Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.     

Mutation at the "exit gate" of the salmonella gyrase a subunit suppresses a defect in the gyrase B subunit

In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

Preferential inhibition of plasmid replication in vivo by altered DNA gyrase activity in Escherichia coli.

The thermosensitive growth phenotype exerted by runaway-mutant plasmids was suppressed by sublethal doses of the DNA gyrase inhibitors novobiocin or nalidixic acid, although the latter drug was less efficient. A novobiocin-resistant gyrB mutant Escherichia coli strain prevented expression of the runaway phenotype at 37 to 42 degrees C in the absence of any drug.     

Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity.

Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling. These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.     

Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations

Abstract We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604 ), jointly with a gyrA mutation (denoted gyrA972 ) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.     

Filamentation promotes F'lac loss in Escherichia coli K12.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.     

Conditional growth of Escherichia coli caused by expression of vaccinia virus DNA topoisomerase I.

Active vaccinia virus topoisomerase I is expressed in Escherichia coli containing plasmid p1940 (S. Shuman, M. Golder, and B. Moss, J. Biol. Chem. 263:16401-16407, 1988). Growth curves showed a decline of 2 to 3 logs in the number of viable cells at 42 degrees C after shift from 30 degrees C because of increased vaccinia virus topoisomerase I level. Mutations in the gyrA and gyrB genes allowed cells to grow equally well at 42 and 30 degrees C. The presence of gyrase inhibitor also improved growth at 42 degrees C.     

DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01

SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M. Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene. Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation. We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A.     

Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus.

We have isolated and cloned the gyrA and gyrB genes from Staphylococcus aureus. These adjacent genes encode the subunits of DNA gyrase. The nucleotide sequence of a 5.9-kb region which includes part of an upstream recF gene, the whole of gyrB and gyrA, and about 1 kb of unknown downstream sequence has been determined. The gyrB and gyrA gene sequences predict proteins of 886 and 644 amino acid residues, respectively, which have significant homologies with the gyrase subunits of Escherichia coli and Bacillus subtilis. Residues thought to be important to the structure and function of the subunits are conserved. These genes have been expressed separately by using a T7 promoter vector. N-terminal sequencing of the cloned gene products suggests that the mature GyrB subunit exists mainly with its initial five residues removed. Protein sequencing also supports the interpretation of our DNA sequencing data, which are inconsistent in several placed with the recently published sequence of the same genes (E. E. C. Margerrison, R. Hopewell, and L. M. Fisher, J. Bacteriol. 174:1596-1603, 1992).     

DNA gyrase: affinity chromatography on novobiocin-Sepharose and catalytic properties.

Novobiocin-Sepharose was prepared by coupling of novobiocin to Epoxy-activated Sepharose 6B and used as an affinity adsorbent. Four novobiocin-binding proteins were isolated from crude extracts of Escherichia coli with molecular weights of 105, 92, 85 and 40 kdal. The two larger proteins were identified as the A subunit (gyrA protein) and the B subunit (gyrB protein) of DNA gyrase topoisomerase II). By this method the two gyrase components can be easily separated and purified in high yield. Although both proteins are involved in the ATP-dependent supercoiling of relaxed plasmid DNA, only the gyrB protein is required for catalyzing the cleavage of ATP. The gyrB protein ATPase activity is competitively inhibited by novobiocin and related coumarin antibiotics. ATP hydrolysis is unaffected by the addition of either gyrA protein or DNA but stimulated in the presence of both.     

A gyrase mutant with low activity disrupts supercoiling at the replication terminus

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.     

Effects of DNA gyrase inhibitors in a polyamine-auxotrophic strain of Escherichia coli

The polyamine content of the Escherichia coli polyamine-auxotrophic strain BGA 8 seemed to influence the effects of nalidixic acid, an antibiotic acting on subunit A of DNA gyrase. The growth rate was more affected under conditions of putrescine depletion and the inhibition could be partially relieved if the polycation was added back to the culture. DNA synthesis was likewise more sensitive to nalidixic acid in cultures grown without polyamine. The expression of some proteins characteristic of the heat-shock response, evoked by the antibiotic, showed a different persistence according to the presence or absence of polyamines. Novobiocin, acting on subunit B of gyrase, also promoted a differential effect depending on the polyamine content, but in this case putrescine-supplemented cells were more sensitive. The described findings suggest a role of polyamines in all the reactions carried out by gyrase, perhaps due to the influence of the polycations on the state of DNA aggregation.     

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.     

New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.     

Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity

DNA gyrase from Bacillus subtilis 168 was purified by affinity chromatography on novobiocin-Sepharose and shown to consist of two subunits, A and B, with molecular weights of 100,000 and 85,000, respectively. The B subunits, which contains novobiocin-sensitive. ATPase activity, could complement the gyrA protein of Escherichia coli. No complementation was detected between the A subunit and the E. coli gyrB protein.     

Increased expression of biodegradative threonine dehydratase of Escherichia coli by DNA gyrase inhibitors

The synthesis of inducible biodegradative threonine dehydratase of Escherichia coli increased several-fold in the presence of the DNA gyrase inhibitors, nalidixic acid and coumermycin. Temperature-sensitive gyrB mutants expressed higher levels of dehydratase as compared to an isogenic gyrB+ strain. Immunoblotting experiments showed increased synthesis of the dehydratase protein in the presence of gyrase inhibitors; addition of rifampicin and chloramphenicol to cells actively synthesizing enzyme preventing new enzyme production. Increased expression of dehydratase by gyrase inhibitors was accompanied by relaxation of supercoiled DNA.