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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/90—Isomerases (5.)

Definitions

• the present invention relates to a new topoisomerase IV, the nucleotide sequences coding for this enzyme, their corresponding vectors and the use of this enzyme to screen biologically active products.
• Topoisomerases are enzymes capable of modifying the topological configuration of DNA rings, of knotting them or of entangling separate rings. They are thus involved in the replication, transcription and recombination of all genetic information (Wang et al., 1990). The mechanism of all these topological conversions is the same: the ring is opened so that a segment of DNA passes through the gap before the ends meet. Two types of topoisomerases are involved in these conversions: type I topoisomerases which cut a single strand of DNA and type II topoisomerases which cut both strands simultaneously.
• Gyrase is an ⁇ 2 ⁇ 2 tetramer whose ⁇ or GyrA and ⁇ or GyrB subunits are encoded respectively by the gyrA and gyrB genes.
• Bacterial gyrases are the only known topoisomerases capable of supercoiling, in the presence of ATP, released DNA rings.
• DNA topoisomerase IV from E. coli, it releases the supercoiled plasmid DNA, unravels the DNA of phage T4 and de-embraces (or decatene) the kinetoplast DNA (Kato et al., 1992 ; Peng et al., 1993).
• the sequence of its corresponding genes, parC and parE from E.coli made it possible to highlight regions of high identity between the subunits of gyrase and those of this topoisomerase IV, respectively ParC with GyrA (35,6 % over the entire sequence) and ParE with GyrB (40.1% over the entire sequence) (Kato et al., 1990).
• the gyrase of E. coli has also been identified as a primary target of fluoroquinolones (Hooper et al., 1993). It has thus been demonstrated that strains of E. coli, mutated at the Ser83 residue in the GyrA subunit, exhibit high resistance to fluoroquinolones (Maxwell, 1992). The fluoroquinolones bind less to mutated DNA-gyrase complexes than to wild-type DNA gyrase complexes. In fact, other point mutations, mapped in the region between residues 67 and 106 of GyrA, lead to strains resistant to fluoroquinolones.
• This region is called QRDR (Yoshida et al, 1990; Cullen et al., 1989). Comparable results have been published with strains of Staphylococcus aureus resistant to fluoroquinolones (Goswitz et al., 1992; Sreedharan et al., 1990). Gyrase is therefore recognized today as the primary target of quinolones. However, a clinical strain of Staphylococcus aureus, having no mutation in the QRDR region of GyrA, has also been described as resistant to fluoroquinolones (Sreedharan et al, 1991).
• the main objective of the present invention is precisely the identification, sequencing and characterization of nucleic sequences coding for subunits of a new topoisomerase, the topoisomerase IV of Staphylococcus aureus, composed of two subunits the Gr1A and the Gr1B.
• the Applicant has found that the primary target of fluoroquinolones in S. aureus is a topoisomerase IV and not gyrase. It has thus demonstrated that clinical strains of S. aureus, whose QRDR region of the GyrA subunit of the gyrase is identical to the wild-type sequence, nevertheless develop resistance to fluoroquinolones due to a mutation they possess in the region of the GrlA subunit of topoisomerase IV, homologous to the QRDR region.
• the present invention first relates to a nucleotide sequence coding for at least one subunit of the topoisomerase IV of Staphylococcus aureus.
• the present invention describes in particular the isolation and characterization of the gr1A and gr1B genes. These genes have been cloned, sequenced and expressed in E. coli, and their enzymatic activity has been characterized. They were isolated from a bank of genomic DNA from Staphylococcus aureus. From the nucleic acid sequence gr1AB (SEQ ID N ° 1), two open phases have been identified, corresponding respectively to the gr1B and grlA genes The gr1A and gr1B genes have been sequenced in SEQ ID K ° 2 and SEQ ID N ° respectively 3.
• the present invention relates to a nucleotide sequence chosen from:
• the term “derivative” means any sequence obtained by one or more modifications and coding for a product retaining at least one of the biological properties of the original protein. Modification means any mutation, substitution, deletion, addition or modification of a genetic and / or chemical nature. These modifications can be carried out by techniques known to those skilled in the art.
• the invention relates to the nucleotide sequences represented by the genes gr1A (SEO ID No. 2) and gr1B (SEQ ID No. 3). It also relates to any gr1A gene having a mutation leading to resistance vis-à-vis molecules of the quinolone family and more particularly fluoroquinolones. As a representative of these mutated genes, mention may more particularly be made of the gr1A gene having a base change from C to A at position 2270 of SEQ ID No. 2. The resulting gene is said to be gr1A (C-2270A) . This mutation leads to a substitution of the Ser-80 residue in Tyr in the Gr1A protein. The resulting protein will be designated as Gr1A (Ser-80 Tyr) .
• Another object of the present invention relates to a recombinant DNA comprising at least one nucleotide sequence coding for a subunit of topoisomerase IV of Staphylococcus aureus. More preferably, it is a
• Recombinant DNA comprising at least one nucleotide sequence as defined above in (a), (b) and (c) and more particularly the gr1A gene (SEQ ID No. 2)
• nucleotide sequences form part of an expression vector which can be autonomously replicating or integrative.
• Another subject of the invention relates to the polypeptides resulting from the expression of the nucleotide sequences as defined above. More particularly, the present invention relates to polypeptides comprising all or part of the polypeptides Gr1A (SEQ ID No. 2) or Gr1B (SEQ ED No. 3) or their derivatives.
• the term derivative designates any molecule obtained by modification of a genetic and / or chemical nature of the peptide sequence. By modification of genetic and / or chemical nature, one can hear any mutation, substitution, deletion, addition and / or modification of one or more residues.
• Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its substrate (s), that of improving its production levels, that of increasing its resistance to proteases, that of increasing and / or modifying its activity, or that of giving it new biological properties.
• chimeric polypeptides comprising an additional heterologous part linked at one end.
• the term derivative also includes polypeptides homologous to the polypeptides described in the present invention, obtained from other cellular sources. Preferably; these are the polypeptides Gr1A (SEQ ID No. 2), GrlB (SEQ ID No. 3) and Gr1A (Ser-80Tyr) .
• the subject of the invention is also any recombinant cell containing a nucleotide sequence, a recombinant DNA and / or a vector as defined above.
• the recombinant cells according to the invention can be both eukaryotic and prokaryotic cells.
• eukaryotic cells which are suitable, mention may be made of animal cells, yeasts, or fungi.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Kluweromyces, Pichia, Schwanniomyces, or Hansenula.
• yeasts mention may be made of yeasts of the genus Saccharomyces, Kluweromyces, Pichia, Schwanniomyces, or Hansenula.
• animal cells mention may be made of COS cells, CHO, C127, Xenopus eggs, etc.
• Micromonospora Aspergillus ssp. or Trichoderma ssp.
• these are prokaryotic cells.
• the following bacteria can more particularly be used Actinomycetes, Bacillus, and more preferably E.coli and Staphylococcus.
• the recombinant cells of the invention can be obtained by any method which makes it possible to introduce a foreign nucleotide sequence into a cell. It may especially be transformation, electroporation, conjugation, fusion of protoplasts, or any other technique known to those skilled in the art.
• the present invention also relates to a process for the preparation of polypeptides as claimed from the culture of one of these recombinant cells.
• the polypeptide or polypeptides thus obtained are recovered according to conventional methods at the end of the culture.
• the invention also relates to an isolated topoisomerase IV, capable of being obtained from the expression of all or part of the gr1A gene (SEQ ID No. 2) and all or part of the gr1B gene (SEQ ID No. 3) or their respective derivatives.
• the term “derivative” is intended to denote the sequences hybridizing with all or part of the gr1A or gr1B gene and coding for a subunit of a topoisomeraselV as well as all of the sequences deriving from a degeneration of the genetic code of these hybrid sequences or of the sequences corresponding to all or part of the gr1A or gr1B gene.
• the present invention is an isolated topoisomerase IV, resulting from the expression of all or part of the gr1A gene (SEQ ID No. 2) and all or part of the gr1B gene (SEQ ID No. 3).
• the present invention relates more particularly to any topoisomerase IV having a primary target behavior with regard to fluoroquinolones.
• the topoisomerase IV of Staphylococcus aureus is the topoisomerase IV of Staphylococcus aureus.
• the topoisomerase IV claimed according to the invention is very particularly useful for screening biologically active products such as, for example, potential antibiotics and in particular molecules of the fluoroquinolone family.
• it can also be used to assay and / or identify products inhibiting the DNA-dependent ATP relaxation reaction and / or products inhibiting the DNA catenan decatenation reaction.
• the Applicant has thus developed an assay for specific enzymatic activity of topoisomerase IV of S. aureus and has shown that this activity is inhibited by antibiotic molecules such as fluoroquinolones.
• the present invention provides a new target for searching for new antibiotics, as well as a screen specific for this target, this screen is described in Example 7.
• This screen makes it possible to demonstrate products inhibiting DNA topoisomerase IV of S. aureus.
• the following can be tested: pure synthetic products or a mixture, natural plant extracts, cultures of bacteria, fungi, yeasts or algae, pure or mixed.
• the test described in the present invention makes it possible to demonstrate both products which stabilize the cleavable complex, the reaction intermediate of the reaction catalyzed by the enzyme, but also inhibitors acting by other mechanisms.
• FIG. 1 Restriction map of the fragment of 4565 bp containing the genes gr1B and gr1A of S. aureus.
• Figure 2 Construction of gr1A and gr1B expression plasmids. The constructions made with gr1A are shown diagrammatically in A and those of gr1B are in B. The DNA of cloned S. aureus is represented by the hatched rectangles, the vectors derived from M13 are in thick black line and the expression vectors are in fine black line, the SstI restriction site is in brackets because it is a cloning site.
• Figure 3 PAGE-SDS electrophoresis gel stained with Coomasie blue. Are deposited total cell extracts, lanes: 1 and 2, XL1-blue, pXL2340, 3 and 4, XL1-blue, pRSETB; 5 and 6, XL1-blue, ⁇ XL2320. The molecular weight markers (in hundreds) are shown to the right of the figure. The arrow shows the overproduced protein. The signs + or - represent induction with or without IPTG.
• FIG. 5 Decatenation activity of the Gr1AB protein. kDNA, kinetoplast DNA; monomers, released and decatenated DNA monomers.
• TopoIV purified E. coli DNA topoisomerase IV (50 ng); Gyrase: purified E. coli DNA gyrase (50 ng);
• Gr1A protein extract of Gr1A (2 ⁇ g);
• GrlB protein extract of GrlB (2 ⁇ g);
• Gr1AB protein extract from Gr1A (2 ⁇ g) mixed with the protein extract from GrlB (2 ⁇ g).
• Example 1- Amplification by PCR of DNA fragments of Staphylococcus aureus internal to the gr1A and gr1B genes.
• This example describes the obtaining of DNA fragments of Staphylococcus aureus internal to the gr1A and gr1B genes. These fragments were obtained after amplification by PCR carried out at 50 ° C with the genomic DNA of the strain of Staphylococcus aureus RN4220 (Novick, 1990) and degenerate oligonucleotides corresponding to the amino acids conserved in the N-terminal regions of the subunits GyrA from E. coli and B. subtilis and ParC from E. coli or GyrB subunits from E. coli and B. subtilis and ParE from E. coli.
• the sense oligonucleotides 2137 and antisense 2135 made it possible to amplify fragments of 255 bp which can code for 85 amino acids which would correspond to positions 39 to 124 on the GyrA sequence of E. coli;
• the sequence of sense oligonucleotide 2137 is 5'-GCGCGAATTCGATGG (A, T) (C, T) T (A, T) AAACC (A, T) GT (A, T) CA-3 '(SEQ ID NO: 4) and that of the antisense 2135 is 5'-CGCGAAGCTTTTC (T, A) GTATA (A, T) C (T, G) CAT (A, T) GC (A, T) GC- 3 '(SEQ ID N ° 5).
• Oligonucleotides 2144 and 2138 led to the amplification of 1 kb fragments which can code for 333 amino acids which would correspond to positions 98 to 430 on the GyrB sequence of E. coli; the sequence of sense oligonucleotide 2144 is 5'-GCGCGAATTCT
• fragment A has 59% identity with the GyrA subunit of S. aureus between positions 44 to 125, fragment A would therefore be a part of a gene for S. aureus gr1A as well. newly identified.
• amino acid sequence that fragment B would encode has 51% identity with the GyrB subunit of S. aureus between positions 105 to 277, fragment B would therefore be a part of an S gene. aureus gr1B thus newly identified.
• This example describes the molecular biology experiments which made it possible to clone and then sequence the gr1A and gr1B genes of Staphylococcus aureus.
• Example 1 The fragments A and B described in Example 1 were used as radioactive probe to identify by hybridization the genes gr1A and gr1B in a genomic DNA library of S. aureus FDA 574 (CE ent + ) constructed in ⁇ gt11 by
• Clontech Laboratories (catalog XL 1501b, lot 0721). Out of a total of 250,000 recombinant phages, twelve phages hybridize with fragment A or fragment B but do not hybridize with specific oligonucleotides of the gyrA or gyrB genes. The size of the EcoRI inserts contained in these phages varies between 0.7 and 3.5 kb and two phages 16 and 111, the insert of which is larger, have been studied.
• the 3.5 kb EcoRI insert from phage 16 was eluted and then digested with Xbal and the two 1.5 and 2 kb fragments were cloned into M13mpl9 and M13mpl 8 (Boehringer Mannheim) to generate pXL2321 and pXL2322.
• the 3.6 kb EcoRI insert from phage 111 was eluted and then digested with PstI and the 2 kb fragment was cloned in M13mp19 to generate pXL2324.
• the inserts contained on the recombinant phages pXL2321, pXL2322 and pXL2324 were sequenced on both strands using the universal primer or internal oligonucleotides using the Sanger method.
• the nucleic sequence gr1AB (SEQ ID No. 1) of 4565 bp, was analyzed by the program of Staden et al., 1982 to identify the coding sequences using a table of codon usage in S. aureus. .
• Two open phases only ORF1 (positions 41 to 2029) and ORF2 (positions 2032 to 4431) were thus determined.
• the coding strand is the upper strand 5 '-> 3'
• the open phase ORF1 starts arbitrarily at ATG position 41 but it can also start at TTG position 17 or 35, this codon already being described as initiation codon in S. aureus; the termination codon of ORF1 overlaps with the initiation codon GTG of O RF2 which is characteristic of translational coupling (Normark et al., 1983); such a coupling has for example been described for the gyrA er gyrB genes of Haloferax sp. (Holmes et al., 1991). These open phases have a GC percentage of 34.5% which is a value in agreement with the values described for the DNA of S.
• fragment B is identical to the sequence described in SEQ ID N ° 1 from position 333 to position 1348 in ORFl and fragment A is identical to the sequence of SEQ ID N ° 1 from position 2137 at position 2394 in ORF2. From the nucleotide sequence, a restriction map is produced with enzymes which cut less frequently, see FIG. 1.
• ORF1 is the gr1B gene and ORF2 the gr1A gene.
• Example 3- Primary structure, expression and function of the proteins Gr1A and GrlB encoded by the genes gr1A and gr1B of Staphylococcus aureus.
• This example describes the primary structure, the expression in E. coli and the function of the Gr1A and GrlB proteins of Staphylococcus aureus.
• This function which corresponds to DNA topoisomerase IV, is based in this example on data of sequence homologies and genetic complementation
• This example describes the computer analysis of the sequence of the gr1A and gr1B genes of Staphylococcus aureus, carried out using the sequence data presented in example 2.
• the gr1B gene codes for a GrlB protein of 663 amino acids (molecular weight 74,318 ); and the gr1A gene codes for a Gr1A protein of 800 amino acids (molecular weight 91,040).
• the coding parts of the gr1B and gr1A genes, the sequences of the Gr1B and Gr1A proteins are presented respectively in SEQ ED No. 3 and SEQ ID No. 2 and the properties of each of these proteins (amino acid composition, isoelectric point, index of polarity) are shown in Tables 1 and 2 below.
• the Kanehisa program described in 1984, was used to align the GrlB and Gr1A proteins with bacterial type II DNA topoisomerases following the gyrases of E. coli, B. subtilis or S. aureus or the topoisomerase IV of E. coli
• the identities see table 3, are high and between 32 and 55%. More specifically, GrlB has more identity with the GyrB subunits of E. coli (49%) and S. aureus (52%) than with E. coli ParE (38%), while Gr1A presents comparable identities with the GyrA subunits of E. coli (32%) and S. aureus (39%) than with E. coli ParE ( 33%).
• the subyrms GyrB of Staphylococcus aureus (Margerrison et al., 1992), Bacillus subtilis (Moriya et al., 1985), and Escherichia coli (Adachi et al., 1987) are named SAGYRB, BSGYRB and ECGYRB respectively, GrlB is appointed
• SAGRLB and ECPARE corresponds to ParE from E coli (Kato et al., 1990).
• a comparable nomenclature is used for the GyrA Gr1A and ParC subunits.
• the numbers under the name of proteins are the numbers of amino acids of these.
• the expression plasmid pXL2320 containing the gr1B gene in the vector pRSETB (Studier et al., 1990; Invitrogen) was constructed by cloning 1) the 1 kb EcoRI-Xbal insert from pXL2321 in pXL2322 to Xbal and EcoRI sites to generate pXL2323; 2) the 1.9 kb Kpnl-EcoRI insert of pXL2323 at the Kpnl and EcoRI sites of the vector pRSETB to generate pXL2319; the 0.5 kb Ndel-Kpnl insert of pXL2325 at the Ndel and Kpnl sites of pXL2319 to obtain pXL2320.
• PXL2325 contains the first 500 bases' of the gene where a CAT sequence has been introduced by mutagenesis, just upstream from the ATG initiation codon to create an NdeI site).
• the expression cassette for the gr1B gene contained in pXL2320 was cloned at the BglII and EcoRI sites of pKT230 (Bagdasarian et al., 1981) to obtain pXL2439.
• the expression plasmid pXL2340 containing the gr1A gene in the vector pRSETB was constructed by cloning 1) the 1.7 kb Ndel-EcoRI insert from pXL2324 at the Ndel and EcoRI sites of the pRSETB vector to generate pXL2338 ; the 0.75 kb Ndel insert of pXL2337 at the Ndel sites of pXL2338 to obtain pXL2340.
• PXL2337 contains the first 750 bases of the gene where a CATATG sequence has been introduced by mutagenesis, in place of the initiation codon GTG to create an NdeI site).
• Plasmids pXL2320, or pXL2340 were introduced into the E. coli XL1-Blue strain (Stratagen) and gene expression was induced when phage T7 RNA polymerase was produced after induction of the gene, coding for T7 phage RNA polymerase, cloned on the M13 / T7 helper phage (Studier et al., 1990, Invitrogen). The cell extracts were analyzed by electrophoresis on PAGE-SDS gel stained with Coomasie blue as has already been described (Denèfle et al., 1987).
• FIG. 3 is shown the production of a protein of i) 79,000 molecular weight, when the gr1B gene is induced in the E. coli XL1-Blue strain, pXL2320; and ii) 90,000 molecular weight, when the gr1A gene is induced in the E. coli XL1-Blue strain, pXL2340.
• the molecular weights measured are in agreement with the molecular weights deduced from the sequence.
• This example describes the heterologous complementation of the mutants of S. typhimurium parCts and parEts by the genes of S. aureus gr1A and gr1B.
• the plasmids pXL2320, pXL2340, pXL2439 or the vector pRSETB were introduced into the strains of S. typhimurium SE7784 ( ⁇ arC281 (Ts) zge-2393 :: Tn10 leu485) or SE8041 (parE206 (Ts) zge-2393 :: Tn10 leu485) (Luttinger et al., 1991).
• thermosensitive phenotype No plasmid complements the thermosensitive phenotype, on the other hand when the plasmids pXL2340 and pXL2439 are introduced simultaneously into the strain SE7784 or into the strain SE8041 the thermosensitive phenotype of the two strains is complementary. Consequently, the coexpression of the gr1A and gr1B genes of S. aureus allows the complementation of the ParC Ts or ParE Ts phenotype of the mutants of S. typhimurium.
• Example 4- DNA topoisomerase IV of S. aureus is the primary target of fluoroquinolones.
• This example describes the presence of a point mutation Ser-80 in the Gr1A subunit with all the clinical strains of S. aureus analyzed resistant to fluoroquinolones while a mutation in the QRDR region (Quinolone Determining Region) (equivalent to the Ser-80 region of Gr1A) in the GyrA subunit does not exist with clinical strains of S. aureus weakly resistant to fluoroquinolones. Therefore the Gr1A subunit is shown to be the primary target of fluoroquinolones in S. aureus.
• the genomic DNA of eight clinical strains of S. aureus and one laboratory strain was prepared and used to amplify at 42 ° C by PCR: i) the first 500 base pairs of gyrA using the 5 'sense oligonucleotides - GGCGGATCCCATATGGCTGAATTACCTCA-3 '(SEQ ID N ° 10) and antisense 5'- GGCGGAAT TCGACGGCTCTCTTTCATTAC-3' (SEQ ID N ° 11); ii) and the first 800 base pairs of gr1A using the sense oligonucleotides 5'- GGCCGGATCCCATATGAGTGAAATAATTCAAGATT-3 '(SEQ ID No.
• the mutations in gyrA exist with strains highly resistant to fluoroquinolones (SA4, SA5 , SA6, S A35, SA42R and SA47; MIC for ciprofloxacin> 16 mg / l); these mutations are a basic change that leads to changes in the amino acids Ser-84 or Ser-85 or Glu-88.
• a mutation in gr1A exists with all strains resistant to fluoroquinolones and corresponds to the change of the residue Ser-80 into Phe or Tyr.
• Example 5 Amplification by PCR (Polymerase Chain Reaction) of the DNA fragment of S. aureus internal to gr1A containing a point mutation which leads to a substitution in Gr1A of Ser-80 in Tyr (Ser-80-> Tyr).
• This example describes the obtaining of the DNA fragment internal to gr1A from a strain of S. aureus. SA2, resistant to fluoroquinolones.
• the gr1A fragment contains a base change from C to A at position 2270 of the wild-type gene (Fig. 1).
• This mutation leads to a substitution of the Ser-80 residue in Tyr in the Gr1A protein. It has been shown that a substitution of the residue Ser-80 for Phe or Tyr exists with all the strains weakly resistant to fluoroquinolones (Example 4).
• the internal fragment to gr1A was obtained after amplification by PCR carried out at 50 ° C. with the genomic DNA of the strain SA2 and of the oligonucleotides 3358 and 3357 which correspond respectively to position 2036 and 3435 on the sequence of gr1A. More specifically, sense oligonucleotides 3358 (SEQ ID No. 12) (Example 4) and antisense 3357 made it possible to amplify a fragment of 1399 base pairs; the sequence of the antisense oligonucleotide 3357 is 5'-
• Oligonucleotide 3358 was also used to introduce, by mutagenesis, a CATATG sequence, in place of the GTG initiation codon to create an NdeI site.
• the amplified gr1A fragment was cloned into the BamHI / SstI cloning sites of pUC18 (Boehringer Mannheim), and 6 clones containing this plasmid, pXL2692, were analyzed after sequencing of their insert. In all cases a CATATG sequence was introduced in place of the initiation codon GTG, and the point mutation at position 2270 of gr1A (C ⁇ A) was found.
• Example 6 Expression in E. coli of the gr1A gene containing a base change corresponding to the change of the Ser-80 residue in Tyr.
• This example describes the construction carried out to express, in E. coli, the mutated gr1A gene under the control of the T7 promoter (Studier et al., 1990).
• the expression plasmid pXL2742, containing the mutated gr1A gene, was constructed by cloning the 0.75 kb insert of pXL2692 into the NdeI site of pXL2338 (Example 3.2).
• the plasmid pXL2742 was introduced into the E. coli XL1-Blue strain and the expression of the gr1A gene was carried out as described in Example 3.2.
• Example 7 DNA topoisomerase IV activity of the Gr1AB protein of S. aureus.
• This example illustrates how an acellular extract containing the Gr1AB protein can be prepared and how the enzymatic activity of the Gr1AB protein present in this extract can be detected and measured.
• the E. coli XL1-blue pXL2340 strain is cultured as follows: 250 ml of LB medium containing ampicillin at 50 mg / l are inoculated at 1 / 100th with an E. coli XL1-blue pXL2340 culture, and incubated at 30 ° C; when the optical density at 600 nm is 0.3, 1 mM IPTG is added; after 30 min of incubation at 37 ° C., the strain is infected with the phage helper M13 / T7 with a multiplicity of infection of 5 pfu per cell for 4 hours.
• the cells obtained from 1.5 liters of culture are resuspended in 20 ml of 50 mM Tris / HCl buffer pH 7.8 containing 10 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.12 % Brij 58 and 0.75 mg / ml of lysozyme. After 30 min at 4 ° C., the mixture is centrifuged for 1 hour at 50,000 x g and the supernatant containing the Gr1A protein is recovered.
• a buffer exchange is carried out on this sample by chromatographing the extract through a column filled with Sephadex G625 (Pharmacia) equilibrated and eluted with Tris / HCl buffer 50 mM pH 7.5 containing 1 mM EDTA, 5 mM DTT, 100 mM NaCl and 10% sacharose.
• a cell-free extract containing the GrlB protein is similarly prepared from the E. coli XL1-blue pXL2320 strain.
• This example illustrates how an enzyme of S. aureus catalyzing the segregation of child chromosomes during the final phase of replication (topoisomerase IV) can be purified.
• the purification of the two subunits Gr1A and GrlB of topoisomerase IV is carried out as described below, using the assay of the decatenation activity described in Example 7.3 to detect the presence of the proteins Gr1A and GrlB throughout of purification, as is commonly used by those skilled in the art.
• the complementation of the fractions containing the Gr1A protein is obtained with 1 ⁇ g of proteins from an extract of the E.
• the purification of the A subunit can be carried out by chromatography, for example by following the following protocol:
• an acellular extract prepared as described in Example 7.1 from approximately 5 g of E. coli XL1-blue pXL2340 cells is chromatographed on a MbnoQ HR 10/10 column (Pharmacia) at a flow rate of 3 ml / min with a linear gradient of NaCl (0.1 M to 0.6 M in 60 min) in a pH 8.0 Tris / HCl 10 mM buffer containing 1 mM EDTA, 1 mM DTT and 10% glycerol (w / v).
• the active fractions are combined and the sample is chromatographed on a Superdex 200 HiLoad 26/60 column (Pharmacia) balanced and eluted with pH 7.5 Tris / 50 mM HCl buffer containing 1 mM EDTA, 5 mM DTT and 0.25 M NaCl.
• the Gr1A protein which is in the form of a symmetrical peak, is co-eluted with the desired activity.
• the preparation has a single visible band on SDS-PAGE after development with silver nitrate, and this band migrates with an apparent molecular weight of approximately 90,000.
• the purification of the subunit B can be carried out by chromatography, for example by following the following protocol:
• an acellular extract prepared as described in Example 5 from approximately 5 g of E. coli XL1-blue pXL2320 cells is injected onto a column of Novobiocin-Sepharose CL-6B (6 ml of gel prepared according to the protocol described by Staudenbauer et al., 1981, Nucleic Acids Research) equilibrated in pH 7.5 Tris / 50 mM HCl buffer containing 1 mM EDTA, 5 mM DTT and 0.3 M NaCl.
• the Gr1B protein is eluted with buffer pH 7.5 Tris / 50 mM HCl containing 1 mM EDTA 5 mM DTT, 2 M NaCl and 5 M urea. This fraction is then chromatographed on a Superdex 200 HiLoad 26/60 gel permeation column (Pharmacia) equilibrated and eluted with buffer pH 7.5 Tris / 50 mM HCl containing 1 mM EDTA, 5 mM DTT and 0.25 M NaCl. The GrlB protein, which is in the form of a symmetrical peak, is co-eluted with the desired activity. After this step, the preparation has a single visible band on SDS-PAGE after development with silver nitrate, and this band migrates with an apparent molecular weight of approximately 80,000.
• the different enzymatic activities of the Gr1AB protein are detected by incubating in the same reaction mixture equal amounts of the two types of extracts prepared using the method described above or any other method making it possible to recover the intracellular enzymatic proteins from the microorganism while preserving their activity, such as for example the processes using the use of presses (such as French Press, X-Press), or the use of ultrasound.
• the reaction is stopped by adding 7 ⁇ l of a 5% SDS and proteinase K mixture at 2.5 mg / ml and the samples are incubated for a second period of 30 min at 37 ° C. and then analyzed by gel electrophoresis. 1% agarose in 0.1M Tris / borate buffer pH 8.3 containing 2 mM EDTA at 6V / cm for 3 h.
• the separation of the released and nickel-plated DNAs (or open circular) is carried out by carrying out an additional electrophoretic migration of 2 hours after addition of ethidium bromide (1 ⁇ g / ml) to the migration buffer.
• the DNA is then quantified by scanning the photo negatives of the gels (Polaroid film type 665) using a Bioimage 50S device (Millipore).
• FIG. 4 shows that the acellular extracts of the E. coli XL1-blue pXL2320 and E. coli XL1-blue pXL2340 strains exhibit, as a mixture, an intense activity of DNA relaxation while each of the extracts is inactive when incubated alone. The reaction is dependent on ATP. In addition, these two extracts, alone or as a mixture, do not exhibit DNA supercoiling activity, typical activity of gyrase.
• the reaction is stopped by adding 7 ⁇ l of a 250 mM EDTA solution (5 min incubation at 37 ° C.), 5 ⁇ l of a 5% SDS and proteinase K mixture at 2.5 mg / ml (incubation 30 min at 37 ° C).
• the mixture is then analyzed by electrophoresis in 1% agarose gel in 0.1 M Tris / borate buffer pH 8.3 containing 2 mM EDTA at 6V / cm for 2 h 30 min.
• DNA staining with ethidium bromide (1 ⁇ g / ml) the DNA is quantified by scanning the photo negatives of the gels (Polaroid film type 665) using a Bioimage SOS device (Millipore) .
• the extracts of the two E. coli XL1-blue pXL2320 and E. coli XL1-blue pXL2340 strains exhibit a mixture of complete decatenation activity of the starting kinetoplast DNA. This activity is evidenced by the appearance of a DNA band of a size of approximately 2.5 kb and by the disappearance of the very large catenary DNA band which penetrates very little into the gel at during electrophoretic migration (Figure 5).
• the E. coli gyrase introduced as a control in this test does not exhibit decatenation activity unlike the E. coli topoisomerase IV DNA which completely decatenates the kinetoplast DNA (FIG. 5).
• Example 8 DNA topoisomerase IV activity of the Gr1AB protein of S. aureus whose subunit Gr1A has a substitution of the residue Ser-80 in Tyr (Ser- 80 ⁇ Tyr).
• This example illustrates how an acellular extract containing the Gr1A protein (Ser-80 ⁇ Tyr) B can be prepared, and how the enzymatic activity of the Gr1A protein (Ser-80 ⁇ Tyr) B can be detected and measured.
• Example 9 Inhibition by fluoroquinolones of DNA topoisomerase IV activity of the wild-type Gr1AB protein of S. aureus and resistance to fluoroquinolones of the protein comprising a Ser-80 ⁇ Tyr transition in the Gr1A subunit.
• the two methods described in Example 7 for the assay of DNA topoisomerase IV activities can be used to demonstrate new molecules acting as inhibitors of topoisomerase IV of S. aureus or to characterize the behavior of topoisomerase IV of S. aureus vis-à-vis molecules already identified as inhibitors of other topoisomerases (for example fluoroquinolones).
• the disappearance or reduction of the DNA band released during the analysis of the reaction mixture after incubation of the Gr1AB protein of S. aureus in the presence of a molecule or of a mixture of several molecules indicates that this molecule (or these molecules) is inhibiting the relaxation activity of Gr1AB, and is therefore potentially antibacterial.
• the incubations are carried out with the purified wild-type Gr1AB protein as described in Example 7, and with the mutant Gr1A (Ser-80 ⁇ Tyr) B protein as described in Example 8
• the two wild-type and mutant Gr1AB proteins are reconstituted by mixing equimolar amounts of their two subunits Gr1A and Gr1B.
• the decatenation test if the disappearance or reduction of the intensity of the decatenated DNA band is observed during the analysis of the reaction mixture after incubation of the Gr1AB protein in the presence of a molecule or a mixture of several molecules, this indicates that this molecule (or these molecules) is inhibitor of the decatenation activity of the protein Gr1AB, and is therefore potentially antibacterial.
• the Gr1AB protein is the primary target for molecules of the fluoroquinolone family, it appears that the fluoroquinolones must act as inhibitors in the decatenation test described in Example 7.
• a fluoroquinolone for example ciprofloxacin
• Ciprofloxacin inhibits 50% of the kinatoplast DNA decatenation activity at a concentration of 4.0 ⁇ g / ml.
• sparfloxacin which is another fluoroquinolone, inhibits 50% of the decatenation activity of kinetoplast DNA at a concentration of 6.0 ⁇ g / ml.
• sparfloxacin inhibits 50% of the DNA decatenation activity of kinetoplast at a concentration of 500 ⁇ g / ml, i.e. a concentration of 80 times greater than that necessary to obtain the same effect with the wild enzyme.
• Norfloxacin inhibits 50% of the kinatoplast DNA decatenation activity at a concentration of 12 ⁇ g / ml with the wild-type Gr1AB enzyme and has the same inhibitory activity at a concentration of 125 ⁇ g / ml with the enzyme Gr1A (Ser-80 ⁇ Tire) B.
• Ofloxacin inhibits 50% of the kinatoplast DNA decatenation activity at a concentration of 10 ⁇ g / ml with the wild-type Gr1AB enzyme and has the same inhibitory activity at a concentration of 250 ⁇ g / ml with the enzyme G ⁇ lA (Ser-80 ⁇ Tyr) B.
• Novobiocin whose mechanism of action is different from that of fluoroquinolones, must therefore in principle exhibit the same inhibitory activity on the two wild-type enzymes Gr1AB and Gr1A (Ser-80 ⁇ Tyr) B in the decatenation test described in l example 7.
• novobiocin inhibits 50% of the DNA decatenation activity of kinetoplast at a concentration of approximately 30 ⁇ g / ml whatever the enzyme used (wild-type Gr1AB or GrlA (Ser-80 ⁇ Tyr ) B mutant)) ABBREVIATIONS
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• QRDR region of the GyrA subunit where point mutations leading to resistance to fluoroquinolones are mapped
• DNA gyrase an enzyme that introduces superhelical turns into DNA Proc Natl Acad Sci USA 73:
• Ciprofloxacin-resistance in coagulase-positive and -négative Staphylococci role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus: and Staphylococcus epidermidis Antimicrob Agents Chemother 35: 2151-2154.

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