroxymethy-7,8-dihydroneopteroate synthase activity is monitored individually or in conjunction with upstream enzymes in folic acid biosynthesis pathway (FolB and/or FolK).

DHPS activity is monitored directly by counting the amount of radioactivity incorporated in 6-hydroxymethy-7,8-dihydroneopteroate when using radioactively labeled p-aminobenzoic acid (pABA). Final product is separated from unreacted pABA by thinlayer chromatography, paper chromatography or on HPLC equipped with radioactivity detector. DHPS substrate (6-hydroxymethyl-7,8-dihydropterin pyrophosphate) is not commercially available, but is quantitatively synthesized in one step from its oxidized precursor available from Schircks Laboratories (Swizerland). DHPS (20 ng) is added in reaction buffer containing 50 mM Tris-HCl, pH 8.0, 20 mM MgCI₂, 0.1 mg/ml BSA, 5 mM dithiothreitol and 0.5 - 10 μM PABA. Reaction is started by addition of stock solution of substrate (6-hydroxymethyl-7, 8-dihydropterin pyrophosphate, 0.05 - 1 μM final concentration). Reaction is terminated by acidification of reaction volume with addition of equal volume of citrate/phosphate or ammonium acetate/acetate buffer, pH 4 containing excess of unlabelled pABA. Quenched reaction is separated by chromatography and the amount of formed product calculated.

DHPS activity is determined in coupled assay with excess of FolB and FolK enzymes. The advantage of coupled assay is that it makes it possible to use commercially available FolB (7,8-dihydro-D-neopterin), or FolK (6-hydroxymethyl-7,8- dihydropterin) substrates, thus forming DHPS substrate in situ.

EXAMPLE 21 ALLOIOCOCCUS OTITIDIS ENCODED FILAMENT ATION TEMPERATURE SENSITIVE GENE A

(FTSA)

The Alloiococcus otitidis ORF-2489 (Seq. ID No. 85) encodes a homolog of E. faecalis FtsA, one of the essential components of bacterial cell division. The "fts" stands for f ilamentation temperature sensitive and has been assigned to most bacterial cell division genes due to the fact that these genes were generally discovered by the isolation of conditional mutants that form filaments at nonpermissive temperature . The ftsA allele was first isolated and identified in E. coli by Ricard and Hirota in 1973, and mapped along with ftsZ in 1980.The protein encoded by this ORF is set forth in Seq. ID No. 86. (see Example 5/Table 4)

Bacterial cell division requires formation of a septum at mid-cell that begins with the polymerization of FtsZ into a ring structure at the nascent division site. FtsZ, another key component of bacterial septation is the first known protein to localize to the division site. In E. coli, shortly after the formation of the FtsZ ring, FtsA and ZipA (another key division component present only in gram-negative bacteria) [7] are independently recruited to the septal ring, most likely through their direct interaction with FtsZ. Subsequent assembly of other division components at the septum requires FtsA as well as FtsZ.

FtsA as a target for anti-infective development

Like FtsZ, FtsA homologs are present and highly conserved in almost all eubacteria. FtsA is essential for cell division and its deletion leads to impaired cell division and sporulation defect. In addition, E. coli cells have to maintain critical ratio of FtsA to FtsZ in order for proper cell division to occur. FtsA belongs to the actin/DnaK/sugar kinase family of proteins. In B. subtilis, FtsA acting as a dimer not only binds ATP but also hydrolyzes ATP. As briefly stated above, in vivo and in vitro evidence have demonstrated that FtsA and FtsZ from various bacterial species directly interact. Taken all together, targeting at FtsA especially at its interaction with FtsZ might lead to identification of broad-spectrum antibacterial agents.

Assays for measuring FtsA activity

ATPase activity of FtsA is assayed by following the formation of ³²Pi from [γ-³²P]- ATP. The reaction mixture containing 50 mM Tris-HCl (pH7.2), 50 mM potassium acetate, 1 mM DTT, 10 mM MgCI₂ and different concentrations of [γ-³²P]-ATP is incubated for 5 minutes at 37°C. The reaction is started by addition of 50 nM purified FtsA of Alloiococcus. The reaction is stopped with 1.5% ammonium molybdate in 0.5N sulfuric acid, and the radioactive Pi extracted into isoamyl alcohol and counted.

Interaction between FtsA and FtsZ is detected quantitatively using yeast two- hybrid system as described. Briefly, Alloiococcus ftsZ is cloned into yeast two-hybrid bait vector pLexA (Clontech) to generate a LexA-FtsZ fusion with DNA-binding property. Alloiococcus ftsA is cloned into the target vector pB42AD (Clontech) to fuse FtsA to the activating domain. Both plasmids are then transformed into a Saccharomycyces cerevisiae strain containing a lacZ reporter under the control of multiple LexA operators. β-Galactosidase activity is determined to quantify relative strength of FtsA-FtsZ interaction.

EXAMPLE 21

ALLOIOCOCCUS OTITIDIS ENCODED FILAMENT ATION TEMPERATURE SENSITIVE GENE Z

(FTSZ)

FtsZ is an essential protein that forms a cytokinetic ring (Z-ring) that drives cell division in bacteria. FtsZ has been identified in most prokaryotic species with the exception of Chlamidia, a Ureaplasma species and Crenarchaea. FtsZ and Z-ring formation are most extensively studied in E. coli. FtsZ is an abundant cytoplasmic protein which is present at ~ 10⁴ copies per cell, and is the first protein to be localized to the division site. Z-ring is required throughout septation and directs the ingrowth of septum in part by recruiting other cell division protein to the division site. Another function is suggested by FtsZ homology to eukaryotic tubulins. Like tubulin, FtsZ is a GTPase and undergoes GTP/GDP-dependent polymerization. Recent studies showed that Z-ring is a very dynamic structure suggesting that GTP-dependent assembly/disassembly of Z-ring might provide constriction force to power cell division. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 83). The protein encoded by the gene is set forth in Seq. ID No. 84.

GTPase activity

FtsZ is a GTPase that contains the tubulin-signature nucleotide-binding motif GGGTGS/TG. Like in DDD-tubulin dimer, the active site for GTP-hydrolysis appears to be shared between two subunits where the GTP-binding pocket is provided by one subunit while the GTPase-activating T7 loop comes from the other subunit. This view is supported by genetic analysis as various mutations that inhibit FtsZ GTPase activity map in the T7-loop region and a conserved Asp-residue in T7-loop is found to be involved in the coordination of the cation involved in GTP hydrolysis. FtsZ GTPase activity is Mg²⁺-dependent and is stimulated by KCl.

Polymerization

In vivo, about 75% of FtsZ is present as multimers. In vitro, FtsZ forms a variety of structures at various conditions. FtsZ assembles into thin protofilaments with GTP and formation of FtsZ polymers is coupled to GTP hydrolysis: when GTP runs out, polymers disassemble. Protofilaments assemble into sheets and bundles in the presence of multimolar amounts of either Mg²⁺ or Ca²⁺ or by addition of DEAE- dextran. In addition, ZipA protein induces bundling of FtsZ polymers. With GDP, FtsZ assembles into curved filaments and minirings.

Interactions with other proteins

In E. coli, at least nine different proteins are localized to the division septum and are required for cell division to proceed. Among them two proteins, ZipA and FtsA, are shown to interact directly with FtsZ. Both of these proteins localize to the division site independently from each other, but require FtsZ for localization. ZipA is an integral membrane protein which is thought to mediate invagination of cell membrane by linking the membrane to constricting Z-ring. Interaction between ZipA and FtsZ is confined to C-terminal portion of ZipA (residues 185-328) and conserved 17-amino acid region on C-terminus of FtsZ. FtsA is an actin-like membrane-associated protein which possesses ATPase activity and might provide energy required for Z-ring dynamics. Interaction between FtsZ and FtsA is not studied in great detail, it is shown that C-terminus of FtsZ is required. The remaining division proteins require both ZipA and FtsA for their localization to Z-ring.

FtsZ as a target for anti-infective development

FtsZ is an essential protein for cell division/bacterial viability. Knock-out ftsZ mutants fail to divide and, as a result, filament and die. The target is widely conserved throughout bacterial kingdom implying that FtsZ-specific inhibitor would have a broad-spectrum antibacterial activity. The potential drawbacks of the target might include the presence and the essential role of a homolog (tubulin) in eukaryotes and an intrinsic difficulty in inhibiting protein-protein interactions by small molecules. Although this target is being studied extensively, no FtsZ-specific compounds are reported up to date.

Assays for measuring FtsZ function

Polymerization of FtsZ is measured by light scattering assay as described previously. FtsZ (12.5 μM) is incubated in 200 μl of polymerization buffer (50 mM MES/NaOH, pH 6.5, 50 mM KCl, 5 mM MgCI₂, 10 mM CaCI₂) in a fluorescence cuvette with a 1 cm path length. The sample is maintained at 30°C, polymerization is induced by addition of 20-500 μM GTP. Light scattering is measured at 90°, both excitation and emission wavelengths are set to 350 nm, slit width is 2 nm. Alternatively, the amount of polymerized FtsZ is analyzed by sedimentation and subsequent quantification of precipitated FtsZ by SDS-PAGE, Coomassie staining and densitometric scanning. In addition, polymers are observed by electron microscopy. This assay represents either primary or secondary/confirmatory assay. GTP binding of FtsZ is monitored by the covalent cross-linking of [γ-³²P]GTP (3000 Ci/mmol) to FtsZ in a previously described competition assay. FtsZ (3 μg) is incubated in 20 μl of 50 mM MES/NaOH, pH 6.5, 100 mM KCl, 4 mM MgCI₂, 1 mM EDTA, 0.1 mM EGTA and 0.5 mM DTT. Various amounts of non-labeled competing nucleotide (GTP or GTP analogs) and 0.1 mM [γ-³²P]GTP are added, samples are incubated at 0°C for 15 min, then UV cross-linked for 5 min and analyzed by SDS- PAGE on 12% gel, autoradiography and densitometric scanning. This assay represents a secondary/confirmatory assay.

The GTP hydrolytic activity of FtsZ is monitored by thin-layer chromatography (TLC) as described previously. Briefly, the reaction mixture consists of 5 mM of [y- ³²P]GTP (40 mCi/mmol), 15 mM magnesium acetate and 0.25-2 mg/ml of FtsZ in reaction buffer (40 mM Tris-acetate, pH7, 200 mM potassium acetate, 2 mM EDTA, 1 mM DTT and 0.5% Triton X-100), aliquots are separated by TLC and amount of GTP converted to GDP is determined by spot-densitometry. Alternatively, GTPase activity is measured either by quantitation of the non-radioactive inorganic phosphate with the malachite green-molybdate reagent as described previously or by quantitation by scintillation counting of radioactive inorganic phosphate released after hydrolysis of [γ-³²P]GTP (26). This assay represents either primary or secondary/confirmatory assay.

Among interactions of FtsZ with various cell division proteins, interaction between FtsZ and ZipA is characterized the best. ZipA -induced bundling of FtsZ is measured by the light scattering assay that is described above, both proteins are used at ≥5 μM.

EXAMPLE 22 ALLOIOCOCCUS OTITIDIS ENCODED GYRA/GYRB (DNA GYRASE, TOPOISOMERASE II)

AND GRLA GRLB (TOPOISOMERASE IV)

DNA topoisomerases: topoisomerases modulate the topological state of DNA in cells. This involves binding to DNA, introducing single or double stranded breaks in the DNA, passing DNA molecules through the break and rejoining the break. This controls the levels of positive and negative supercoiling of DNA and functions in catenation/decatenation. Controlling the topological state of DNA is critical to the fundamental processes of transcription, recombination, replication and partitioning of the chromosome. There are two main categories of topoisomerases, type I and type II. Type I topoisomerases introduce single stranded breaks in DNA whereas type II enzymes introduce double stranded breaks. GyrA/GyrB (gyrase) and GrlA/GrlB (topoisomerase IV) are both type II enzymes that are essential for cell viability.

DNA gyrase (GyrA/GyrB) is a type II topoisomerase that functions to control the degree of supercoiling in double stranded DNA. It is essential for viability and plays central roles in replication, repair, recombination and transcription of DNA. Gyrases have the ability to introduce double stranded breaks in DNA molecules while remaining bound to the DNA through phosphotyrosine bonds, pass uncut DNA through the break and then rejoin the breaks, with repeated cycles being driven by the hydrolysis of ATP. Gyrase has the unique ability to introduce negative supercoils in closed circular DNA and also functions to catenate/decatenate DNA duplexes. The generation of negative supercoiling is important for initial stages in replication. DNA gyrase from Escherichia coli has been studied in detail. It is a complex of two subunits of GyrA (encoded by gyrA) and two subunits of GyrB (encoded by gyrB) (ie. A₂B₂ complex). The subunits are organized in discreet domains. An N-terminal domain of GyrB harbors ATPase activity while the C-terminal domain is thought to interact with the GyrA subunit, and is involved in DNA binding. The N-terminal domain of GyrA is apparently involved in DNA strand breakage-ligation reactions while the C-terminal segment is involved in DNA binding. Crystal structures of the DNA strand breakage/reunion domain of E. coli GyrA, and the N-terminal ATPase domain of E. coli GyrB have been determined. DNA gyrase has also been purified and characterized from gram positive organisms such as S. aureus. Comparison of DNA gyrases from several bacteria reveal a high degree of conservation of important domains. Topoisomerase IV (GrlA/GrlB) is a type II topoisomerase but unlike gyrase it does not possess negative supercoiling activity. Its primary role in replication appears to be in the decatenation of multiply linked daughter chromosomes, important for terminal stages of the replication process. Topoisomerase IV has been purified and characterized from gram negatives eg. E. coli, (where the GrlA/GrlB subunit homologs are designated ParC and ParE), and gram positives eg S. aureus. Homologs of thse gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID Nos 17 and 19). The proteins encoded by the genes are set forth in Seq. ID Nos. 18 and 20. GyrA/GyrB (Gyrase) and GrlA GrlB (topoisomerase IV) as targets for anti- infective development:

Alloiococcus otitidis is an infectious organism associated with disease, and consequently, novel antimicrobials to combat these infections are desirable. DNA gyrase and Topoisomerase IV is essential for bacterial viability and is a well- established and validated antibacterial target.

Purification of DNA gyrase and topoisomerase IV from Alloiococcus otitidis

Genes encoding the GyrA/GyrB and GrlA/GrlB subunits or their functional domains are obtained using polymerase chain reaction amplification of the genomic region encoding them. The genes are then subcloned into standard expression vectors, with or without affinity tags. The enzyme is then overexpressed in Escherichia coli and purified using a standard tag system or conventional chromatography.

Measurement of gyrase and topoisomerase IV by kinetoplast DNA decatenation assay: Type II topoisomerases introduce double stranded breaks in DNA and mediate catenation/decatenation of DNA. Topoisomerase IV activity is readily determined with decatenation assays using as substrate kinetoplast DNA (KDNA) from Crithidia fasciculata. The DNA isolated in this procedure is a highly networked series of catenated double stranded minicircles and is easily be pelleted by centrifugation. The activity of topoisomerase II enzymes results in the release of decatenated DNA minicircles from the networked KDNA. These have a high mobility in agarose gels and migrate into the gel ahead of the networked material, which has very low mobility, allowing for determination of decatenation activity using ethidium bromide stained agarose gel electrophoresis. Alternatively, using radiolabeled KDNA, the level of decatenation activity is measured by counting radioactivity remaining in reaction supernatants following centrifugation to pellet the networked material. Typical conditions used for assaying decatenation activity of S. aureus and E coli topoisomerase IV activity are as follows: C. fasciculata KDNA (0.9 mg/ml) is incubated in 40 μl of reaction buffer (50 mM Tris- HCl, pH 7.7, 5 mM MgCI₂, 5 mM DTT, 50 μg/ml bovine serum albumin, 1.5 mM ATP and 350 mM potassium glutamate) with appropriate amounts of the Grl subunits, for 1 hour at 37° C. If non radiolabeled KDNA is used, these reactions can be stopped and analyzed by agarose gel electrophoresis, or for radioassays, the reaction is stopped by gentle mixing with 10 μl of stop solution (50 % glycerol, 50 mM EDTA (pH 8.0), 2.5 % SDS and 0.1 % bromphenyl blue) and centrifuged at 15 000 x g for 5 min at 20° C. Decatenation activity is determined by counting radioactivity in 25 μl of the supernatant in a scintillation counter. Alternatively, a modified assay employing flow injection fluorometry of 4', 6-diaminidino-2-phenylindole (DAPI) treated supematants has been described that could be suitable for moderate throughput non radioactive assays, or filtration of the reactions through appropriate filters may efficiently separate the decatenated species from KDNA. Although the above described assays were used for topoisomerase IV, modified decatenation reactions using KDNA isolated from Leishmania donovani reveal significant decatenation activity by gyrase from E. coli and Mycobacterium smegmatis, indicating the applicability of the assay to prokaryotic gyrases.

DNA Supercoiling/relaxation assays.

DNA gyrase function is directly assayed using a simple supercoiling assay typified by that described for the measurement of Escherichia coli DNA gyrase activity. Briefly, incubation of relaxed closed circular plasmid DNA (pUC18, 7.5 nM) in the presence of DNA gyrase (approximately 10 nM) in 40 mM Tris-HCl (pH 8.0) buffer containing 25 mM KCl, 4 mM MgCI2, 2.5 mM spermidine and 1.4 mM ATP buffer results in the introduction of supercoils in the plasmid DNA. Changes in DNA supercoiling status are readily observed by the alteration of mobility of the DNA in agarose gels stained with ethidium bromide and comparison to the mobility of relaxed and supercoiled plasmid template. This strategy is employed for screening for DNA gyrase inhibitors.

Topoisomerase IV activity is assayed by measuring relaxation of supercoiled plasmid DNA. A typical relaxation assay used for S. aureus topoisomerase IV activity is as follows: topoisomerase IV enzyme and supercoiled plasmid DNA (pBR322, 0.6 μg) is incubated in 40 μl 50 mM Tris-HCl, pH 7.7, containing 5 mM MgCI₂, 5 mM DTT, 50 μg/ml bovine serum albumin, 1.5 mM ATP, 5 mM spermidine and 20 mM KCl, for 30 min at 37°C. Changes in DNA supercoiling status can be readily observed by the alteration of mobility of the DNA in agarose gels stained with ethidium bromide and comparison to the mobility of relaxed and supercoiled plasmid template

The ATPase activity of topoisomerases is measured using a coupled spectrophotometric ATPase assay described for the GyrB subunit of E. coli. ATPase activity is assayed in 300 μl of 40 mM Tris-HCl (pH 8.0), containing 25 mM KCl, 2.5 mM spermidine, 4 mM MgCI2, 400 μM phosphoenolpyruvate, 250 μM NADH, 3 μl of pyruvate kinase /lactate dehydrogenase mix and ATP (0.5 - 3.5 mM). The reaction is started by the addition of truncated N-terminal derivatives of the GyrB protein (5 μM) containing the ATPase domain. ATPase activity is reflected as a decrease in absorbance of light at 340 nanometer wavelength.

DNA cleavage assay.

Quinolone drugs interfere with the DNA strand breakage-ligation cycle activity of many topoisomerases. Incubation of topoisomerase and linear or supercoiled pBR322 plasmid DNA, or small linear DNA fragments, in the presence of quinolones and magnesium results in the trapping of a complex of topoisomerase, DNA with a double stranded break and the drug. The topoisomerase remains bound to the cleaved DNA, however treatment with a denaturant such as SDS or proteinases remove/degrade the gyrase, releasing the cut DNA. Certain consensus sequences representing preferred cut sites of E. coli gyrase in plasmid pBR322 have been identified in template DNA molecules used in these assays. This assay is useful for mode of action studies of inhibitors of gyrase/topoisomerase IV activity and in particular of the strand breakage-ligation function. Cleavage reactions are performed with linear or supercoiled DNA. A typical cleavage reaction using linear DNA to measure cleavage by E. coli and S. aureus gyrase and topoisomerase IV in the presence of drugs is as follows: gyrase/ topoisomerase IV is incubated in 20 μl 25 mM Tris-HCl (pH 7.5) containing 0.5 mM EDTA, 0.5 mM DTT, 3 μg bovine serum albumin per ml, 10 mM MgCI₂, 120 mM KCL 10 mM ATP, 10 000 dpm of 3' end labeled linear pBR322 plasmid DNA and drug for 1 hour at 37°C. (Note: for S. aureus, KCl is replaced with 0.7 M potassium glutamate). Reactions are terminated by adding 5 μl 2.5% SDS-2.5 mg proteinase K per ml and incubating at 37°C for 30 minute, then adding 5 μl 30% glycerol-1% SDS-50 mM EDTA-0.05 % bromophenol blue. Cleavage products are resolved on 1% agarose gels and visualized by autoradiography.

Additional cleavage assays are also used that measure 1 ) the linearization of supercoiled plasmid DNA (pBR322), with linearization measured using scanning densitometry of DNA species separated on 1 % agarose gels, or 2) the cleavage of small linear DNA molecules of approximately 100 bp encompassing the preferred cleavage sequence 5'- GGCTGGATGGCCTTCCCCAT - 3' from position 990 in plasmid pBR322. In the latter case, the fragment is produced by PCR and radiolabeled with γ-³²P ATP at the 5' end of the top strand. This DNA is incubated with 1.3 pmol DNA gyrase in a total volume of 10 μl 35 mM Tris-HCl (pH 8.0), 24 mM KCl, 2 mM spermidine, 4 mM MgCI2 and inhibitor compound at 37°C for 10 min. Reactions are stopped by addition of 8 mM EDTA and 1 % SDS, then treated with 500 μg/ml proteinase K for 2 hours at 37°C. The DNA is then cleaned by phenol- chloroform extraction and ethanol precipitation, resuspended in TE buffer (pH 8.0), and loaded and resolved on 12 % sequencing gels containing 7M urea. In the presence of inhibitors of the strand breakage-ligation function, radioactive cleavage products are detectable by autoradiography. Modifications of this assay whereby one strand of the DNA substrate is labeled with an affinity tag such as biotin and the other is radiolabeled or fluorescently labeled should facilitate rapid separation and detection of cleavage products using streptavidin coated columns or plates, resulting in higher assay throughput.

GYRASE ACTIVITY ASSAYS: DNA REPLICATION:

Early work by Fuller and Kornberg revealed that a partially purified crude soluble fraction derived from Escherichia coli cells (designated fraction II) contained the components necessary for replication of plasmids containing oriC (E. coli chromosomal origin of replication). Replication mediated by this fraction specifically required supercoiled plasmids. Although the exact makeup of the protein complex mediating the replication was not known, the replication reaction was inhibited by 1 ) rifampicin, and 2) nalidixic acid and novobiocin, indicating essential roles for both RNA polymerase and DNA gyrase, respectively. Subsequently the reaction was reproduced using replication machinery reconstituted from purified protein HU, DnaA, DnaC, DnaB, single stranded binding protein (SSB), primase, DNA polymerase holoenzyme, RNA polymerase holoenzyme and GyrA/GyrB.

The requirement for gyrase activity for replication is exploited for the identification of gyrase inhibitors using a replication-based high throughput screen. Gyrase specific inhibitors are identified from the overall pool of replication inhibitors using the secondary assays detailed below. Screening for inhibitors of gyrase in a setting where gyrase is participating in an overall reaction that is essential in bacteria might better select physiologically relevant inhibitors

An assay suitable for high throughput screening of inhibitors of replication (including gyrase and DnaA inhibitors) is based on the replication reaction of Kaguna and Kornberg. This reaction was set up as follows; standard reaction in 25 μl: 40 mM Hepes (pH 7.6), 2 mM ATP, 0.5 mM GTP, CTP and UTP, 50 μg/ml bovine serum albumin, 6 mM phospho creatine, 100μM dATP, dGTP, dCTP and dTTP, γ-³³P dTTP (50-150 cpm/pmol total nucleotides) 1 1 mM magnesium acetate,100 μg/mL creatine kinase,85 ng SSB, 48 ng DnaB, 40 ng DnaC, 20 ng primase, 160 ng DNA polymerase III holoenzyme, 800 ng RNA polymerase, 150 ng GyrA, 350 ng GyrB, 120 ng DnaA, 2.5 units topoisomerase 1, 190 ng HU, 0.15 ng Rnase H 200 ng supercoiled plasmid template. The reaction is assembled at 0 °C and initiated by incubation at 30°C. Replication reactions are terminated by the addition of EDTA to 20 mM. Incorporation of nucleotides into DNA is measured by filtration through 96 well DEAE filter plates and counting retained radioactivity.

Compounds inhibiting gyrase activity in Alloiococcus otitidis are found as part of a larger program directed at replication. This reaction described above uses the replication machinery of a gram-negative organism, which differs somewhat from the replication machinery of gram positives such as Staphylococcus aureus with respect to the specific protein subunits involved. Therefore a similar system specific to Alloiococcus otitidis is assembled from the relevant proteins purified from Alloiococcus otitidis. Several techniques are then utilized to determine the interaction of inhibitors with Gyr A and GyrB. These are described in the literature and include a) Nuclear magnetic resonance; and b) Capillary electrophoresis. Example 23 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYMES MURA

Bacterial cell wall peptidoglycan (murein) is a large macromolecule of periodic structure whose basic unit, a disaccharide-peptapeptide, is polymerized linearly via the disaccharide motif and cross-linked laterally via the peptide motif. The process of bacteria cell wall biosynthesis starts from the transferase MurA, which transfers the addition of an enolpyruvyl moiety to the 3'-hydroxyl-UDP-N-acetyl glycosamine (UDP-GluNAc). Subsequently, the reductase MurB reduces the enol ether to the lactyl ether, utilize one equiv. of NADPH and a solvent proton to form UDP-Λ/-acetyl muramic acid (UDP-MurNAc). Next a series of ATP dependent amino acid ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the pentapeptide side chain using the newly synthesized carboxylate as the first acceptor site. Each enzyme is responsible for the addition of one more residue except MurF, catalyzes D-ala-D-ala. MurE in gram negative bacteria catalyzes the meso-2, 6- diaminopimelate (DAP), while in gram positive bacteria MurE catalyzes L-lysine. The product of MurF, UDP-NAM pendapeptide is the final product of the cytoplasm enzymes and is the most important precusor for further peptidoglycan biosynthesis. UDP-MurNAc pendapeptide is then and catalyzed at the plasma membrane by the membrane bound enzymes such as the translocase MraY and transferase MurG.

UDP-Λ/-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in bacterial cell wall biosynthesis. The enzyme transfers an enolpyruvyl group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDP-GluNAc) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. MurA product enolpyruvate UDP-Λ/-acetylglucosamine (EP-UNAG) is a precursor to UDP- N-acetylmuramate (UDP-MurNAc), an essential building block for the bacterial cell wall. MurA is conserved across both gram-positive and gram-negative bacterial species: gram- negative bacteria have one copy of the murA and gram-positive bacteria have two copies. Alloiococcus otitidis murk was identified as described in Example 5/Table 4 and its genomic structure set forth in Seq. ID No. 101. The amino acid sequence of the protein encoded by this gene is set out in Seq. Id No. 102. Alloiococcus otitidis murA as a target for anti-infective development

MurA in E. coli and Streptococcus pneumoniae has been shown to be essential by gene deletion technique. The essentiality of MurA in gram-positive bacteria such as Streptococcus pneumoniae was demonstrated in that its deletion is fetal. No mammalian homolog to MurA has been reported. MurA is specifically inhibited by the natural product antibiotic fosfomycin. Thus the importance of MurA in peptidoglycan biosynthesis makes it an attractive target for the design of novel antibacterial agent.

Assays for measuring MurA function

Phosphate detection:

MurA activity is detected by quantitating the UDP-GluNAc-dependent Pi from PEP and assayed by Lanzetta's malachite Green-ammonium molybdate assay. Pi is quantitated by measuring the optical density at A660 nm.

Coupled assay with MurB:

A coupled assay in access of MurB, which reduces the MurA product EP- UNAG G to UDP-MurNAc, couples the MurA transferase activity with NADPH oxidation. The oxidation of NADPH is monitored at 340 nm and is stoichometric with the production of EP-UNAG.

Fluorescence experiments

Fluorescence experiments to detect murA are performed using the hydrophobic fluorescence probe 8-anilino-1 -naphthalene sulfonate (ANS). The fluorescence quenching of MurA/ANS solutions upon addition of UDP-GlcNAc or pyruvate-P is concentration dependent and in a saturating manner.

Isothermal titration calorimetry The binding of UDP-GluNAc to MurA is studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDP-GluNAc and also in its absence as demonstrated by MALDI mass spectrometry. Novel Fosfomycin analogs and other antibiotics that bind to murA are also identifiable using isothermal titration chemistry.

Capillary electrophoresis-based enzyme assay A capillary electrophoresis-based enzyme assay for MurA is described by Dai and colleagues . This method, based on UV detection, provides baseline separation of one of the reaction products, EP-UNAG, from substrates PEP and UDP-GlcNAc within 4 min. The other product, phosphate, is not detectable by UV at 200 nm. Quantitation of individual components, substrates or product, is be accomplished based on the separated peaks. This assay is also used to detect novel antibiotics, which inhibit murA activity.

EXAMPLE 23 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYMES MURB

MurB, the UDP-Λ/-acetyl enolpyruvyl glucosamine reductase, commits the second step of bacterial cell wall biosynthesis in cytoplasm and is responsible for the reduction of the enol ether to the lactyl ether, utilizes one equiv. of NADPH and a solvent proton. The product of MurB is UDP-N-acetylmuramic acid (UDP-MurNAc), the linker of the peptide and glycan portions of cell wall precursor UDP muramyl-pentapeptide. MurB from E. coli is a 342 amino acid protein, which has a distinctive yellow color characteristic of bound flavin as its co-factor. The biochemistry characterization and X-ray crystal structure of MurB in E. coli, in Staphylococcus aureus and Streptococcus pneumoniae have been studied extensively. The gene Alloiococcus oitidis murB was identified as disclosed as described in Example 5, and is set out in Seq. ID No. 39. The amino acid sequence of the protein encoded by this gene is set out in Seq. ID No. 40.

Alloiococcus oitidis murB as a target for anti-infective development

The essentiality and unique function of MurB in prokaryotic cells and the absence of homologue in eukaryotic cells make it an attractive novel antibacterial target. To date, no small molecule inhibitors of MurB have been reported. Alloiococcis oititidis ORF-1263 (murB ) (Seq. ID No. 39) encodes enzyme UDP-Λ/-acetylenolpyruvylglucosamine Reductase (MurB) as shown by sequence homology.

Assays for measuring MurB activity

Spectrophotometric assay monitoring NADPH consumption:

MurB activity is typically monitored by its biochemical reaction in which NADPH reduces the bound FAD and resulting decrease in absorbance at 340 nm. Enzyme is maximally activated in the presence of K+, NH⁴ at cation concentrations between 10-50 mM.

Coupled assay with MurC:

In designing an end point assay for high through put screen (HTS), a novel coupled assay in access of UDP-MurNAc L-alanine synthase (MurC) was developed at Wyeth. This assay utilizes the biochemically synthesized MurA product EP-UNAG as substrate, coupled with limited MurB and excess MurC in the reaction with all other substrates/components involved. In this assay, MurB is responsible for the reduction of the enol ether to the lactyl ether, and the follow up enzyme MurC catalyzes the ATP dependent ligation of the first of the five amino acids of UDP- peptapeptide with a release of one molecule of phosphate. After 60 minutes of incubation, color reagent malachite green was added and phosphate was detected spectrophotometrically.

Fluorescence binding assay A fluorescence method developed at Wyeth is used to determine the binding potency (Kd value), stoichiometry and nature of binding site of substrates and inhibitors interactions with MurB enzymes. This assay is based on changes in intrinsic fluorescence of inhibitor and/or enzyme, upon formation of enzyme-inhibitor complex. Oxidized form of MurB consists of two fluorescent groups, namely tryptophan residues and the cofactor FAD. Upon binding inhibitor or substrate, local changes in the solvent environment of these groups or overall conformational and electronic changes occur in the enzyme due to which the fluorescence emission is altered. For instance, inhibitor binding significantly quenched the fluorescence and altered the solvent environment of FAD to a less polar environment. The changes in the fluorescence of the FAD moiety are used to estimate binding constants for MurB inhibitors. Binding experiments are set up in which a fixed concentration of enzyme is titrated with increasing concentrations of the inhibitor. In typical inhibitor binding experiments, the fluorescence emission of the FAD moiety is quenched due to specific interactions of the inhibitor with MurB enzymes and the binding site was saturated at micromolar concentrations of inhibitor. The changes in the fluorescence are fitted to mathematical binding models to determine binding affinity.

Temperature-jump isothermal denaturation procedure

Temperature-jump isothermal denaturation procedure with various methods of detection is used to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism- as well as differential scanning calorimetry are used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence are used to quantitate the dose dependencies of the ligand effects.

NMR studies

NMR studies are performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms are assigned for 334 residues (96%). For NADP+-complexed MurB, one or more backbone atoms are assigned for 313 residues. The strategies used for obtaining resonance assignments are known. Localizing the NADP+ binding site on the MurB enzyme is also studied by NMR methodology.

EXAMPLE 25 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYME, MURC

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the third chemical step of bacterial cell wall biosynthesis. This enzyme is a nonribosomal peptide ligase which utilize ATP to form an amide bond between L-alanine and UDP- N-acetylmuramic acid (UDP-MurNAc). This ATP-dependent ligation adds the first of five amino acids to the sugar moiety of the peptidoglycan precursor. Also, in this reaction, ATP is converted to ADP with release of one molecule of inorganic phosphate. Thus MurC reaction is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. The genetic, biochemistry analysis and crystal graphic studies of MurC in gram-negative bacteria E. coli have been extensively studied. Characterizations of MurC in other pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa have also been documented.

Alloiococcis otitidis encoded MurC as a target for anti-infective development

The Alloiococcis otitidis ORF-2602 (murC, Seq. ID No. 95) encodes enzyme UDP-MurNAc:L-alanine ligase (MurC) as determined by sequence homology. This enzyme presents a target for the development of novel anti-infectives to treat the disease(s) caused by this pathogen. Novel compounds identified using combinatorial chemistries are assayed for their inhibitory effect on MurC activity using one of the asssays set out below.

Assays for measuring MurC activity Spectrophotometric assay detecting phosphate release:

MurC activity is detected by the inorganic phosphate production. Typically the reaction mixture contains substrates ATP, L-alanine, UDP-MurNAc, DTT, MgCI₂ and MurC enzyme. After 20 minutes incubation, the reaction is quenched with the addition of malachite Green-ammonium molybdate for a colored reaction. Absorbance at 660 nm is read 5 minutes after the quench. Absorbance values are converted to concentration of Pi with standard curves using KH₂PO₄, which is prepared under identical conditions without the enzyme MurC.

Spectrophotometric assay detecting formation of ADP Due to the conversion of ATP to ADP in MurC reaction, the production of

ADP is monitored in coupled enzymes spectrophotometrically. In this reaction, in addition to MurC substrate UDP-MurNAc, L-alanine and ATP, NADH, phosphoenolpyruvate, MgCI₂ and (NH₄)₂SO₄, two other coupled enzymes pyruvate kinase and lactase dehydrogenase are also presented. Reaction mixtures without ATP and MurC are incubated at 37°C for 10 min before ATP is added for another minute. Reaction is then started by the addition of MurC. The decrease of NADH absorbance at 340 nm is monitored spectrophotometrically. One unit of activity corresponds to 1 umol of ADP formed per hour.

L-Alanine radio-labeled assay:

The MurC enzyme activity in this assay is measured as endpoint using ¹⁴C-L- alanine and ATP incubated with MgCI₂, and (NH₄)₂SO₄ in 100 mM Tris/HCI, pH 8.0. Reaction is initiated by the addition of the catalytic amounts of MurC. Samples of the reaction mixture are then mixed with glacial acetic acid and then stored at 4°C. Remaining ¹⁴C -L-alanine is separated from ¹⁴C -UDPMurNAc on SCX columns run under vacuum. Quenched reaction samples are supplemented with equilibration buffer and counted using a liquid scintillation counter.

EXAMPLE 26 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYMES MURD

Bacterial UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD), a cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes the fourth step of bacterial cell wall biosynthesis. In this reaction, MurD catalyzes ATP-dependent addition of D- glutamate to an alanyl residue of the UDP-N-acetylmuramyl-L-alanine (UDP- MurNAc-L-Ala) precursor, generating the UDP-MurNAc-dipeptide. The formation of a peptide linkage between the amino function of D-glutamate and the carboxy terminius of UDP-N-acetylmuramuamyl-L-alanine is generated through this reaction. The stoichiometric consumption of ATP supplies the energy needed for this peptide bond formation with concomitant generation of ADP and orthophosphate. The murD genes were cloned and characterized from gram-positive bacteria of Staphylococcus aureus and Streptococcus pyogenes, and gram-negative bacteria from Escherichia coli, Haemophilus influenzae, Bacillus subtilis. Structures of MurD from E. coli and MurD complexed with its substrate UDP-MurNAc-L-Ala have been solved to 2.0 A resolution. The role of specific amino acids at the active site of MurD have been extensively studied using the ortholog and paralog amino acid invariants. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 89). The protein encoded by the gene is set forth in Seq. ID No. 90.

Alloiococcus otitidis encoded MurD as a target for anti-infective development

Due to its high specificity and essentiality, MurD is an attractive target for the development of novel antimicrobial agents. Alloiococcis otitidis ORF-2494, by sequence homology, has been shown to encode enzyme UDP-N-acetylmuramyl-L- alanine:D-glutamate ligase (MurD) (Seq. ID. No. 89). Inhibition of MurD activity is used to identify novel antimicrobial agents.

Assays for measuring MurD activity

Spectrophotometric assay detecting phosphate release: MurD activity in the presence or absence of a putative inhibitory molecule of

MurD is detected by the orthophosphate production in test tube or in 96-well format. Typically the reaction mixture contains substrates ATP, D-glutamine, UDP-MurNAc- L-Ala, DTT, MgCI2 and MurD enzyme. After 20 minutes incubation, the reaction is quenched with the addition of malachite Green-ammonium molybdate for a colored reaction. Absorbance at 660 nm is read 5 minutes after the quench using Molecular Devices SpectraMax 250 plate reader. Absorbance values are converted to concentration of Pi using orthophosphate standards, which are prepared under identical conditions without the enzyme MurD.

Spectrophotometric assay for detecting formation of ADP in the presence or absence of a putative inhibitory mollecule of MurD:

Due to the conversion of ATP to ADP in MurD reaction, the production of ADP is monitored with coupled enzymes of pyruvate kinase and lactase dehydrogenase spectrophotometrically. In this reaction, in addition to MurD substrate UDP-MurNAc-L-ala and ATP, MgCI₂ and (NH₄)₂SO₄, there is also in significant access of NADH, phosphoenolpyruvate, and two coupled enzymes pyruvate kinase and lactase dehydrogenase. This protocol monitors ADP formation in the MurD catalyzed reaction, in the presence or absence of a putative inhibitory mollecule of MurD, by the decrease of NADH absorbance at 340 nm.

L-Glutamate radio-labeled assay:

The MurD enzyme activity in the presence or absence of putative inhibitors of MurD is also measurable using D-¹⁴C- glutamate as an endpoint assay. The reaction mixture contains D-¹⁴C- glutamate UDP-MurNAc-L-Ala, ATP, MgCI₂, (NH₄)₂SO₄ in 100 mM Tris/HCI, pH 8.0. An HPLC assay with online UV and flow scintillation detects the formation of UDP-MurNAc-L-Ala-D-¹⁴C Glu and ADP in each reaction.

EXAMPLE 27 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYME, MURE

The fifth step in the cytoplasmic peptidoglycan biosynthetic is catalyzed by MurE. In this step, the monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L- alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso- diaminopimelic acid or L-lysine, respectively. Therefore, MurE from E. coli is the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-diaminopimelic acid ligase, and MurE from S. aureus is the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase. Thus represents the major difference of MurE from other murein enzymes in cytoplasm. The amino acid residues catalyzed by MurE plays a key role in the integrity of sacculus since it is directly involved in the peptide cross-linkage. MurE reaction is also ATP-dependent, which supplies the energy needed for the peptide bond formation with concomitant generation of ADP and orthophosphate.

The essentiality of MurE has been well documented in E. coli, in S. aureus, as well as other pathogens such as Haemophilis influenzae, Vibrio cholerae and Corynebacterium glutamicum. Gene murE has been shown to be essential in bacteria. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 25). The protein encoded by the gene is set forth in Seq. ID No. 26.

Alloiococcus otitidis MurE as a target for anti-infective development Alloiococcis otitidis ORF-851 , by sequence homology encodes enzyme UDP- N-acetylmuramyl-L-alanine-D-glutamate ligase: meso-diaminopimelic acid/or L- Lysine (MurE) (Seq. ID No 25). MurE activity in the presence or absence of a putative inhibitory molecule of MurE activity is used to identify novel antimicrobial I agents, which may be used ti treat disease caused by Alloiococcis otitidis.

Assays for measuring MurE activity

Radio labeled substrate assay: meso-A2pm-adding activity Activity of MurE from Alloiococcis otitidis in the presence or absence of a putative inhibitory molecule of MurE activity is measured by using radio-labeled meso-¹⁴C A2pm mixing with ATP, MgCI_2, UDP-MurNAc-L-Ala-D-Glu, DTT in 100 mM Tris/HCI and MurE from Alloiococcis otitidis .

Radio labeled substrate assay: L-lysine adding activity

Activity of MurE from Alloiococcis otitidis in the presence or absence of a putative inhibitory molecule of MurE activity is measured by using radio-labeled UDP- MurNAc-L-Ala-D-14C-Glu mixing with ATP, MgCI_2, DTT, L-lysine in 100 mM Tris/HCI and MurE from Alloiococcis otitidis. In both cases, mixtures are incubated at 37°C for 30 min, and reactions stopped by the addition of acetic acid. Reaction product is separated by high votage electrophoresis in 2% formic acid for 45 min. The radio active spots corresponding to substrate and reaction product are detected by overnight autoradiography, or with radio scanner. The spots are also cut out and counted using liquid scintillation counter.

Example 28

ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYME, MURF

The D-alanyl-D-alanine-adding enzyme MurF encoded by the murF gene catalyzes is the last step of the cytoplasmic peptidoglycan biosynthesis. MurF performs the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu- meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-pentapeptide). The product of MurF, UDP-MurNAc pendapeptide, is the final product of the cytoplasm enzymes and is the most important precusor for further peptidoglycan biosynthesis. UDP-MurNAc pendapeptide is then catalyzed by the plasma membrane bound enzymes such as the translocase MraY and transferase MurG. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 3). The protein encoded by the gene is set forth in Seq. ID No. 4.

Alloiococcus otitidis MurF as a target for anti-infective development

Due to its high specificity, essentiality, and importance of its product UDP- MurNAc pentapeptide, MurF is attractive as. an antibacterial target. The Alloiococcis otitidis ORF-48, by sequence homology,encodes enzyme UDP-N-acetylmuramyl-L- alanine-D-glutamate ligase: meso-diaminopimelic acid/or L-Lysine -alanyl-D-alanine- adding enzyme (MurF) (Seq. ID No. 3). MurF activity in the presence or absence of a putative inhibitory molecule of MurF activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcis otitidis.

Assays for measuring MurF activity

Spectrophotometric assay detecting phosphate release:

Activity of MurF from Alloiococcis otitidis in the presence or absence of a putative inhibitory molecule of MurF activity is detected by the inorganic phosphate release in the ATP dependent MurF reaction. This assay detects nonomole amount of Pi in the reaction mixture contains substrates ATP, D-ala-D-ala, UDP-MurNAc- tripeptide, DTT, MgCI₂and MurF enzyme. After 5 minutes incubation, the reaction is quenched with the addition of malachite Green-ammonium molybdate for a colored reaction.

Coupled spectrophotometric assay detecting formation of ADP

Due to the conversion of ATP to ADP in MurF reaction, the production of ADP in the presence or absence of a putative inhibitory molecule of MurF activity, is monitored with coupled enzymes of pyruvate kinase and lactase dehydrogenase spectrophotometrically. In this reaction, the decrease at 340 nm is observed as NADP is consumed in MurF reaction process. The reaction typically contains tris buffer, substrates ATP, D-ala-D-ala, UDP-MurNAc-tripeptide, DTT, MgCI₂, phosphoenopyruvate, NADPH and MurF enzyme.

EXAMPLE 29 ALLOIOCOCCUS OTITIDIS ENCODED CELL WALL BIOSYNTHETIC ENZYME, MURG

MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase. MurG catalyzes the transfer of Λ/-acetyl glucosamine from UDP to the C4 hydroxyl of a lipid-linked N- acetyl muramic acid derivative (lipid I) to form lipid II. Lipid II is a linked disaccharide that is the minimal subunit of peptidoglycan. Once lipid II is formed, this disaccharide is translocated across the bacterial membrane where it is polymerized and cross- linked to form the peptidoglycan layers. MurG has been shown to be essential for bacterial survival. The inactivation of MurG gene rapidly inhibits peptidoglycan synthesis in exponential growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 87). The protein encoded by the gene is set forth in Seq. ID No. 88.

Alloiococcus otitidis MurG as a target for anti-infective development

MurG is shown to be associated with the inner face of cytoplasmic membrane, and establishing that the entire peptidoglycan monomer unit assembled before being transferred across the membrane. MurG is a key enzyme at the border line between cytoplasmic and membrane of pepdidoglycan synthesis, thus makes it an attractive target for novel antibacterial agent. Further, no mammalian analogues of MurG have been identified. Due to its high specificity, essentiality, and importance, MurG is attractive as an antibacterial target.

The Alloiococcis otitidis ORF-2492 has been shown to encode, by sequence homology, glycosyltransferase (MurG) (Seq. ID No ). MurG activity in the presence or absence of a putative inhibitory molecule of MurG activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcis otitidis. Assays for measuring MurG function

Radiolabeled reaction

Activity of MurG from Alloiococcis otitidis in the presence or absence of a putative inhibitory molecule of MurG activity is measured by using ¹⁴C labeled N- UDP-GluNAc in the reaction containing UDP-MurNAc-pentapeptide, MgCI₂, ATP and MurG protein. The reaction is stopped after 30 min incubation and by boiling for 3 min. The reaction mixtures are applied to a Whatman I filter paper and subject to descending chromatography overnight. Radioactivity is located and countered with a scanner. This assay is also used to identify the specificity of inhibitor of MraY or MurG, based on the detection of radiolabeled ¹⁴C GluNAc incorporated into membrane precursors.

Fluorometric assay Based on the decrease in NADPH fluorescence at 465 nm, MurG reaction is also monitored in a reaction mixture of HEPES buffer, MgCI₂, Triton, phosphoenolpyruvate, and coupled enzymes of lactic dehydrogenase and pyruvate kinase, UDP-GluNAc and synthesized lipid I analogue in the presence or absence of putative inhibitors of MurG activity. One micromolar UDP corresponds to 500- fluorescence unit under the instrument setting.

EXAMPLE 30 ALLOIOCOCCUS OTITIDIS ENCODED BY HMG CoA REDUCTASE (MVAA)

Two pathways for isopentenyl diphosphate (IPP) synthesis have been described in bacteria: the mevalonate pathway and the non-mevalonate (MEP or GAP-pyruvate) pathway. The mevalonate pathway predominates in the archaebacteria, gram-positive organisms, yeast and mammals; whereas the MEP pathway is found in gram-negative organisms, B. subtilis, chlamydia, and mycobacterium. The first HMG CoA reductase gene to be sequenced was cloned from P. mevalonii, in which HMG CoA reductase permits growth on mevalonate as a sole carbon source. A number of genes of the mevalonate pathway were identified in S. aureus, S, epidermidis, S. pyogenes, S. pneumoniae, E. faecalis and E. faecium. One of the genes, which encodes for HMG-CoA reductase (mvaA), when deleted severely attenuated for virulence in a mouse model indicating that mvaA is essential. Due to its high specificity, essentiality, and importance, mvaA is attractive as an antibacterial target. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 37). The protein encoded by the gene is set forth in Seq. ID No. 38.

HMG-CoA reductase (MvaA) as a target for anti-infective development

The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, HMG-CoA reductase (mvaA) (Seq. ID No 37). MvaA activity in the presence or absence of a putative inhibitory molecule of HMG-CoA reductase (mvaA) activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.

Assays for measuring HMG-CoA reductase (mvaA) activity

MvaA is purified by standard methods using widely available molecular tags following expression at high level from E. coli. Enzymatic activity is monitored in the presence or absence of a putative inhibitory molecule of HMG-CoA reductase activity by following oxidation of NADPH to NADP spectrophotometrically at 340 nm. The assay is carried out in the following buffer: 0.25 mM NADPH, 0.25 mM HMG-CoA, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 25 mM KH₂PO₄ (pH 7.5). The assay is amenable to HTS in high density screening microtiter plates.

Forward reaction: Activity of HMG-CoA reductase (mvaA) from Alloiococcus otitidis in the presence or absence of a putative inhibitory molecule of HMG-CoA reductase activity is measured by reductive deacylation of HMG-CoA to mevalonate as measured the consumption of NADPH to NADP. Unlike other class II HMG Coa reductases, MvaA from Alloiococcus otitidis, like S. aureus, can use either NADPH or NADH cofactor in the reaction. The following kinetic data describe the reaction: K_m(HMGCoA) = 40 μM, K_m(NADp_H) = 70 μM, K_m(NADp) = 100 μM (12). This assay is inhibitable by the statin drug fluvastatin; the Ki was measured at 320 μM, which is four orders of magnitude higher than the Ki for class I HMG-Coa reductases. Reverse reaction: The oxidative acylation of mevalonate to HMG-CoA in the presence or absence of a putative inhibitory molecule of HMG-CoA reductase activity is also monitored. The following kinetic data describes the reaction: K_m(_mevaio_nate) = 670 μM, K_m(GoAsH) = 390 μM, K_m(NADP) = 580 μM (12).

EXAMPLE 31

ALLOIOCOCCUS OTITIDIS ENCODED DIPHOSPHOMEVALONATE DECARBOXYLASE (Mv D)

Diphosphomevalonate decarboxylase, encoded by mvaD, the final enzyme acting in the mevalonate pathway of IPP synthesis was cloned from S. aureus by Wilding ef a/ in 2000. Insertional inactivation of mvaD could only be accomplished when the strains were supplemented with mevalonate, indicating that mvaD is essential. The final step of the mevalonate pathway leading to IPP is the decarboxylation and dehydration of mevalonate-5-pyrophosphate to form isopentenyl diphosphate by MvaD (diphosphomevalonate decarboxylase).

MvaD homologues are well represented in gram-positive organisms (10). Phylogenetic analysis revealed that the cluster of gram-positive enzymes (39-80% identity) were well separated from the eukaryotic homologues, suggesting utility as an antibacterial target. The Alloiococcis otitidis ORF- 1275b has been shown to encode, by sequence homology, diphosphomevalonate decarboxylase (MvaD (Seq. ID No. 43). MvaD activity in the presence or absence of a putative inhibitory molecule of diphosphomevalonate decarboxylase (MvaD activity is used to identify novel antimicrobial agents, which may be used to treat the disease(s) caused by Alloiococcus otitidis. The protein encoded by the gene is set forth in Seq. ID No. 44.

Example 32 ALLOIOCOCCUS OTITIDIS ENCODED HMG CoA SYNTHASE (MVAS)

The second step of the mevalonate pathway leading to IPP is the irreversible condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA by MvaS (HMG CoA synthase). It has been shown that mvaS knockout mutant of S. pneumoniae was attenuated for virulence. Due to its high specificity, essentiality, and importance, mvaS is attractive as an antibacterial target. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 35). The protein encoded by the gene is set forth in Seq. ID No. 36.

HMG COA SYNTHASE (MVAS) AS A TARGET FOR ANTI-INFECTIVE DEVELOPMENT

The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, MvaS (HMG CoA synthase) (Seq. ID No. 35). MvaS activity in the presence or absence of a putative inhibitory molecule of HMG-CoA synthase (mvaS) activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.

Assays for measuring MvaS function

MvaS is purified by standard methods using widely available molecular tags following expression at high level from E. coli. HMG-CoA synthase activity in the presence or absence of a putative inhibitory molecule of HMG-CoA synthase (mvaS) is assayed by measuring the loss of the enolate form of acetoacetyl-CoA spectrophotometrically. The reaction is carried out in a buffer containing 50 mM Tris (pH 9.75), 5.0 mM MgCI₂, 500 μM acetyl-CoA, 20 μM acetoacetyl-CoA and enzyme. The enolate formed is monitored at 302 nm; therefore, as the acetoacetyl-CoA is consumed the signal is depleted. Using this assay the following kinetic data is measured: K_m(acetyi-co_A) = 350 μM; K_m ^app _(acetoaoe_tyi-c_oA) = 10 μM. This assay is amenable to HTS in high- high density screening microtiter plates. Example 33

ALLOIOCOCCUS OTITIDIS ENCODED NICOTINAMIDE ADENINE DINUCLEOTIDE ADENYLYL

TRANSFERASE (NADD)

Nicotinamide adenine dinucleotide (NAD) is an essential molecule in all living cells. NAD is synthesized via a multi-step de novo pathway or via a pyridine salvage pathway. The enzyme nicotinic acid mononucleotide adenylyl transferase (NaMN AT, EC2.7.7.18) catalyzes the conversion of ATP and nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD). The nadD gene, encoding bacterial NaMN AT, is essential for NAD biosynthesis and bacterial cell survival. NadD contains well-conserved the nucleotidyl transferase consensus sequence (GXFXXXHXGH). The adenylyl transferase encoded by the nadD gene prefers NaMN over nicotinomide mononucleotide (NMN) as substrate. Due to its high specificity, essentiality, and importance, nadD is attractive as an antibacterial target. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 91). The protein encoded by the gene is set forth in Seq. ID No. 92.

NICOTINAMIDE ADENINE DINUCLEOTIDE ADENYLYL TRANSFERASE (NADD) AS A TARGET FOR ANTI-INFECTIVE DEVELOPMENT

The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, niotinomide adenine dinucleotide adenyl transferase (NadD) (Seq. ID No. 91). NadD activity in the presence or absence of a putative inhibitory molecule of NadD activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.

Assays for measuring NadD function Discontinuous assay NadD activity in Alloiococcus otitidis is measured in the presence or absence of a putative inhibitory molecule of NadD activity. NadD converts nicotinic acid mononucleotide (NaMN) and adenosine triphosphate (ATP) to nicotinic acid dinucleotide (NaAD) and pyrophosphate (PP,). Each PPi molecule produced by the NadD reaction is then converted to two phosphate (Pi) molecules in the presence of inorganic pyrophosphatase (PPase). The Pi molecules present are quantitated with a malachite green reagent at 660 nm.

HPLC-based assay: Enzyme activity is measured by HPLC quantitation of the reaction products. A neutralized aliquots from the reaction described above was injected into an HPLC system utilizing a 250 x4.6 mm Supelcosil LC-18 5μm reversed-phase column. The elution conditions: 9 min at 100% buffer A (0.1 M potassium phosphate buffer, pH6.0,6 min at up to 12% buffer B (buffer a, containing 20% methanol, 2.5 min at up to 45% buffer B, 2.5 min at up to 100% buffer B, and hold at 100% buffer B for 5.5 min. The eluate absorbance was monitored at 254 nm.

Continuous assay

In bacteria, NadD combines nicotinic acid mononucleotide (NaMN) and adenosine triphosphate (ATP) to form nicotinic acid adenine dinucleotide (NaAD). NadE then converts NaAD into nicotinamide adenine dinucleotide (NAD) in the presence of ammonia and ATP. In the assay, the NAD product is reduced to NADH with alcohol dehydrogenase (ADH) and ethanol, thus permitting direct spectrometric detection of NADH at 340 nm wavelength. The coupled reaction above also includes inorganic pyrophosphatase (PPase) to prevent accumulation of the pyrophosphate byproduct from the consumption of ATP.

EXAMPLE 34

ALLOIOCOCCUS OTITIDIS ENCODED NICOTINAMIDE ADENINE DINUCLEOTIDE SYNTHASE

(NADE)

NAD is a central compound in cellular metabolism. The final metabolic step in the pathway is conversion of nicotinamide adenine dinucleotide - product of NadD reaction -to NAD, a step catalyzed by the enzyme NAD synthetase (NadE). NaMN - substrate for NadD - can be formed by three different enzymatic reactions: in the de novo pathway from quinolinate, in Preiss-Handler salvage pathway from nicotinic acid, and in the nucleoside salvage pathway by deamindation of nicotinamide mononucleotide. In bacteria, there are no known alternatives for the metabolic steps between NaMN and NAD. Mutants blocked in these steps cannot be recovered as auxotrophs since the required metabolites are not taken up by cells. In the bacterial cells, the second substrate for NadE is ammonium, as opposed to glutamine for eukaryotes. NadE is an essential and conserved protein in the eubacterial nicotinamide adenine dinucleotide (NAD) biosynthesis pathway. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 49). The protein encoded by the gene is set forth in Seq. ID No. 50.

Assays for measuring NadE function:

The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, niotinomide adenine dinucleotide adenyl synthase (NadE) (Seq. ID No. 49). NadE activity in the presence or absence of a putative inhibitory molecule of NadE activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.

DISCONTINUOUS ASSAY:

In assay, NadE converts nicotinic acid adenine dinucleotide (NaAD) into nicotinamide adenine dinucleotide (NAD) in the presence of ammonia and ATP. Each PPi molecule produced by the NadE reaction can then be converted to two phosphate (Pi) molecules in the presence of inorganic pyrophosphatase (PPase). The P| molecules present can then be quantitated with a malachite green reagent at 660 nm.

HPLC-based assay: Enzyme activity can be measured by HPLC quantitation of the reaction products. A neutralized aliquots from the reaction described above was injected into an HPLC system utilizing a 250 x4.6 mm Supelcosil LC-18 5μm reversed- phase column. The elution conditions: 9 min at 100% buffer A (0.1 M potassium phosphate buffer, pH6.0,6 min at up to 12% buffer B (buffer a, containing 20% methanol, 2.5 min at up to 45% buffer B, 2.5 min at up to 100% buffer B, and hold at 100% buffer B for 5.5 min. The eluate absorbance was monitored at 254 nm (1). Continuous assay:

Coupled NadD-NadE assay. NadD and NadE can be detected in one continuous coupled assay. In first reaction, NadD combines nicotinic acid mononucleotide (NaMN) and adenosine triphosphate (ATP) to form nicotinic acid adenine dinucleotide (NaAD). NadE then converts NaAD into nicotinamide adenine dinucleotide (NAD) in the presence of ammonia and ATP. In the assay, the NAD product is reduced to NADH with alcohol dehydrogenase (ADH) and ethanol, thus permitting direct spectrometric detection of NADH at 340 nm wavelength. The coupled reaction above also includes inorganic pyrophosphatase (PPase) to prevent accumulation of the pyrophosphate byproduct from the consumption of ATP (this method can be use as HTS format).

NadE assay. In assay, NadE converts NaAD into nicotinamide adenine dinucleotide (NAD) in the presence of ammonia and ATP. The NAD product is reduced to NADH with alcohol dehydrogenase (ADH) and ethanol, thus permitting direct spectrometric detection of NADH at 340 nm wavelength. The reaction above also includes inorganic pyrophosphatase (PPase) to prevent accumulation of the pyrophosphate byproduct from the consumption of ATP (this method can be use as HTS format).

EXAMPLE 35 ALLOIOCOCCUS OTITIDIS ENCODED PUTATIVE MEMBRANE PROTEIN NORA

An efflux transporter NorA that was originally identified in Staphylococcus aureus belongs to the family of multidrug resistance (MDR) transporters. NorA is encoded by chromosomally-located norA gene, it has broad substrate specificity and mediates resistance to various lipophilic and monocationic compounds such as ethidium bromide (EtBr), cetrimide, benzalkonium chloride, rhodamine 6G, tetraphenylphosphonium (TPP), chloramphenicol as well as some hygrophilic quinolones such as norfloxacin, ciprofloxacin and oxafloxacin. Increased levels of norA expression are associated with single nucleotide changes upstream of norA in a putative promoter/operator region and lead to increased pleiotropic resistance. NorA is a putative membrane protein with 12 predicted membrane-spanning domains and is classified as a member of major facilitator superfamily (MFS), a subgroup of MDR transporters characterized by the presence of 12-14 transmembrane segments and the use of proton motive force as an energy source for drug efflux. NorA homologs that belong to MFS family include Bmr and Bit of Bacillus subtilis, EmeA of Enterococcus faecalis and PmrA of Streptococcus pneumonia. The expression of bm gene in B. subtilis is upregulated by the product of adjacent bmR gene in the presence of inducers (rhodamine 6G and TPP), and there is an evidence that expression of norA in S. aureus is regulated by AlrS-AlrR two-component regulatory system.

It remains unknown whether the efflux of various toxins is a primary function of NorA. When overexpressed in E. coli, norA produces resistance to a broad range of substrates including fluoroquinolones. Everted membrane vesicles prepared from nor>4-expressing E coli exhibit energy-dependent transport of norfloxacin, the transfer is abolished by cyanide m-chlorophenylhydrazone (CCCP) and nigericin but not by valinomycin indicating that NorA-mediated transfer is coupled to the proton gradient of cell membrane. Norfloxacin uptake in everted vesicles as well as NorA- associated resistance phenotype is inhibited by reserpine and verapamil that also inhibit other MDR transporters and are toxic to mammalian cells. Histidine-tagged NorA (NorA-His) was recently overexpressed and purified from E. coli, reconstituted into both everted membrane vesicles and proteoliposomes and was shown to function as a self-sufficient efflux pump using fluorescent dye Hoechst 33342. Due to its high specificity, essentiality, and importance, norA is attractive as an antibacterial target. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 67). The protein encoded by the gene is set forth in Seq. ID No. 68.

NORA AS A TARGET FOR ANTI-INFECTIVE DEVELOPMENT

The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, NorA (Seq. ID No. 67). NorA activity in the presence or absence of a putative inhibitory molecule of NorA activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.. Because of broad substrate specificity of NorA, NorA inhibitors should be particularly useful against pathogens that possess multiple drug resistance. Whole-cell high-throughput screen (HTS) assay that measures NorA activity in the presence or absence of a putative inhibitory molecule of Alloiococcis otitidis NorA activity is used to identify potential inhibitors of NorA activity. The assay utilizes B. subtilis strain (ΛΔNA) that has both Bmr and Bit genetically inactivated while Alloiococcis otitidis NorA is supplied on the plasmid expression vector. The screen is based on the reversing of the resistance of ΔΔNA to EtBr. The exponentially growing cells are inoculated into the wells of a 96-well plate to OD₆₀₀=0.001 , the compounds are added at 20 μg/ml and EtBr is added at 10 μg/ml. Plates are incubated for 18 hrs at 37°C and examined for growth. Compounds that inhibit growth are subsequently tested in the presence/absence of EtBr for toxicity and effectivity. The efflux of EtBr from cells is monitored as described previously. The exponentially growing cells are loaded with EtBr at a concentration of 10 Dg/ml for 20 min at 37°C in the presence of reserpine (20 Dg/ml). Cells are centrifuged, resuspended to an OD₆oo=0.2 in a minimal medium GM1 alone or in the presence of inhibitor compound. Fluorescence of EtBr is monitored on a fluorimeter at an excitation D of 530 nm and emission D of 600 nm..

MONITORING OF HOECHST 33342 EFFLUX

The efflux of fluorescent dye Hoechst 33342 from either everted membrane vesicles prepared from Alloiococcus otitidis His-NorA overexpressing E. coli or a proteoliposomes reconstituted with Alloiococcus otitidis His-NorA is also used to monitor NorA activity in the presence or absence of putative inhibitors of NorA. Everted membrane vesicles are diluted into 2 ml of 50 mM potassium HEPES (pH 7.2), 8.5 mM NaCl, 2 mM magnesium sulfate at a final protein concentration of 40 μg/ml. NorA is activated by the addition of either 0.5 mM lactate or 0.1 mM Mg²⁺- ATP. Hoechst 33342 is used in a range of 12.5 to 200 nM. Inhibitors are added at various concentrations prior to the addition of Hoechst 33342. Fluorescence change is monitored at excitation and emission wavelenghths of 355 and 457 nm respectively in a FluoroMax spectrofluorimeter. For proteoliposome assay, the His- NorA proteoliposomes are diluted into a cuvette containing 2 ml of 20 mM potassium phosphate, 50 mM potassium sulfate, 2 mM magnesium sulfate (pH 7.0) at a protein concentration of 10 μg/ml. The inhibitor compounds and Hoechst 33342 are added at various concentrations and the fluorescence is measured as described previously. EXAMPLE 36 ALLOIOCOCCUS OTITIDIS ENCODED OBG GTPASE

The obg gene is the second gene in a two-gene operon along with the stage-

O sporulation gene spoOB in B. subtilis. SpoOB is central to the phospho-relay signal cascade that initiates sporulation. Obg is a member of the GTPase superfamily by virtue of homology throughout a small portion of the protein that in other members of the family is responsible for nucleotide (GTP/GDP) binding. Obg is essential for growth. Initiation of sporulation is thought to be triggered by changes in the GTP content of the cell; therefore, the presence of a GTP binding protein in an operon with a central player in the process is suggestive of a role for Obg in sensing GTP levels and transmitting a signal to SpoOB.

It has been shown that Obg is involved in activation of the σ^B transcription factor in B. subtilis in response to environmental stress. Cells were depleted of Obg utilizing a construct that put obg under the control of an inducible (P|_ao) promoter. Depletion of IPTG resulted in bacteria that failed to activate σ^B. These studies further showed by yeast-two-hybrid analysis that Obg interacted with several known σ^B regulators, the so-called Rsb proteins. The' role Obg plays in transmitting signals important for sporulation and activation of the stress sigma factor may be indicative of the activities that small GTP binding proteins carry out in triggering cell division in response to GTP levels. Due to its high specificity, essentiality, and importance, obg is attractive as an antibacterial target. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 71 ). The protein encoded by the gene is set forth in Seq. ID No. 72.

OBG AS A TARGET FOR ANTI-INFECTIVE DEVELOPMENT

Obg is essential for bacterial viability. Conditional lethal alleles revealed that

Obg is required for early events in sporulation and is involved in transmitting signals require for activation of the stress sigma factor. The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, obg (Seq. ID No.71). Obg activity in the presence or absence of a putative inhibitory molecule of Obg activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis..

Nucleotide binding Obg binding to nucleotide in the presence or absence of putative antimicrobials, which inhibit Obg activity, is monitored by a simple filter-binding assay. Alloiococcus otitidis Obg (1 -5 μg) is incubated with α³²P-GTP (0.2 μCi) in a buffer consisting of 50 mM Tris (pH 8.5), 1.5 mM MgCI₂, 0.1 mM EDTA, 200 mM KCl, 10% glycerol for 30 minutes to 3 hours at 37^°C. A portion of the reaction mix is spotted on nitrocellulose membrane, washed (50 mM Tris (pH 8.5), 1.5 mM MgCI₂, 1 mM DTT) and dried. The membrane is then exposed to X-ray film. Alternatively, the spots are excised and counted. This assay is directly amenable to HTS using filter plates.

GTPase activity

The GTP hydrolytic activity of Obg is monitored using thin-layer chromatography (1 , 2, 10). Obg and α³²P-GTP are incubated in 50 mM Tris (pH 8.5), 1.55 mM MgCI₂, 0.1 mM EDTA, 200 mM KCl, 10% glycerol for 30 minutes at 37°C. An aliquot of the reaction is placed on PEI cellulose and the strip developed with 0.5 M KH₂P0₄, 1.0 M NaCl (pH 3.7). The spots conforming to GDP and GTP are identified by UV shadowing, excised and counted. .

Alternatively, the hydrolysis of γ³²P-GTP is monitored by assaying for liberated Pi (12). Obg and α³²P-GTP are incubated in 50 mM Tris (pH 8.5), 1.5 mM MgCI₂, 0.1 mM EDTA, 100 mM KCl, 10% glycerol for 30 minutes to 3 hours at 37°C. The reaction is stopped by the addition of a slurry of charcoal in 1 mM Kpi (pH 7.5), which selectively binds the GTP and GDP. The liberated Pi in the supernatant is monitored by Cerenkov counting. Free P, is also monitored with the Malachite Green reagent.

Autophosphorylation

Obg autophosphorylation is monitored by incubating Obg with γ³²P-GTP in 50 mM Tris (pH 8.5), 1.5 mM MgCI₂, 0.1 mM EDTA, 100 mM KCl, 10% glycerol for 30 minutes at 37°C. Samples are analyzed following separation on SDS polyacrylamide gels, drying the gel and exposure to film.

EXAMPLE 37 RPOA, RPOB, RPOC, AND RPOD, THE GENES ENCODING THE SUBUNITS COMPRISING

ALLOIOCOCCUS OTITIDIS RNA POLYMERASE: ALPHA, BETA, BETA', AND SIGMA.

RNA polymerase is an enzyme comprised of multiple highly conserved subunits which catalyzes the DNA template directed polymerization of ribonucleic nucleotides into ribonucleic acid. It is composed of a core enzyme, D2,D,D', along with a fifth subunit present in stoichiometric amounts, DDDwhich can catalyze RNA synthesis non-specifically. Holoenzyme is formed by the introduction of the subunit DDD, which enhances gene promoter recognition and allows specificity. Homologs of the genes identified in Alloiococcus otitidis are described in Example 5/Table 4 (Seq. ID Nos 7, 9, 11 , and 13). The amino acid sequence of the protein encoded by these genes are set forth in Seq. ID Nos. 8, 10, 12 and 14.

Functions for the individual subunits have been defined biochemically, and interactions between them have now been deduced structurally by crystallographic analysis of the enzyme from Thermatoga thermophila, and to a lesser extent, Escherichia coli. The alpha subunit, encoded by rpoA, is required for enzyme assembly. It also interacts with transcription factors and with DNA elements involved in enhanced promoter strength. Beta, encoded by rpoB, is involved in initiation and elongation of the polymerization product. Beta' (encoded by rpoC), is responsible for binding of the enzyme to the DNA template. Omega is required to restore denatured RNA polymerase to function in vitro. Finally, sigma, encoded by rpoD, directs the enzyme to promoters on the template to enhance specificity of transcription (polymerization).

ALLOIOCOCCUS OTITIDIS RNA POLYMERASE: ALPHA, BETA, BETA', AND SIGMA AS A

TARGET FOR ANTI-INFECTIVE DEVELOPMENT

Bacterial RNA polymerase is a validated target for antimicrobial chemotherapy in that several inhibitors have been identified and at least one, rifampin, is in use clinically. Alloiococcus otitidis RNA polymerase holoenzyme is essential for bacterial viability. The Alloiococcis otitidis ORFs- have been shown to encode, by sequence homology, RNA polymerase holoenzyme (Seq. ID Nos. 7, 9, 11 and 13). Alloiococcus otitidis RNA Polymerase activity in the presence or absence of a putative inhibitory molecule of Alloiococcus otitidis RNA Polymerase activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.

Assays for the activity of RNA polymerase

Genes encoding the subunits of Alloiococcus otitidis RNA polymerase can be obtained using polymerase chain reaction amplification of the genomic region encoding them. The genes are subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme are overexpressed in Escherichia coli and purified using a standard tag system or conventional chromatography . Because RNA polymerase catalyzes the incorporation of single ribonucleotides into RNA, the incorporation of radiolabelled nucleotides into larger oligonucleotides is monitored to measure activity of the enzyme in the presence or absence of putative inhibitors of RNA polymerase activity. An automated high throughput filtration assay has been previously described for E. coli polymerase which uses filterplates containing a hydrophobic membrane and DEAE beads to capture polymerized RNA. G-less supercoiled DNA is used as a template at 6 ug/ml. Reaction contained 0.5 mM ATP, 0.1 mM UTP, 0.3 mM CTP, approximately 100,000 counts per minute (per 100 ul) [γ-³³P] CTP (2000 Ci/mmol, NEN/DuPont), 4 % polyethylene glycol, 4 mM DTT, 10 mM MgCI₂, in 50 mM Tris-acetate (pH 7.8), and 100 mM potassium acetate. The reaction is carried out at 34 degrees C for 40 minutes, with 10% DMSO present in all reactions. The reaction was stopped by adding 100 ul 15% DEAE-Sephacel bead slurry in 50% methanol, 20 mM EDTA, and 0.02% NP-40. The reaction was incubated for 40-60 minutes at room temperature without shaking, and then transferred to a unifilter plate on a filtermate cell harvester. The wells were washed six times with 2X PBS and 0.1% NP-40. After washing the bottom of the plate was sealed, and 50 ul scintillation counting liquid was added. Radioactivity was counted using a microplate scintillation counter.

Deconvolution assays are carried out by measuring the inhibition of sigma activity. Because sigma is required only for promoter specificity, polymerization may occur non-specifically if sigma is inhibited. Consequently a second assay is described above that is used to deconvolute activity against sigma.

The binding of putative inhibitory compounds to core enzyme. Several techniques are utilized to determine the interaction of inhibitors with individual subunits and include nuclear magnetic resonance and capillary electrophoresis.

EXAMPLE 38 YPHC, ENCODING A SMALL GTPASE OF UNKNOWN FUNCTION FROM ALLOIOCOCCUS

OTITIDIS

The yphC was initially identified in Bacillus subtilis in a collaboration between Wyeth and Millennium pharmaceuticals as being essential for growth by insertional mutagenesis. Subsequently it was determined that YphC, the encoded protein, contained two GTPase domains and had some homology to era. It was further identified in Thermatoga maritima and Escherichia coli . While no function has yet been determined for yphC, it appears that the carboxy terminal may contain an RNA binding site. In addition, site directed mutagenesis of four amino acids in the carboxy region were found to be lethal (unpublished results, Millennium). Under non- permissive conditions, strains carrying temperature sensitive alleles of the gene in E. coli become elongated, and chromosome segregation becomes abberrant, suggesting a role in cell division. Homologue of this gene identified in Alloiococcus otitidis is described in Example 5/Table 4 (Seq. ID No 73). The protein encoded by the gene is set forth in Seq. ID No. 74. YphC from Alloiococcus otitidis as a target for antimicrobial chemotherapy

YphC is an essential protein in Bacillus subtilis and E. coli, and is conserved among bacteria including Alloiococcus otitidis. The Alloiococcis otitidis ORF- has been shown to encode, by sequence homology, YphC (Seq. ID No. 73). YphC activity in the presence or absence of a putative inhibitory molecule of YphC activity is used to identify novel antimicrobial agents, which may be used to treat disease caused by Alloiococcus otitidis.. Consequently it is proposed here that an assay which identified inhibitors of YphC from Alloiococcus would result in small molecules which can be developed into effect antimcrobial agents. Additionally, because of the conservation of the enzyme among bacteria, inhibitors of the protein's function from this organism should have broad spectrum activity.

Assays for the GTP hydrolysis by YphC The YphC gene from Alloiococcus otitidis is obtained using polymerase chain reaction amplification of the genomic region encoding it. The gene is subcloned into a standard expression vector either containing an amino acid tag for ease of purification or not. The enzyme is then overexpressed in Escherichia coli and purified using a standard tag system or conventional chromatography. Activity of YphC in the presence or absence putative antimicrobial agents is monitored using the assay system described below.

GTP hydrolysis - detection by thin layer chromatography: Reaction is carried out in a 50 ul reaction of 50 mM Tris-Cl (pH 7.5), 400 mM KCl, 5 mM MgCI2, 1 mM DTT, 10 uM [a-32P] GTP, and 10 ug purified YphC, at 37 degrees for 10 minutes. The reaction is terminated by transfer of 5 ul samples to 10 ul of ice-cold 20 mM EDTA. Portions are spotted onto polyethyleneimine-cellulose thin layer chromatography plates, which are developed in 0.75 KH2P04 (pH 3.65). The plate is autoradiographed to identify hydrolysis products. BIBLIOGRAPHY

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Claims

WHAT IS CLAIMED IS:

1. A purified or isolated Alloiococcus otitidis nucleic acid sequence comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, wherein expression of said nucleic acid is essential for the proliferation of a cell.

2. A purified or isolated nucleic acid of Alloiococcus otitidis comprising a fragment of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105 said fragment selected from the group consisting of fragments comprising at least 10, at least 20, at least 25, at least 30, at least 50 and more than 50 consecutive nucleotides of one of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

3. A purified or isolated antisense nucleic acid comprising a nucleotide sequence complementary to at least a portion of an intragenic sequence, intergenic sequence, sequences spanning at least a portion of two or more genes, 5' noncoding region, or 3' noncoding region within an operon comprising a proliferation-required gene of Alloiococcus otitidis whose activity or expression is inhibited by an antisense nucleic acid and selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

4. A purified or isolated nucleic acid comprising a nucleotide sequence having at least 70% identity to a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, fragments comprising at least 25 consecutive nucleotides selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, the nucleotide sequences complementary to one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, and the sequences complementary to fragments comprising at least 25 consecutive nucleotides of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

5. A vector comprising a promoter operably linked to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence of any one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

5. A purified or isolated polypeptide of Alloiococcus otitidis comprising a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a fragment selected from the group consisting of fragments comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of one of the said polypeptides.

6. A purified or isolated Alloiococcus otitidis polypeptide comprising a amino acid sequence having at least 25% amino acid identity to a polypeptide whose expression is inhibited by a nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or at least 25% amino acid identity to a fragment comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or more than 60 consecutive amino acids of a polypeptide whose expression is inhibited by a nucleic acid comprising a nucleotide sequence selected from the group consisting of one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

7. A purified or isolated Alloiococcus otitidis polypeptide comprising selected from one of the even numbered sequences set forth in Seq. ID Nos: 2 to Seq. ID Nos: 106, wherein the polypeptide is essential for the proliferation of a cell.

8. A method of producing an Alloiococcus otitidis polypeptide comprising introducing into a cell a vector comprising a promoter operably linked to a nucleic acid comprising a nucleotide sequence encoding a polypeptide whose expression is essential for the proliferation and viability of Alloiococcus otitidis, and which is inhibited by an antisense nucleic acid, and which is selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105.

9. A method of inhibiting the proliferation of Alloiococcus otitidis in an individual comprising inhibiting the activity or reducing the amount of a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105 or inhibiting the activity or reducing the amount of a nucleic acid encoding said gene product.

10. A method for identifying a compound which influences the activity of an Alloiococcus otitidis gene product , which is required for proliferation, said gene product comprising a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, said method comprising:

(a) contacting said gene product with a candidate compound; and

(b) determining whether said compound influences the activity of said gene product.

11. A method for identifying a compound or an antisense nucleic acid having the ability to reduce activity or level of a Alloiococcus otitidis gene product, which is required for proliferation, said gene product comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, said method comprising the steps of:

(a) contacting a target gene or RNA encoding said gene product with a candidate compound or antisense nucleic acid; and

(b) measuring the activity of said target.

13. A method for inhibiting cellular proliferation of Alloiococcus otitidis comprising introducing an effective amount of a compound with activity against a gene whose activity or expression is essential for cellular proliferation, and which is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a compound with activity against the product of said gene into a population of Alloiococcus otitidis cells expressing said gene.

13. A composition comprising an effective concentration of an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, or a proliferation- inhibiting portion thereof in a pharmaceutically acceptable carrier.

14. A method for identifying a compound having the ability to inhibit proliferation of Alloiococcus otitidis cell comprising:

(a) identifying a homologue of a gene or gene product whose activity or level is inhibited by a nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, in a test cell, wherein said test cell is not Alloiococcus otitidis;

(a) identifying an inhibitory nucleic acid sequence which inhibits the activity of said homologue in said test cell;

(b) contacting said test cell with a sublethal level of said inhibitory nucleic acid, thus sensitizing said cell;

(c) contacting the sensitized cell of step (c) with a compound; and (d) determining the degree to which said compound inhibits proliferation of said sensitized cell relative to a cell which does not contain said inhibitory nucleic acid.

16. A method for identifying a compound having activity against a biological pathway required for proliferation comprising:

(a) sensitizing a cell by providing a sublethal level of an antisense nucleic acid complementary to a nucleic acid encoding a gene product required for proliferation, wherein the activity or expression of said gene product is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, in said cell to reduce the activity or amount of said gene product; (a) contacting the sensitized cell with a compound; and

(b) determining the degree to which said compound inhibits the growth of said sensitized cell relative to a cell which does not contain said antisense nucleic acid.

17. A method for identifying a compound having the ability to inhibit one of the Alloiococcus otitidis polypeptides encoded by a polynucleotide selected from one of odd numbered sequences set forth in Seq. ID Nos: 1 to Seq. ID Nos: 105, and which is essential for cellular proliferation comprising:

(a) contacting a cell which expresses the polypeptide with the compound; and

(b) determining whether said compound reduces proliferation of said contacted cell by acting on said gene product.

18. A method for identifying a compound having the ability to inhibit one of the purified and isolated Alloiococcus otitidis polypeptides selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq. ID No.: 106, and which is essential for cellular proliferation comprising: (c) contacting the purified and isolated polypeptide with the compound in vitro in the presence or absence of a substrate, which is essential for the activity of the polypeptide; and

(d) determining the effect of the compound on the polypeptide by measuring the effect of the polypeptide on the substrate.

19. A compound which interacts with an Alloiococcus otitidis polypeptide selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq. ID No.: 106 and inhibits its activity.

20. A method for manufacturing an antimicrobial compound comprising the steps of screening one or more candidate compounds to identify a compound that reduces the activity or level of an Alloiococcus otitidis polypeptide selected from one of the even numbered sequences set forth in Seq. ID No.: 2 to Seq.

ID No.: 106, said polypeptide comprising a gene product whose activity or expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105; and manufacturing the compound so identified.

21. A compound which inhibits proliferation of Alloiococcus otitidis by interacting with a gene encoding a polypeptide that is required for proliferation or with a polypeptide required for proliferation, wherein said polypeptide is selected from the group consisting of a gene product having at least 70% nucleotide sequence identity from one of the odd numbered sequences set forth in Seq.

ID No.: 1 to Seq. ID No. 105, polypeptide encoded by a nucleic acid having at least 70% nucleotide sequence identity to a nucleic acid encoding a polypeptide whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105, a polypeptide having at least 25% amino acid identity to a gene product whose expression is inhibited by an antisense nucleic acid comprising a nucleotide sequence selected one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105, a polypeptide encoded by a nucleic acid comprising a nucleotide sequence which hybridizes to a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105 under stringent conditions, a gene product encoded by a nucleic acid comprising a nucleotide sequence which hybridizes to a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105 under moderate conditions, and a gene product whose activity may be complemented by the gene product whose activity is inhibited by a nucleic acid selected from one of the odd numbered sequences set forth in Seq. ID No.: 1 to Seq. ID No. 105.