Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV.
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The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified as P. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities of P. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-type P. aeruginosa. (+info)
A mutation in QRDR in the ParC subunit of topoisomerase IV was responsible for fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.
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Forty-one strains of Streptococcus pneumoniae were isolated at Seoul National University Children's Hospital from 1991 to 1997. Isolates were divided into six groups based on MICs of three quinolones, ciprofloxacin, ofloxacin and norfloxacin. Sequencing showed that the isolates which were intermediately resistant to three quinolones or resistant to at least one kind of quinolone had one missense mutation, Lys137-->Asn(AAG-->AAT) substitution in the ParC subunit of topoisomerase IV without additional mutation in QRDR of the GyrA subunit of DNA gyrase. In conclusion, the ParC subunit of DNA topoisomerase IV is the primary target site for fluoroquinolone in S. pneumoniae and Lys137-->Asn substitution renders the quinolone resistance in S. pneumoniae. (+info)
Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli.
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Isogenic mutants derived from quinolone-susceptible isolate WT by introducing gyrA (S83L, D87G) and parC (S80I, E84K) mutations associated with quinolone resistance were characterized with respect to quinolone resistance, growth rate, and degree of global supercoiling. The latter was determined by use of a pair of reporter plasmids carrying supercoiling-dependent promoters pgyrA and ptopA, respectively, transcriptionally fused to the reporter gene bla coding for TEM-1 beta-lactamase. The quotient (Qsc) of the beta-lactamase specific activity determined for a mutant carrying either plasmid was taken as a measure of the degree of global supercoiling. These Qsc data were comparable to results obtained from the separation of topoisomers of plasmid pBR322 on chloroquine-containing agarose gels and indicate a reduced degree of negative supercoiling in resistant mutants relative to the parent, WT. The S83L mutation in gyrA had the strongest influence on quinolone resistance while leaving other parameters nearly unaffected. The gyrA double mutation (S83L plus D87G) had an effect on quinolone resistance similar to that of a single mutation. Phenotypic expression of the parC mutation (S80I) was dependent on the presence of at least one gyrA mutation. Expression of high-level fluoroquinolone resistance (ciprofloxacin MIC, > 4 micrograms/ml) required a combination of the gyrA double mutation and one parC mutation (S80I or E84K). Such mutants showed considerable alterations of growth rate, global supercoiling, or both. Introduction of a parC mutation affected neither the doubling time nor the degree of supercoiling, while the presence of the gyrA D87G mutation was associated with a significant reduction in the degree of DNA supercoiling. (+info)
Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium.
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High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis. (+info)
Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis.
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Five clinical isolates of Mycoplasma hominis from three different patients were examined for resistance to fluoroquinolones; some of these isolates were probably identical. All five isolates harbored amino acid substitutions in the quinolone resistance-determining regions of both DNA gyrase (GyrA) and topoisomerase IV (ParC or ParE). Furthermore, the novobiocin MIC for three isolates showed a significant increase. This is the first characterization of fluoroquinolone-resistant clinical mycoplasma isolates from humans. (+info)
In vitro activities of 13 fluoroquinolones against Staphylococcus aureus isolates with characterized mutations in gyrA, gyrB, grlA, and norA and against wild-type isolates.
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The in vitro activities of 13 fluoroquinolones (FQs) were tested against 90 Staphylococcus aureus clinical isolates: 30 wild type for gyrA, gyrB, grlA and norA and 60 with mutations in these genes. Clinafloxacin (CI-960), sparfloxacin, and grepafloxacin were the most active FQs against wild-type isolates (MICs at which 90% of isolates were inhibited, 0.06 to 0.1 microgram/ml). Mutations in grlA did not affect the MICs of newer FQs. grlA-gyrA double mutations led to higher MICs for all the FQs tested. Efflux mechanisms affected the newer FQs to a much lesser extent than the less recently developed FQs. (+info)
Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones.
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Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones-ciprofloxacin, sparfloxacin, and clinafloxacin-which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway. (+info)
Sequence analysis of the gyrA and parC homologues of a wild-type strain of Vibrio parahaemolyticus and its fluoroquinolone-resistant mutants.
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Vibrio parahaemolyticus causes seafood-borne gastroenteritis in humans. It is particularly important in Japan, where raw seafood is frequently consumed. Fluoroquinolone is one of the current drugs of choice for treating patients infected by V. parahaemolyticus because resistant strains are rarely found. To study a possible fluoroquinolone resistance mechanism in this organism, nucleotide sequences that are homologous to known gyrA and parC genes have been cloned from V. parahaemolyticus AQ3815 and sequenced by amplification with degenerate primers of the quinolone resistance-determining region (QRDR), followed by cassette ligation-mediated PCR. Open reading frames encoding polypeptides of 878 and 761 amino acid residues were detected in the gyrA and parC homologues, respectively. The V. parahaemolyticus GyrA and ParC sequences were most closely related to Erwinia carotovora GyrA (76% identity) and Escherichia coli ParC (69% identity) sequences, respectively. Ciprofloxacin-resistant mutants of AQ3815 were obtained on an agar medium by multistep selection with increasing levels of the quinolone. One point mutation only in the gyrA QRDR was detected among mutants with low- to intermediate-level resistance, while point mutations in both the gyrA and parC QRDRs were detected only in strains with high-level resistance. These results strongly suggest that, as in other gram-negative bacteria, GyrA and ParC are the primary and secondary targets, respectively, of ciprofloxacin in V. parahaemolyticus. (+info)