MXPA01003289A - Methods of identifying and characterizing mutations within bacterial dna gyrase and fabi - Google Patents

Abstract

The instant invention allows for the simultaneous creation and identification, or identification of mutations that confer resistance to antibacterial compounds.

Description

, _ > METHOD OF IDENTIFICATION AND CHARACTERIZATION OF MUTATIONS IN THE BACTERIAL DNA GYPSY AND FABI BACKGROUND OF THE INVENTION 5 The discovery and development of an antibacterial agent is aided by the knowledge by which the compound inhibits cell growth. A common technique to elucidate this information is to isolate mutants that alter the susceptibility of the organism to the compound and identify which mutation is responsible for the phenotype. By identifying which gene the mutation is in, or affecting expression, one can often learn which cellular pathway the compound inhibits, which compounds bind to affect growth, and obtain information about how the compound binds to the target molecule.

Random mutagenesis followed by phenotypic selection, such as resistance to an antibacterial agent, has shown to be an effective technique for establishing structure-function relationships for proteins in yeasts, viruses and bacteria (Kwan T., Gros P., Biochemistry, 1998; 37: 3337-3350; Loeb DD et al., Nate, 1989; (6232): 397-400; Rolli V. et al., Biochemistry 1997; 36: 12147-12154). The success of such experiments is determines, in part, by the randomness of the mutagenic method, the ability to select mutants of interest, the amounts of mutant cells that can be generated, and the ability to identify the mutation that is responsible for the phenotype of a selected mutant. This procedure has been used to isolate gene mutants that are cloned into plasmids or other extrachromosomal elements. While this may work in some cases, the technique is labor intensive and is complicated in cases where the strain is diploid for the gene of interest or toxic when the gene of interest is expressed from a multicopy plasmid.

Recently, Kok et al have shown that combining mutagenesis of a fragment defined by PCR with natural transformation is a way to identify mutations that abolish the function of PobR in the Acinetobacter region (Kok R., D'Argenio D., and Ornston LN, Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulatory gene, population J. Bac, 1997; 179: 4270-4276). This technique exploits the ability of Acinetobacter to take PCR products and, through homologous recombination, replace the pop chromosomal gene with one that was amplified by PCR. If a PCR product contains a mutation that results in a dysfunctional gene product and this mutation is incorporated into the chromosome, the resulting organism would not be able to convert 4-hydroxybenzoate into a toxic metabolite and would be viable in the presence of hydroxybenzoate The detailed experiments subsequently demonstrated the use of combining random mutagenesis of genes encoding known targets of antibacterial compounds with homologous recombination to generate and identify point mutations resulting in resistance to antibacterial compounds. By using long-error-prone PCR with oligonucleotide primers designed to generate long overlap products (approximately 10,000-15,000 base pairs) that span the entire genetic material of an organism, the chromosome of an organism can be randomly mutated into defined fragments. After transformation and homologous recombination, the ability of each of these products, and subsequently each region of the chromosome, to contain a mutation that results in an altered phenotype, such as resistance to an antibacterial compound, can be examined. By comparing the DNA sequence of the region of a mutant organism corresponding to the PCR product used to generate the mutant with the analogous region of the wild-type chromosome, the mutations responsible for the phenotype can be identified.

PCR amplification of the complete genetic material of an organism can also be used to identify a mutation in a chromosome that results in an altered phenotype generated by any other technique. For these experiments, the complete chromosome of the mutant organism would be segregated into PCR fragments of 10,000-15,000 base pairs that overlap. The ability of each region to restore the altered phenotype of a wild-type strain after transformation and homologous recombination could then be used to isolate the location of the mutation in a region defined by the product used to recreate an organism with the mutant phenotype. The DNA sequence of this region could then be examined from the mutant organism and compared to the analog region of the wild-type chromosome to identify the mutation responsible for the phenotype. This technique will be useful to identify mutations responsible for antibacterial resistance in spontaneous mutants and mutants generated using agents that damage DNA.

PREVIOUS ART OF THE INVENTION This current invention is a method for identifying molecular targets in bacteria treated with an antibacterial compound. The method is based on creating and identifying mutations in bacteria that confer altered susceptibility to an antibacterial compound. Mutations provide valuable information about the molecular target of the compound and how the compound and target interact. The generated bacterial strains can be used to provide information that could be useful for identifying and characterizing compounds that could be used or developed to treat bacterial infections of humans, other animals and plants.

Using Neisseria gonorrhoeae, gyrA or tabl was subjected to site-specific and random nucleotide mutagenesis to identify mutations confering resistance to ciprofloxacin or diphenylethers, respectively. These experiments identified new and previously described mutations associated with resistance to these compounds. These experiments also demonstrate the ability to create and identify mutations in Neisseria gonorrhoeae associated with resistance to antibacterial compounds by combining mutagenesis random with phenotypic selection.

The current invention is a system that allows the creation and simultaneous identification of mutations that confer resistance to antibacterial compounds. 20 This technology is for the identification, or isolation and identification, of mutations responsible for the altered susceptibility of various bacteria to chemicals (or any other selectable phenotype). This invention can be used in any The bacterium that can be transformed with DNA can carry out homologous recombination and for which the genome sequence can be determined. Examples of these include, but are not limited to: Neisseria gonorrhoeae, Haemophilus influenzae, Streptococcus pneumoniae, Acinetobacter, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, Bacillus subtilis, and Helicobacter pylorri.

This invention is a process for identifying and characterizing drug-target interactions comprising: a) Generate a defined set of overlapping PCR products (approximately 10 kilobases of bases per product) using chromosomal DNA of Neisseria gonorrhoeae strain N400 as a template which, taken together, comprise the complete DNA composition of the body chromosome. The PCR reactions are run in such a way that they introduce, in a random fashion, changes in the nucleotide composition that some of the resulting DNA fragments have one or more changes in the nucleotide composition compared to the DNA template. These changes occur almost randomly as a function of the PCR conditions and the DNA polymerase used. b) Transform the N400 with groups of the PCR products of -10 kilobase pairs, which corresponds to approximately 100 kilobase bases of the chromosome. c) Isolate new strains of Neisseria gonorrhoeae from the transformation showing altered susceptibility to a chemical. Since the PCR fragments are generated by pairs of primers corresponding to portions of the chromosome for which the DNA sequence has been determined, the mutation or mutations responsible for the altered susceptibility can be assigned to a particular region of the chromosome. d) Transforming Neisseria gonorrhoeae N400 separately with each of the PCR products comprising the "b" group to identify the region of the chromosome that is flanked by a pair of primers carrying the mutation or mutations responsible for the altered susceptibility; e) Design new primers based on the sequence of the region defined in "d" to generate smaller overlap PCR products (approximately 3 kilobases of bases for each PCR product) and use to amplify chromosomal DNA of the strain isolated in "d" . f) Transform the N400 with the "e" PCR products to define the region of approximately 2 kilobase pairs or smaller of the chromosome that has the mutation or mutations responsible for the altered susceptibility; g) make a DNA sequence of the region of approximately 2 kilobase pairs or smaller defined in h) Compare the DNA sequence with the DNA sequence of the same N400 region. If a simple change in the order of the nucleotides is found, this change is defined as a mutation which confers altered susceptibility to the compound. If more than one change is observed, additional sets of primer design, PCR amplification, transformations and selections are executed so that the contribution of each mutation to the phenotype can be determined.

This invention can also be used to identify mutations that confer altered susceptibility to a chemical in strains of Neisseria gonorrhoeae that have been previously isolated using other methods. In this case, Stage "a" above would be: a) Generate a defined set of overlapping PCR products (approximately 10 kilobases of bases per product) using chromosomal DNA from a mutant strain of Neisseria gonorrhoeae as a template that has been previously generated and demonstrated to be more or less susceptible to a chemical than N400 The PCR products, taken together, comprise the complete DNA composition of the chromosome of the mutant organism.

The steps b-h would be identical to that described above.

Additionally, the invention is a process for identifying and characterizing drug-target interactions using Neisseria gonorrhoeae which comprises: a) randomly mutating a defined region of the chromosome that can alter the susceptibility to chemical compounds. This region can encompass i) a single codon using splicing by extension of overlap with degenerate oligonucleotides at a specific codon, ii) 20 to 100 base pairs using site-specific mutagenesis mediated by the oligonucleotide with a degenerate oligonucleotide, or iii) a gene or complete region defined as defined in "f" above using low fidelity PCR; b) introducing mutations generated from "a" into a wild-type base such that the wild-type region is replaced by the mutant region; c) isolating organisms with an altered phenotype such as resistance to a chemical compound; d) sequencing and comparison of the entire region transformed into "b" to identify the mutation or mutations responsible for the phenotypic alteration as described in steps g and h above; d) using strains or purified proteins with mutations identified in steps "a-d" to help understand the mechanism of action, define the binding site and / or identify resistant forms of antibacterial compound targets.

The invention also pertains to a process for identifying and characterizing a mechanism of action of an antibacterial compound comprising: generating DNA fragments by DNA amplification by polymerase chain reaction of a complete genome of a bacterium under conditions that allow the mutation of the fragments; allow one or more of the fragments of DNA generated within the chromosome of a bacterium by homologous recombination; isolate bacteria that demonstrate resistance to an antibacterial compound; and identify the mutation contained in the fragment of DNA The invention also pertains to a process for identifying mutations contained in the chromosome of a bacterium that result in an identifiable phenotype comprising: (a) generating DNA fragments by amplification of the bacterial chromosome by polymerase chain reaction corresponding to regions of the chromosome bacterial that may contain a mutation; (b) allowing one or more of the DNA fragments to be incorporated into the chromosome of a bacterium that does not exhibit the phenotype identifiable by homologous recombination; (c) isolate the bacteria that demonstrate the identifiable phenotype; and repeat the steps from a to c until a fragment of Single minor DNA of approximately 10 kilobases in length is identified as being responsible for the mutation; and identify the mutation contained in the DNA fragment.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the generation of site-specific mutants using splicing by extension overlap. A- RAPID GENERATION OF SPECIFIC SITE CHROMOSOMIC MUTANTS; B-PRIMER A1, PRIMER B1, CEVER A2, PRIMER B2, * = SPECIFIC SITE MUTATIONS; C-GENERATE TRANSLATION FRAGMENTS CONTAINING MUTATION BY MEANS OF PCR; D-FRAGMENT 5 ', FRAGMENT 3'; E-COMBINE FRAGMENTS 5 'AND 3' WITH EXTERNAL CEBATORS AND PCR TO GIVE COMPLETE LENGTH PRODUCT; F- TRANSFORMATION, G-PRODUCT PCR, CROMOSOMA; H-SELECTION FOR RESISTANCE TO ANTIBACTERIAL OR IDENTIFYING MUTANTS NOT SELECTED BY ENZYMATIC MODIFICATION OF RESTRICTION.

Figure 2 shows the generation of Neisseria gonorrhoeae with mutations resistant to quinolone in gyrA. A-RANDOMASTESIS MUTAGENESIS AND IDENTIFICATION OF THE MUTAGENESIS RESPONSIBLE FOR THE RESISTANCE OF QUINOLONE; B-PCR UNDER LOW FIDELITY CONDITIONS; C- ALLEE EXCHANGE OF RANDOM GROUP ON CROMOSOMA; D-ISOLATE QUINOLONE RESISTANT BACTERIA; E-SEQUENCE HEAVY DUTY TO IDENTIFY MUTATION.

Figure 3 shows transformation efficiencies and isolated genotypes of PCR-mediated mutagenesis of gyrA. A-TEMPLATE PCR; B-CIPROFLOXATION; C-WITHOUT DRUG, D-GENOTYPES gyrA; E-DILUTION Figure 4 illustrates an overview of rapid elucidation of antimicrobial target. A- RAPID ALUCIDACION ANTIMICROBIAL OBJECTIVE (RATE); GENERATE ONE LIBRARY OF RANDOM CROSS-SCREEN POINT MUTATIONS BY PCR WITH LOW FIDELITY CONDITIONS; C-TRANSFORM GROUPS OF 12 PCR PRODUCTS (1000 kB) IN A NATIVE TYPE BASE AND INSULATE RESISTANT COMPOUND STRAINS; D- NATIVE TYPE CHROMOSOME (2.2 MB); E-RETRANSFORMING WITH INDIVIDUAL PRODUCTS (10 kB) OF GROUPS CONTAINING RESISTANCE TO IDENTIFY FRAGMENTS WITH MUTATIONS; F-GENERATE SMALLEST PCR PRODUCTS (1 kB) TO ADDITIONALLY MAPE MUTATIONS RESPONSIBLE FOR PHENOTYPE; G SEQUENCE DNA FROM THE REGION CONTAINING RESISTANCE OF RESISTANT ISOLATION.

DETAILED DESCRIPTION OF THE INVENTION It is beneficial to understand the mechanism of inhibition of antibacterial compounds for the discovery and development of an effective antibiotic. The natural competence and highly recombinant nature of Neisseria gonorrhoeae makes this organism ideal for identifying and characterizing drug-target interactions. Neisseria gonorrhoeae was used to demonstrate the utility of this invention, however, the system is applied to any other bacterial species that is capable of transforming with DNA and can carry out homologous recombination.

To demonstrate the use of Neisseria gonorrhoeae to identify mutations that lead to resistance of antibacterial compounds, gyrA or fabl was mutated using site-specific mutagenesis, domain, and region. These mutations were then introduced into Neisseria gonorrhoeae strain N400 and mutations associated with resistance to ciprofloxacin, clinafloxacin, dihydroxyphenylether (DHDPE) or triclosan were identified.

PCR products that contain mutations based on mutations previously described in gyrA (which encode a mutation of Ser91 in Phe, and Asp95 in Gly) and parC (which codes for mutation of Ser88 in Pro, and Glu91 in Lys) associated with resistance to quinolone (Belland RJ, Morrison SG, Ison C, and Huang WM, Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates, Mol.Micromol., 1994; 14: 371-380) were generated using splicing by overlap extension (Figure 1). These mutant PCR products were then recombined on the chromosome by transformation and strains with mutant alleles were identified by their decrease in susceptibility to ciprofloxacin. These experiments resulted in the creation of strains NG-2693 (gyrA S9IF, D95G and parC S88P, E91K) and NG-2709 (gyrA S91F, D95G).

To identify other mutations in the region that determines quinolone resistance (QRDR) associated with quinolone resistance, the DNA sequence corresponding to residues 88-103 of gyrA was mutated randomly using site-specific mutagenesis with a degenerate oligonucleotide. These mutations were then transformed into a wild-type base in which the ciprofloxacin-resistant mutants were selected after homologous recombination. DNA sequencing of gyrA QRDR from resistant mutants confirmed that Ser91 and Asp95 are independently involved in the inhibition by quinolone of DNA gyrase (Belland R.J. et al, 1994; Deguchi T., Yasuda M., Nakano M., Ozeki S., Ezaki T., Saito I., and Kawado Y., Quinolone-resistant Neisseria gonorrhoeae: Correlation of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV with antimicrobial susceptibility profiles, Antimicrob. Agents Chemoter., 1996; 40: 1020-1023) and revealed various mutations not previously reported in this region that confer resistance to ciprofloxacin in Neisseria gonorrhoeae as shown in Table 1. Strains generated as a result of these experiments are NG-2691, NG-2698, GC 156 and GC 158. TABLE 1. Mutations Resistant to Cyrrofloxacin from gyrA identified by Random Mutagenesis QRDR with a Degenerate Oligonucleotide Genotypes resistant to quinolone HGDSAVYDTIVRMAQN (Sec.1) EF G C N H EA To identify other mutations that lead to resistance to ciprofloxacin that may not be located between residues 88 and 103 of gyrA, we performed PCR of a fragment of 8.8 kilobase bases containing gyrA to create a group of PCR products with random nucleotide substitutions distributed throughout the entire region (Kok R. et al, 1997). This group was subsequently introduced into N400 by transformation and strains with mutations leading to ciprofloxacin resistance were isolated (Figure 3).

Colonies resistant to Ciprofloxacin were observed at a frequency of 10"2 for bacteria transformed with the library generated by PCR, this frequency was at least 4 orders of magnitude higher than that observed for cells that did not transform, indicating that the mutations were probably generated as a result of PCR amplification and in the chromosome region that corresponds to the 8.8 kilobase base PCR product used in the transformation.

To identify where the mutation of the 8.8 kilobase fragment responsible for the resistance was located, pairs of oligonucleotide primers were designed to PCR amplify a product of 800 base pairs of the 5 'portion of gyrA containing the QRDR. Each PCR product was then used to transform strains sensitive to ciprofloxacin and, in all cases, was able to generate ciprofloxacin-resistant colonies at high frequencies. Therefore, all ciprofloxacin resistant strains generated using a 8.8 kilobase base random library contain a mutation in the 800 base pair region containing the 5 'region of gyrA.

The DNA sequence corresponding to the first 200 amino acids of GyrA of each ciprofloxacin-resistant strain was determined to identify which mutation was responsible for the resistance. The amino acid sequence of GyrA determined from the wild-type strain is shown below with all mutations associated with ciprofloxacin resistance generated by random PCR show the respective wild-type residue below. The QRDR (residues 75-114) is framed. These experiments also identified new and previously identified mutations (Belland et al., Mol Micro., 1994; 14: 2; Deguchi et al., Antimicrob Agents Chemother., 1995; 39: 561-563) associated with quinolone resistance.

Amino Acid Sequence of IM400 GyrA and Quinolone Resistant Mutants 60 MTDATI RHDHKFALETLPVSL EDEMR SYLDYAMS VI VRRALPDVRDGLKPVHRRVLYAM H G 135 161 DGLAAAAMRYTE I RMAKl SHEMLADI EEETVNFGPNYDGSEHEPLVLPTRF PT (Sec.2) V Q G K To ensure that other mutations responsible for ciprofloxacin resistance were not present, a PCR product corresponding to the sequenced region was generated from each resistant strain. These PCR products were then transformed again into a sensitive strain, and resistant bacteria were isolated. Since high transformation frequencies were observed for each product, and DNA sequencing confirmed that all resistant strains contained identical mutations as seen in their respective pattern, it was concluded that these mutations were solely responsible for the resistance phenotype. The names of the strains and the coalterations responsible for the amino acid substitutions generated by all the mutagenesis procedures of gyrA are summarized in Table 2.

Deposits have been made to meet the requirements for the Budapest Treaty for the purposes of patent procedure. The ATCC numbers will be provided.

Table 2: Strains of Neisseria gonorrhoeae To evaluate the phenotypes of some of these mutants, the minimum inhibitory concentrations (MICs) of a panel of antibacterial compounds were determined. By comparing the effect that these mutations have on the efficiency of the antibacterial compound used to generate the resistant strain and related compounds, one can predict how effective these compounds will be against the existing resistant strains and determine if the compounds are inhibiting cell growth through similar mechanisms. For example, Table 3 demonstrates that ciprofloxacin, clinafloxacin, enoxacin, trovafloxacin and ofloxacin all inhibit cell growth by affecting DNA gyrase because single-point mutations in gyrA confer resistance to all these compounds. Although ciprofloxacin, trovafloxacin and clinafloxacin show similar activities against the wild-type strain, the efficiency of clinafloxacin is more than ten times better against the high-level quinolone-resistant strain (GC19). This information is very important to determine the efficiency of a compound when it is known that existing resistant species are present in the environment. It can also be included that residues 93 and 100 are probably not critical for the interaction between quinolones and DNA gyrase in Neisseria gonorrhoeae since the alterations in these residues have no effect on the susceptibilities to the tested quinolones. It is important to note that factors affecting the intracellular concentration of compounds (flow pumps, modifying enzymes) are controlled in these experiments because all strains are isogenic except for the mutations noted, so that all changes in susceptibility they are a direct result of the observed amino acid alteration.

Table 3. Susceptibility of N. gonorrhoeae gyrA and parC mutants to a panel of quinolones THE These experiments have identified resistance mutations to most residues that have been associated with resistance to quinolone (a field studied for more than 30 years) of gram-negative and gram-positive bacteria. From this it can be expected that the novel mutations observed in the gyrA gene of Neisseria gonorrhoeae can also be expected to confer resistance in analogous regions of type ll topoisomerases in bacteria with at least 30% of the residues identical to Neisseria gonorrhoeae GyrA QRDR (residues 75- 114 of GyrA). Also, a new region of the A subunit of Neisseria gonorrhoeae DNA Girasa located near the QRDR, based on the crystal model of E. coli, involved in quinolone resistance has been identified. The region partially comprises residues 62-65 and 161 of the Neisseria GyrA protein. The precise mechanism by which these mutations confer resistance to quinolones is currently unknown, but elucidating this information can provide valuable information for developing new inhibitors of ll-type topoisomerases and defining binding sites for quinolone. Since bacterial strains carrying amino acid changes in these positions reveal information about the function of the enzyme and its interaction with inhibitors, they can be used to identify and characterize compounds for use in the treatment of bacterial infections.

In a similar series of experiments, strains of Neisseria gonorrhoeae that are less susceptible to the chemical dihydroxydiphenylether (DHDPE) and related compounds, which include the commercially used antibacterial compound triclosan, were isolated by PCR amplification of the Fabl gene of Neisseria gonorrhoeae strain N400. The PCR products were used to transform the N 400 strain of Neisseria gonorrhoeae and strains that were less susceptible to DHDPE or related compounds were isolated. The fabl gene of each of the resulting strains was amplified by chromosomal DNA PCR and the DNA sequence was determined. The subsequent alignment shows the amino acid sequence of the Wild-type Fabl protein with all mutations that have been identified using PCR-mediated mutagenesis in Neisseria gonorrhoeae below its respective wild-type residue. These mutations are located throughout the entire gene and are concentrated in residues located in close proximity to the active site as predicted by the crystallographic structure of the E. coli enzyme.

Amino Acid Sequence of Fabl and DHDPE or Triclosan Resistant Mutations 30 60 MG FL QO K K 1 L. I TGM 1 S ER S I A YG I AKAC R EQGA E LA F T Y V VDK L E ER VR KMAA E L D S E LV V T S V T 90 120 FR C D VA S DD E I NQV F ADLG KHWDG LDG L V H S G F A P K E A L S G D L D S 1 S R E A F N T A H E i S T C V G H S V 150 ISO A Y S L P A L A K A A R P MM R G R N S A I V A L Y Y L G A V R A I P N Y V M G A K A S L E A G 1 R F T A A C L G K V H A I V 210 2A0 E G I R C N G I S A G P I K T L A S S G U A D F G K L L G H V A A H N P L R N V T I E V G N T A A F L L D S S G N S V Y S T cr, V V L V I V 26 f T G E I T Y V D G G Y-S I N A L S T E G. N (Sec.3) Strains and codon substitutions that resulted in an altered Fabl amino acid sequence are shown in Table 2. These strains help to understand the mechanism of the enzyme and how the enzyme inhibitors work. Thus, they are useful in the discovery of chemicals that can be used to treat bacterial infections. Mutations in analogous codons in other bacteria that have a Fabl gene that is similar in sequence to Neisseria gonorrhoeae fabl, which includes, but is not limited to, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis would be expected to alter the susceptibility of these proteins to the related inhibitors. In fact, mutations have been observed in all residues previously shown to confer resistance to triclosan or DHDPE in E. coli using this technique in Neisseria gonorrhoeae (McMurry et al., Nature 1998 394: 531-2; Heath et al, J. Biol, Chem. 1999 274: 11110-11114;. These mutant strains and purified proteins would be useful for discovering new chemicals to treat bacterial infections.

Previous experiments show that by using permutations of existing technologies to randomly mutate a DNA fragment and use this DNA fragment to replace the wild type analogue region of a bacterial chromosome it is possible to isolate strains that are resistant to chemical agents. The present invention is an expansion of these mutagenic procedures for the complete chromosome of an organism that allows for the 1) simultaneous generation and identification of; or 2) the identification of mutations that result in an altered phenotype such as altered susceptibility to a chemical compound.

The system involves generating a library of DNA fragments from defined regions of the chromosome in such a way that the fragments contain mutations. This can be achieved by PCR amplification of the entire genome in overlapping fragments of approximately 10 kilobase pairs. The DNA fragments can represent the complete chromosome of Neisseria gonorrhoeae or various portions of them. The fragments are introduced into Neisseria gonorrhoeae and mutants are isolated that have products introduced into the chromosome that results in an altered sensitivity to a chemical. Isolates that have a decreased susceptibility are identified and isolated based on their ability to grow in the presence of the chemical. The result of this is that the mutation maps to a specific region of the chromosome as defined by the DNA that lies between the primers used to generate the library of DNA fragments used in the transformation that created the mutant strain. Once the mutation has been mapped to a reasonably sized portion of the chromosome, for example less than 3 kilobases of bases, using an interactive process of primer design, PCR amplification, transformation and selection of bacteria with altered susceptibility to the chemical, the DNA of the mutant region carrying the mutation can be made a sequence. In this way the mutation responsible for the altered susceptibility can be identified. This identifies the gene or genes involved in the mechanism by which the chemical affects the growth of bacteria.

This system can also be used to identify mutations in a bacterial chromosome that has been generated by other means and results in a phenotypic alteration. Examples of this are I) strains that carry extra chromosomal elements that result in a detectable phenotype, such as loss of virulence, fluorescence by means of green fluorescence protein (GFP) or resistance to an antibacterial compound; or 2) mutant strains that contain point mutations that result in resistance to antibacterial compounds with known or unknown targets. In the latter case, PCR products containing the complete genome can be systematically subjected to in vitro mutagenesis, where any external DNA fragment can be randomly inserted into the PCR product using the GPS system of New England Biolabs. The resulting PCR products can then be transformed into the wild-type strain, the extrachromosomal material recombined into the chromosome, and the mutants containing the desired phenotype identified and isolated. In the latter case, the resistant mutants can be generated using chemical means such as ethylenemethanesulfonate, DNA damaging agents such as UV irradiation, or simply by isolating spontaneous mutants growing on the plates containing a concentration of the chemical compound which prevents the growth of the original strain. Once a strain carrying the detectable phenotype has been generated, it can be performed on PCR of the complete chromosome of the mutant organism in defined regions and identify the location of the mutation as described above.

This invention allows some to identify genes and gene products that can be mutated and result in an altered phenotype such as changing the susceptibility of an organism to a particular chemical. This can be done without any previous information about where such mutations would have to occur in the chromosome to confer the altered susceptibility or without any previous information about how the chemical affects the bacterium. The result of the use of this invention is that new information can be obtained regarding the interaction of the compound and the bacteria that can be used in a program to discover and develop a new antibacterial compound to treat human infections., other animals and vegetables.

This invention can be used in bacteria other than Neisseria gonorrhoeae. Specifically, strains of bacteria that can be transformed with DNA, by any method, that are capable of carrying out homologous recombination and for which the complete genetic sequence can be determined. Particular examples include, but are not limited to, Haemophilus influenzae, Streptococcus pneumoniae, Acinetobacter, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, and Bacillus subtilis.

MATERIAL AND METHODS Generation of isogenic Neisseria gonorrhoeae strains with altered gyrA and parC alleles PCR products containing mutations in gyrA (serine in residue 91 by phenylalanine and aspartate in residue 95 by glycine, hereinafter referred to as S91F and D95G) and parC (serine in position 88 by proline and glutamate in position 91 by lysine, hereinafter referred to as S88P and E91K) were created using splicing by overlapping extension (SOEing) (Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR, Site-directed mutagenesis by overlap extension using the polymerase chain reaction , Gene, 1989; 77: 51-59). All PCR reactions were performed using genomic DNA isolated from strain N400, a derivative of MS11. The construction of the strains was done in 2 stages. The first step was to generate a DNA fragment by polymerase chain reaction (PCR) containing the desired mutation. The second stage was to recombine the mutations within the chromosome, resulting in the replacement of the wild-type allele by which it contains the desired mutation (allele replacement).

Generation of the PCR product that contains site-specific gyrA mutations.

A gyrA PCR product of 480 bp was generated using primers A (d-GTCCGCCATGGCAGGTTTCTCGACAAAC-S ') (Sec. 4) and B (5'-CATACGGACGATGGTGCCGTAAACTGCGAAATCGCCGTGGGGGTG -3') (Sec. 5) (underlined altered restriction sites). A 570 bp gyrA PCR product that overlap the other gyrA product was generated using the primers C (5'-CACCCCCACGGCGATTTCGCAGTTTACGGCACCATCGTCCGTATG-3 ') (Sec. 6) and D (5-CAACTTGAATTCGTTGACCTGATAGGG-3) (Sec. 7). The resulting PCR products were purified and combined with primers A and D in a PCR reaction to produce a 1050 bp fragment (called the SD-FG gyrA PCR product) containing the desired gyrA mutations.

Generation of the Random Library of the Region that Determines the Resistance to Quinolone An 800 bp PCR product containing the first 600 bp of gyrA from N. gonorrhoeae was amplified using the oligonucleotides GC gyrA 5 'Ncol (5'-GTCCGCCATGGCAGGTTTCTCGACAAAC-3') (Sec. 8) and GC gyrA 3 'Hindlll ( 5-CCCAAGCITGATGGTGTCGGTGAGGTTG-3 ') (Seq. 9) (mutant residues in bold, restriction sites of underlined enzymes). The resulting fragment and pAlterEX-2 (Promega) were digested with Ncol and Hindlll, and ligated to create pAlt-gyrA.

To generate a group of isolated random insertions to gyrA QRDR (which encodes residues 88-103), the oligonucleotide g / Tl-random (5'-cacggcgattccgcagtttacgacacAa.tcgtccgtatag.cgcaaaatTTCGC-3 ') (Sec. 10) was synthesized by Integrated DNA Technologies (lower case nucleotides were synthesized using phosphoramidite standard solutions contaminated with 0.7% of each non-wild type phosphoramidite, the destroyed Xcml site is underlined). The resulting group of oligonucleotides contained an average of one random mutation per oligonucleotide. This 53-mer was used for site-specific mutagenesis of pAlt-gyrA by the manufacturer's protocol (Altered Sites, Promega). To ensure that all colonies resulting from mutagenesis were not wild-type, a silent C by A mutation generated in the primer (shown in bold) was generated which destroyed a unique Xcml site. This allowed all the plasmids to be digested with Xcml to eliminate non-recombinant plasmids. All colonies (-4000) isolated from the mutagenesis reactions were pooled together to generate a collection of plasmids containing substitutions of random single base pairs in gyrA corresponding to residues 88-103. This random library was then used as donor DNA in transformation experiments and isolates resistant to ciprofloxacin were isolated at .002 μg / mL. The first 600 base pairs of gyrA were sequenced to identify the mutation responsible for the resistance.

Generation of region-specific random mutagenesis by means of long low-fidelity PCR To evaluate the capacity of libraries generated from long low-fidelity PCR to generate quinolone-resistant Neisseria gonorrhoeae, oligonucleotides gyrA-F1 (5'-TCCATCCCGACAAATTCG-3 ') (Sec. 11) and gyrA-B1 (5'- TTGCGGTAGTGTTCGACCAG-3 ') (Sec. 12) to amplify an 8.8 kb fragment that contained the complete gyrA gene of N400, a derivative of N. gonorrhoeae (Tonjum et al., Mol. Micro., 1995; 16: 451-464 ) of rec / A-inducible MS11. This PCR product, and one generated using the resistant allele of NG-2709 (gyrA S91F, D95G) as a template, was transformed into the main strain by means of spot transformation and incubated 24 hours as previously described (Forest et al, Mol Microbio 1999 31 (3): 743-752). Ten microliters of 10-fold dilutions in series were then plated onto common plates and plates containing 0.002 μg of ciprofloxacin per milliliter. The bacteria were incubated after 40 hours at 37 ° C in a 5% C02 atmosphere, and the frequency of ciprofloxacin-resistant colony-forming units (CFUs) was determined by dividing the number of ciprofloxacin-resistant CFUs per milliliter by the total number of transformed cells per milliliter. Identification of mutations responsible for ciprofloxacin resistance The ciprofloxacin-resistant colonies isolated by the above procedure, using PCR libraries generated from wild-type DNA, were used as template DNA for PCR reactions using the GCs gyrA 5 'Ncol and gyrA 3' Hindll to generate a product of 800 bp which contains the 5 'portion of gyrA. These products were then used as donor DNA for another transformation and selection series to assess whether the resistance mutation was located within this region of the chromosome. The transformation frequencies of all the products were determined and the DNA sequencing of the donor DNA and the corresponding region of the newly created ciprofloxacin-resistant mutant were determined.

Generation of Fabl mutants resistant to DHDPE Random mutations were generated in Fabl as previously described (Kok et al.) Using the PCR product generated with Gc7 (5'-GGAATTCCATATGCGTATTTGAAACGTCCAATGCC-3 ') (Sec. 13) and Gc8 (5'-GCACCTGCAGCAATGCGG TAC-3) ( Sec. 14) using 10 ng of N400 genomic DNA as a template. The PCR reactions were performed with Taq polymerase (GIBCO-BRL) or the XL PCR kit (Perkin-Elmer). Ten independent PCR reactions were performed using each polymerase with the following reaction mixtures: 10 μl of 10x buffer (supplied with enzyme), 10 ng of N400 genomic DNA as template, 20 pmoles of primers, 200 μM of dNTP, and 1.5 mm of MgCl2 (for Taq) or 2.0 mm of Mg (OAc) 2 (for XL PCR) (final volume of 100 μl). The 20 reactions were pooled after 35 cycles of 95 ° C for 15 seconds, 58 ° C for 30 seconds and 72 ° C for 1 minute. The resulting PCR products were precipitated in ethanol and resuspended at 0.5 μg / mL in H2O for the subsequent transformation of the gonococcal strains.

The N400 was transformed with mutant PCR products using the spot transformation technique in solid medium or liquid transformation as previously described. The cells were then plated onto solid GC medium containing 0.5, 2 or 10 μg of DHDPE per mL to select DHDPE resistant bacteria. The isolated colonies were passed 2 times on solid GC medium to ensure homogeneity. The Fabl alleles were amplified by PCR directly from the colonies using Gc7 and Gc8 and sequenced. All PCR products containing N400 mutations were used to transform the N400 and the selection process was repeated. If the frequency of resistant mutants was at least 100 times that when using a PCR product generated using N400 DNA as a template, it was concluded that the mutation responsible for the resistant phenotype was fabl.

Generation of random library by means of mutagenic PCR of large regions of the chromosome.

PCR primers were designed using home software, PRIMER, together with BIGPRIME (a modification by the Genetics Computer Group of its PRIME program to allow products of up to 25 kb). PRIMER uses the BIGPRIME program to interactively design a list of oligonucleotide pairs to amplify regions of DNA spanning the received nucleic acid sequence. This region may consist of all or part of a genetic material of organisms. The oligonucleotides were designed to identify pairs of primers based on the following criteria; i) its ability to generate large PCR products of up to 20 kilobase bases in length, ii) the resulting products would contain at least one copy of the GC uptake sequence, and iii) each product contained at least 300 base pairs of sequence of overlap with adjacent products. The result of this process is a list of pairs of generated primers that will result in the PCR amplification of the region supplied with products that overlap each other in at least 300 base pairs, contains the uptake sequence, and are ideally 10 kilograms of bases in length.

Ten independent PCR products from each pair of primers was generated using the Gen-amp XL PCR kit (Perkin-Elmer) as described by the manufacturer with the following specifications: 100 μL reacts with 30 pmol of each pair of primers, 10 ng of Chromosomal DNA FA-1090, 200 μM dNTP, 1 mM Mg (OAc) 2, and standard regulatory equipment. Each reaction was subjected to 35 cycles of amplification on a PE 9600, in the PCR amplification was evaluated using agarose gel electrophoresis. The 10 analogous reactions were pooled to create a library of diverse random point mutations covering 8-12 kb of a defined portion of the chromosome of N. gonorrhoeae.

To confirm that the PCR products corresponding to the region of the predicted chromosome to be amplified, restriction digestion analysis was performed. Groups of PCR products were subjected to restriction digestions using EcoRI or HindIII and the fragments were separated using agarose gel electrophoresis. The fragment sizes were compared with the predicted digestions of the identical region based on the FA-1090 sequence. Primers that were unable to generate one or which resulted in patterns inconsistent with the predicted pattern were resigned using home software.

Generation and Isolation of Resistant Mutants PCR products from 12 adjacent regions of chromosome were then pooled (representing approximately 100 kb) were introduced into a wild-type strain by transformation (Zhang et al., PNAS, 1992; 89: 5366-5370). Briefly, 5 μg (1 μg / mL) of each group was stained on a freshly striated N. gonorrhoeae-containing plate, and the cells were incubated overnight to allow uptake and recombination of the mutant PCR products. The cells from each spot were then resuspended in 150 μL of GC and 5 μL of 10"\ 10" 2, and 10"3 dilutions were used to inoculate 96-well plates containing 100 μL of GC medium supplemented with Isovitalex and an inhibitory concentration of the antibacterial agent After 2-4 days of incubation at 37 ° C with 5% CO2 the wells containing viable bacteria were striated on common plates and individual colonies were isolated.

Identification of Mutations that Confer Resistance To identify the mutation responsible for the resistance phenotype, DNA from the resistant mutant was amplified in 12 independent reactions using pairs of primers corresponding to the region containing the resistance mutation. These products were then used as the donor DNA in transformation experiments as described above, and the PCR product containing the resistance mutation was identified by its ability to restore the resistance phenotype. By generating smaller PCR products (1-kb) that span the 8-12 kb PCR product that confers resistance, the transformation and selection process was repeated and the mutation was mapped to a fragment of 1-2 kb of DNA. The DNA sequence of this fragment was determined using fluorescence tinsion sequencing on an ABI 377 and analyzed using the SEQUENCHER program (Genecodes). The resulting sequence was compared to the analog region of wild-type DNA to identify any mutation fx LIST OF SEQUENCES < 110 > Dunham, Steven Olson, Eric < 120 > A MODEL SYSTEM FOR THE SUPPLY OF DRUGS < 130 > PR.3021.001 60 / 105,965 DRUG DISCOVERY < 140 > 60 / 105,965 < 141 > 1998-10-28 < 160 > 14 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 16 < 212 > PRT < 213 > Neisseria gonorrhoeae < 400 > 1 His Gly Asp Ser Wing Val Tyr Asp Thr Me Val Arg Met Wing Gln Asn 1 5 10 15 > L * < 210 > 2 < 211 > 173 < 212 > PRT < 213 > Neisseria gonorrhoeae < 400 > 2 Met Thr Asp Ala Thr Me Arg His Asp His Lys Phe Ala Leu Glu Thr 1 5 10 15 Leu Pro Val Ser Leu Glu Asp Glu Met Arg Lys Ser Tyr Leu Asp Thr 20 25 30 Wing Met Ser Val Me Val Arg Arg Wing Leu Pro Asp Val Arg Asp Gly 35 40 45 Leu Lys Pro Val His Arg Arg Val Leu Tyr Ala Met His Glu Leu Lys 50 55 60 Asn Asn Trp Asn Ala Ala Tyr Lys Lys Ser Ala Arg Me Val Gly Asp 65 70 75 80 Val Me Gly Lys Tyr His Pro His Gly Asp Ser Wing Val Tyr Asp Thr 85 90 95 e Val Arg Met Wing Gln Asn Phe Wing Met Arg Tyr Val Leu Me Asp 100 105 110 Gly Gln Gly Asn Phe Gly Ser Val Asp Gly Leu Wing Wing Wing Met 115 120 125 Arg Tyr Thr Glu Me Arg Met Wing Lys Me Ser His Glu Met Leu Wing 130 135 140 Asp Me Glu Glu Glu Thr Val Asn Phe Gly Pro Asn Tyr Asp Gly Ser 145 150 155 160 Glu His Glu Pro Leu Val Leu Pro Thr Arg Phe Pro Thr 165 170 < 210 > 3 < 211 > 261 < 212 > PRT < 213 > Neisseria gonorrhoeae < 400 > 3 Met Gly Phe Leu Gln Gly Lys Lys Me Leu Me Thr Gly Met Me Ser 1 5 10 15 Glu Arg Ser Me Wing Tyr Gly Me Wing Lys Wing Cys Arg Glu Gln Gly 20 25 30 Wing Glu Leu Wing Phe Thr Tyr Val Val Asp Lys Leu Glu Glu Arg Val 35 40 45 Arg Lys Met Ala Ala Glu Leu Asp Ser Glu Leu Val Phe Arg Cys Asp 50 55 60 Val Ala Ser Asp Asp Glu Me Asn Gln Val Phe Wing Asp Leu Gly Lys 65 70 75 80 His Trp Asp Gly Leu Asp Gly Leu Val His Being Me Gly Phe Wing Pro 85 90 95 Lys Glu Ala Leu Ser Gly Asp Phe Leu Asp Ser Me Ser Arg Glu Ala 100 105 110 Phe Asn Thr Ala His Glu Me Ser Ala Tyr Ser Leu Pro Ala Leu Ala 115 120 125 Lys Ala Ala Arg Pro Met Met Arg Gly Arg Asn Ser Ala Me Val Ala 130 135 140 Leu Ser Tyr Leu Gly Wing Val Arg Wing Me Pro Asn Tyr Asn Val Met 145 150 155 160 f * X "Gly Met Wing Lys Wing Wing Leu Glu Wing Gly Me Arg Phe Thr Wing Wing 165 170 175 Cys Leu Gly Lys Glu Gly and Arg Cys Asn Gly e Ser Gly Wing Pro 180 185 190 Me Lys Thr Leu Wing Wing Ser Gly Me Wing Asp Phe Gly Lys Leu Leu 195 200 205 Gly His Val Ala Ala His Asn Pro Leu Arg Arg Asn Val Thr Me Glu 210 215 220 Glu Val Gly Asn Thr Ala Ala Phe Leu Leu Ser Asp Leu Ser Gly 225 230 235 240 Me Thr Gly Glu Me Thr Tyr Val Asp Gly Gly Tyr Ser Me Asn Ala. 245 250 255 Leu Ser Thr Glu Gly 260 < 210 > 4 < 211 > 28 < 212 > DNA < 213 > Neisseria gonorrhoeae * < 400 > 4 gtccgccatg gcaggtttct cgacaaac 28 < 210 > 5 < 211 > 45 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 5 catacggacg atggtgccgt aaactgcgaa atcgccgtgg gggtg 45 < 210 > 6 < 211 > 45 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 6 cacccccacg gcgatttcgc agtttacggc accatcgtcc gtatg 45 < 210 > 7 < 211 > 27 < 212 > DNA < 213 > Neisseria gonorrhoeae caacttgaat tcgttgacct gataggg 27 < 210 > 8 < 211 > 28 < 212 > DNA < 213 > Neisseria gonorrhocae < 400 > 8 gtccgccatg gcaggtttct cgacaaac 28 < 210 > 9 < 211 > 28 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 9 cccaagcttg atggtgtcgg tgaggttg 28 < 210 > 10 < 211 > 53 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 10 cacggcgatt ccgcagttta cgacacaatc gtccgtatgg cgcaaaatttcgc 53 < 210 > 11 < 211 > 18 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 11 tccatcccga caaattag 18 < 210 > 12 < 211 > 20 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 12 ttgcggtagt gttcgaccag 20 < 210 > 13 < 211 > 35"• > * <212> DNA <213> Neisseria gonorrhoeae < 400 > 13 ggaasttccat atgcgtattt gaaacgtcca atgcc 35 < 210 > 14 < 211 > 21 < 212 > DNA < 213 > Neisseria gonorrhoeae < 400 > 14 gcacctgcag caatgcggta c 21

Claims (36)

42 [CLAIMS

1. A process for identifying and characterizing mutations leading to a selectable phenotype comprising: a) generating a defined set of overlapping 10 kb PCR products containing random point mutations spanning the entire chromosome of an organism; b) transform groups of 12 PCR products corresponding to 100 kb of the chromosome into a wild-type base; c) isolating strains of bacteria resistant to the compound; d) re-transforming sensitive bacteria with individual products, from 10 kb resistant strains, thereby identifying a reaction with one or more mutations; e) generate smaller PCR products, 1 kb, to promote map mutations responsible for the phenotype; and f) making a DNA sequence from the region that confers resistance to identify the chromosomal mutation.

2. A process for identifying and characterizing quinolone-DNA gyrase interactions using Neisseria gonorrhoeae comprising: a) randomly mutating the region determining the quinolone resistance (QRDR) of gyrA using site-specific mutagenesis mediated by an oligonucleotide with a 43 degenerate oligonucleotide or mutate the entire gene using low fidelity PCR; b) transform these random mutations into a wild type base; c) selecting isogenic quinolone-resistant mutants after analogous recombination; d) make a sequence of the gyrA QRDR confirming that Ser91 and Asp95 are independently involved in the inhibition by quinolone of Girasa DNA; e) identify the following new mutations associated with quinolone resistance in N. gonorrhoeae: Asp90 to Glu, Ser91 to Cys, Asp95 to His, Glu161 to Gly, Glu161 to Lys, Asn65 to His, AspdO to Gly, and Glu62 to Lys; and f) using these mutants to help understand the mechanism of action of quinolones, and other inhibitors of topoisomerase type IV.

3. Mutations in GyrA of Neisseria gonorrhoeae associated with quinolone resistance selected from: Asp90 in Glu, Ser91 in Cys, Asp95 in His, Glu161 in Gly, Glu161 in Lys, Asn65 in His, AspdO in Gly, and Glu62 in Lys.

4. The process according to claim 1, characterized in that to identify and characterize drug-target interactions. 44

5. A process for identifying and characterizing a mechanism of action of an antibacterial compound comprising: generating DNA fragments by DNA amplification by polymerase chain reaction of bacteria under conditions that allow the mutation of the fragments; allowing one or more of the generated DNA fragments to be incorporated into the chromosome of a bacterium by homologous recombination; isolate bacteria that demonstrate resistance to an antibacterial compound; and identify the mutation contained in the DNA fragment.

6. A process for identifying mutations contained in the chromosome of a bacterium that results in an identifiable phenotype characterized in that it comprises: a) generating DNA fragments by amplification of the bacterial chromosome by polymerase chain reaction corresponding to regions of the bacterial chromosome that can contain a mutation; b) allowing one or more of the DNA fragments to be incorporated into the chromosome of a bacterium that does not exhibit the phenotype identifiable by homologous recombination; Four. Five c) isolate bacteria that demonstrate the identifiable phenotype; repeat the steps from a to c until a fragment of the single minor DNA of approximately 10 kilobases in length is identified as being responsible for the phenotype; and identify the mutation contained in the DNA fragment.

7. The process according to claim 5, characterized in that the gene fragments generated collectively encode the complete genome of the bacteria.

8. The process according to claim 1, characterized in that the bacteria are from the group of genera Neisseria, Haemophilus, Streptococcus, Staphylococcus, or Escherichia.

9. The process according to claim 5 or 6, characterized in that the bacteria are from the group Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, or Escherichia coli. 46

10. The process according to claim 5, characterized in that the antibacterial compound is a fluoroquinolone.

11. The process according to claim 5, characterized in that the antibacterial compound is ciprofloxacin.

12. The process according to claim 5, characterized in that the antibacterial compound is clinafloxacin.

13. The process according to claim 5, characterized in that the compound is dihydroxydiphenylether (DHDPE).

14. The process according to claim 5, characterized in that the antibacterial compound is Triclosan.

15. The process according to claim 6, characterized in that the DNA fragments are generated using bacteria that contain a mutation necessary for resistance to an antibacterial compound. 47

16. The process according to claim 1 or 2, characterized in that the antibacterial compound inhibits the growth or subsistence of the bacteria under any condition.

17. The process according to claim 1 or 2, characterized in that the antibacterial compound inhibits the growth or subsistence of the bacteria in culture.

18. The process in accordance with the claims 1 or 2, characterized in that the antibacterial compound inhibits the growth or subsistence of the bacteria in an animal host.

19. The process according to claim 1 or 2, characterized in that the antibacterial compound is a type II topoisomerase inhibitor.

20. The process according to claim 1 or 2, characterized in that the antibacterial compound is a Fabl inhibitor.

21. The process according to claim 1 or 2, characterized in that the antibacterial compound is an enzyme inhibitor involved in fatty acid biosynthesis. 48

22. The process according to claim 6, characterized in that a strain of bacteria carrying the mutation was isolated from a culture that had been treated as a chemical mutagene.

23. The process according to claim 6, characterized in that a strain of bacteria carrying the mutation was isolated from a culture that had been treated with ultraviolet light.

24. The process according to claim 6, characterized in that a strain of bacteria carrying the mutation was isolated from a culture in which the bacteria had been subjected to a mutagenic protocol consisting of the insertion of DNA into the chromosome of the bacteria.

25. Bacteria comprising a protein in which a contiguous dilation of 40 amino acids is at least 30% identical to residues 75 to 114 of GyrA Neisseria gonorrhoeae and the analogous residue for: 62 is lysine or 63 is arginine or glutamic acid or 65 is histidine or 135 is valine or 161 is glutamic acid or Msina or glycine. 49

26. Strains of Escherichia coli comprising a GyrA protein in which the amino acid analogue of the GyrA amino acid of Neisseria gonorrhoeae 62 is lysine or 63 is arginine or glutamic acid or 65 is histidine or 135 is valine or 161 is glutamic acid or lysine or glycine

27. The Neisseria gonorrhoeae strains comprising a GyrA protein in which the amino acid residue 62 is lysine or 63 is arginine or glutamic acid or 65 is histidine or 80 is alanine or glycine, or 90 is arginine or glutamic acid, or 91 is tyrosine or alanine or cysteine, or 92 is proline, or 95 is arginine or alanine or valine or tyrosine or histidine or glycine, or 114 is histidine, or 135 is valine, or 161 is glutamic acid or lysine or glycine. fifty

28. Strains of Neisseria gonorrhoeae selected from the group consisting of NG-2707, GC318, NG-2721, NG-2711, NG-2706, NG-2717, NG-2687, GC 158, NG-2690, GC219, GC291, NG- 2691, NG-2720, NG-2723, GC156, NG-2698, NG-2709, NG2716, NG-2719, and NG-2712.

29. A protein comprising of which a contiguous dilation of 40 amino acids that is at least 30% identical to residues 75 to 114 of the Neisseria gonorrhoeae of GyrA and the analogous residue to: 62 is lysine or 63 is arginine or glutamic acid or 65 is histidine or 135 is valine or 161 is glutamic acid or lysine or glycine.

30. Neisseria gonorrhoeae GyrA protein comprising amino acid substitutions when residue 62 is lysine, or 63 is arginine or glutamic acid, or 65 is histidine, or 80 is alanine or glycine, or 90 is arginine or glutamic acid, or 91 is tyrosine or alanine or cysteine, or 92 is proline, or 51 95 is arginine or alanine or valine or tyrosine or histidine or glycine, or 114 is histidine, or 135 is valine, or 161 is glutamic acid or lysine or glycine.

31. Bacteria comprising a protein that are at least 30% identical to the sequence of the Neisseria gonorrhoeae Fabl protein in which the amino acid residue corresponding to 15 is valine, or 20 is threonine, or 23 is glycine, or 25 is valine , or 51 is threonine, or 91 is threonine, or 93 is cysteine or serine, or 95 is valine, or 104 is leucine, or 105 is histidine, or 144 is valine, or 147 is histidine, or 159 is alanine, or 160 is isoleucine, or 162 is valine, or 52 193 is asparigin or valine, or 201 is valine, or 203 is tyrosine or valine, or 204 is serine or leucine or isoleucine or valine, or 212 is threonine or valine, or 247 is asparagine.

32. A strain of Neisseria gonorrhoeae selected from the group consisting of NG-2669, NG-2654, NG-2651, NG-2670, NG-2660, NG-2641, NG-2639, NG-2638, NG-2640, NG-2648 , NG-2657, NG-2656, NG-2653, NG-2658, NG-2663, NG-2642, NG-2671, NG-2652, NG-2661, NG-2644, NG-2667, NG-2665, NG -2655, NG-2643, NG-2666, NG-2664, NG-2647, NG-2646, NG-2650, NG-2649, NG-2645, NG-2659, NG-2662, and NG-2672.

33. A strain of Escherichia coli comprising a Fabl protein with the amino acids analogous to those described in claim 31, with the exception of mutations resulting in the change of residue 93 in alanine or serine or cysteine or valine, 159 in threonine or, 203 in leucine. 53

34. A protein comprising at least 30% identical to the sequence of the Neisseria gonorrhoeae protein in which the amino acid residue corresponding to 15 is valine, or 20 is threonine, or 23 is glycine, or 25 is valine, or 51 is threonine or 91 is threonine, or 93 is cysteine or serine, or 95 is valine, or 104 is leucine, or 105 is histidine, or 144 is valine, or 147 is histidine, or 159 is alanine, or 160 is isoleucine, or 162 is is valine, or 193 is asparigin or valine, or 201 is valine, or 203 is tyrosine or valine, or 204 is serine or leucine or isoleucine or valine, or 212 is threonine or valine, or 247 is asparagine. 54

35. A Fabl Neisseria gonorrhoeae protein comprising the amino acid corresponding to the residue: 15 is valine, or 20 is threonine, or 23 is glycine, or 25 is valine, or 51 is threonine, or 91 is threonine, or 93 is cysteine or serine or 95 is valine, or 104 is leucine, or 105 is histidine, or 144 is valine, or 147 is histidine, or 159 is alanine, or 160 is isoleucine, or 162 is valine, or 193 is asparigin or valine, or it is valine, or 203 is tyrosine or valine, or 204 is serine or leucine or isoleucine or valine, or 212 is threonine or valine, or 247 is asparagine. 55

36. The process for selecting compounds for antibacterial activity comprising: generating DNA fragments by DNA amplification by polymerase chain reaction of a complete genome of bacteria under conditions that allow the fragments to mutate; allowing one or more of the generated DNA fragments to be incorporated into the chromosome of bacteria by homologous recombination; isolation of bacteria that demonstrate resistance to an antibacterial compound; identify the mutation contained in the DNA fragment; contact the bacteria with compounds; and evaluate the compounds for antibacterial activity.