Management of E. coli sister chromatid cohesion in response to genotoxic stress.

Aberrant DNA replication is a major source of the mutations and chromosomal rearrangements associated with pathological disorders. In bacteria, several different DNA lesions are repaired by homologous recombination, a process that involves sister chromatid pairing. Previous work in Escherichia coli has demonstrated that sister chromatid interactions (SCIs) mediated by topological links termed precatenanes, are controlled by topoisomerase IV. In the present work, we demonstrate that during the repair of mitomycin C-induced lesions, topological links are rapidly substituted by an SOS-induced sister chromatid cohesion process involving the RecN protein. The loss of SCIs and viability defects observed in the absence of RecN were compensated by alterations in topoisomerase IV, suggesting that the main role of RecN during DNA repair is to promote contacts between sister chromatids. RecN also modulates whole chromosome organization and RecA dynamics suggesting that SCIs significantly contribute to the repair of DNA double-strand breaks (DSBs).

Topoisomerase IV is required for partitioning of circular chromosomes but not linear chromosomes in Streptomyces.

Filamentous bacteria of the genus Streptomyces possess linear chromosomes and linear plasmids. Theoretically, linear replicons may not need a decatenase for post-replicational separation of daughter molecules. Yet, Streptomyces contain parC and parE that encode the subunits for the decatenase topoisomerase IV. The linear replicons of Streptomyces adopt a circular configuration in vivo through telomere-telomere interaction, which would require decatenation, if the circular configuration persists through replication. We investigated whether topoisomerase IV is required for separation of the linear replicons in Streptomyces. Deletion of parE from the Streptomyces coelicolor chromosome was achieved, when parE was provided on a plasmid. Subsequently, the plasmid was eliminated at high temperature, and ΔparE mutants were obtained. These results indicated that topoisomerase IV was not essential for Streptomyces. Presumably, the telomere-telomere association may be resolved during or after replication to separate the daughter chromosomes. Nevertheless, the mutants exhibited retarded growth, defective sporulation and temperature sensitivity. In the mutants, circular plasmids could not replicate, and spontaneous circularization of the chromosome was not observed, indicating that topoisomerase IV was required for decatenation of circular replicons. Moreover, site-specific integration of a plasmid is impaired in the mutants, suggesting the formation of DNA knots during integration, which must be resolved by topoisomerase IV.

Transferable quinolone resistance in Vibrio cholerae.

Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutation in parC, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosomes of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.

Cohesion by topology: sister chromatids interlocked by DNA.

Sister chromatid cohesion is coupled with chromosome replication and influences chromosome segregation and intra-S repair. Specialized proteins, the cohesins, together with other pathways contribute to tether sister chromatids. In this issue of Genes & Development, Wang and colleagues (pp. 2426-2433 demonstrate that TopoIV, a type II DNA topoisomerase, modulates cohesion in Escherichia coli, by removing interlocked DNA junctions between sister chromatids. They propose that DNA precatenanes, arising during replication fork progression, hold sister chromatids together.

Modulation of Escherichia coli sister chromosome cohesion by topoisomerase IV.

A body of evidence supports the idea that newly replicated Escherichia coli chromosomes segregate progressively as replication progresses, with spatial separation of sister genetic loci occurring approximately 15 min after their replication. We show that the time of this cohesion can be modulated by topoisomerase IV (TopoIV) activity. Impairment of TopoIV prevents segregation of newly replicated sister loci and bulk chromosome segregation, whereas modest increases in TopoIV decrease the cohesion time substantially. Therefore, we propose that precatenanes, which form as replication progresses by interwinding of newly replicated sister chromosomes, are responsible for E. coli sister chromosome cohesion.

Disminución de la actividad bactericida del ciprofloxacino en mutantes de Salmonella generados tras exposición repetida a diversas fluoroquinolonas / Decrease in bactericidal activity of ciprofloxacin in Salmonella mutants generated following repeated exposure to various fluorquinolones

Después de generar in vitro mutantes de Salmonella spp. con sensibilidad disminuida a las fluoroquinolonas tras su exposición repetida a concentraciones subinhibitorias de estos fármacos y caracterizar las alteraciones del gen gyrA, hemos determinado la modificación de la actividad bactericida que presenta el ciprofloxacino sobre los mutantes obtenidos. La pérdida de la actividad bactericida del ciprofloxacino se detecta en todos los mutantes, pero es mayor en los provenientes de cepas resistentes al ácido nalidíxico. Esto puede ayudar a explicar los fracasos terapéuticos observados con algunos tratamientos con fluoroquinolonas frente a este tipo de cepas. Además, nuestro modelo evalúa las primeras interacciones que se producen entre el microorganismo y los antibióticos, y pone de manifiesto que la pérdida de actividad bactericida del ciprofloxacino es un proceso que se produce tras la exposición a cualquiera de las fluoroquinolonas probadas

Salmonella mutants with reduced fluoroquinolone susceptibility were generated in vitro following repeated exposure to subinhibitory concentrations of the drugs and the alterations in the gyrA gene were characterized. Afterwards, the change in bactericidal activity exhibited by ciprofloxacin against the resulting mutants was determined. A decrease in the bactericidal activity of ciprofloxacin was found in all the mutants, but was more pronounced in mutants of nalidixic acid-resistant strains. This may help to explain the therapeutic failures sometimes described when fluoroquinolones are used in the treatment of these strains. In addition, our model evaluates the first interactions produced between the microorganism and the antibiotics, and demonstrates that the loss of bactericidal activity of ciprofloxacin occurs following exposure to all of the fluoroquinolones tested

[Decrease in bactericidal activity of ciprofloxacin in Salmonella mutants generated following repeated exposure to various fluorquinolones]. / Disminución de la actividad bactericida del ciprofloxacino en mutantes de Salmonella generados tras exposición repetida a diversas fluoroquinolonas.

Salmonella mutants with reduced fluoroquinolone susceptibility were generated in vitro following repeated exposure to subinhibitory concentrations of the drugs and the alterations in the gyrA gene were characterized. Afterwards, the change in bactericidal activity exhibited by ciprofloxacin against the resulting mutants was determined. A decrease in the bactericidal activity of ciprofloxacin was found in all the mutants, but was more pronounced in mutants of nalidixic acid-resistant strains. This may help to explain the therapeutic failures sometimes described when fluoroquinolones are used in the treatment of these strains. In addition, our model evaluates the first interactions produced between the microorganism and the antibiotics, and demonstrates that the loss of bactericidal activity of ciprofloxacin occurs following exposure to all of the fluoroquinolones tested.

Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica.

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.

Quinolone resistance mechanisms in pneumococci.

Quinolones are widely used in the treatment of respiratory infections, in large part because of their activity against Streptococcus pneumoniae and other commonly encountered respiratory tract pathogens. Pneumococcal isolates that are resistant to these "respiratory quinolones" have now begun to emerge. Resistance is attributable to mutations affecting the intracellular targets of these drugs, topoisomerase IV and DNA gyrase; drug efflux contributes to quinolone resistance in some isolates. Most commonly, strains fully resistant to the newer quinolones have one or more mutations affecting DNA gyrase and topoisomerase IV. Although various agents of this class exhibit selectivity in primarily targeting one or the other of these enzymes, the passage of isolates in the presence of any agent can result in selection of mutations affecting both enzymes. Quinolone resistance in S. pneumoniae has arisen in heterogeneous genetic backgrounds but, ominously, has now appeared in strains that are well adapted for regional and global transmission.

[Resistance to quinolones and beta-lactams in Salmonella enterica due to mutations in topoisomerase-encoding genes, altered cell permeability and expression of an active efflux system]. / Resistencia a quinolonas y betalactámicos en Salmonella enterica, y su relación con mutaciones en las topoisomerasas, alteraciones en la permeabilidad celular y expresión de un mecanismo de expulsión activa.

BACKGROUND: The mechanisms of resistance to fluoroquinolones and beta-lactams were studied in isolates of Salmonella enterica resistant to both antimicrobial groups, isolated over time from two patients treated with fluoroquinolones. METHODS: The clonal relationships among the various strains was established by serotyping and pulsed-field gel electrophoresis. MICs for beta-lactams, quinolones, chloramphenicol and tetracycline were determined. Presence of beta-lactamases was ruled out by a colorimetric assay. Quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes were sequenced, and the relevance of the mutations in these regions was evaluated by complementation assays. Outer membrane protein profiles, the effect of phenylalanyl-arginyl-naphthylamide (PAN, 20 mg/l) on the MICs of several quinolones, and norfloxacin accumulation in the absence and in the presence of a metabolic inhibitor were also determined. RESULTS: The following mutations were found: gyrA (Asp87 --> Gly; Ser83 --> Phe; Asp87 --> Lys), gyrB (Ser463 --> Phe) and parC (Glu84 --> Gly). Altered outer membrane protein profiles, including decreased expression of a porin equivalent to OmpF from Escherichia coli was observed. Active efflux of norfloxacin was proved in both a clinical isolate and a mutant obtained in vitro. In the presence of PAN, nalidixic acid MICs decreased 4-32 times (except in one strain), pefloxacin MICs decreased 4-16 times for 5 out of 9 evaluated strains, and MICs of both norfloxacin and ciprofloxacin did not change or changed within a single dilution step. CONCLUSIONS: Quinolone-resistance is the consequence of a combination of mutations in topoisomerase-encoding genes, altered permeability and active efflux. Altered permeability and active efflux would also contribute to decreased susceptibility to beta-lactams.

Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204.

OBJECTIVES: To study the role of TolC and of parC mutation in high-level fluoroquinolone resistance in clonal clinical strains of Salmonella enterica serotype Typhimurium phage type DT204 (S. Typhimurium DT204). METHODS: Deletion of the tolC gene (DeltatolC) was first performed in a susceptible S. Typhimurium DT104 strain lacking target gene mutations involved in fluoroquinolone resistance. P22 transduction was further used to transduce DeltatolC from this strain to a high-level fluoroquinolone-resistant S. Typhimurium DT204 strain carrying several target gene mutations, including one in parC (ciprofloxacin MIC of 32 mg/L). RESULTS: Deletion of tolC in the high-level fluoroquinolone-resistant S. Typhimurium DT204 strain resulted in the same decrease in resistance levels (16- to 32-fold) as shown previously for an acrB mutant of the same strain, suggesting that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. In some S. Typhimurium DT204 DeltatolC transductants, concomitant loss of the parC (Ser-80-->Ile) mutation, located approximately 9.3 kb upstream of tolC, resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones and thus a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L). CONCLUSION: The AcrAB-TolC efflux system, together with multiple target gene mutations, including the parC mutation, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin.

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.

PriA is essential for viability of the Escherichia coli topoisomerase IV parE10(Ts) mutant.

The parE10(Ts) mutation, which renders Escherichia coli thermosensitive for growth by inactivation of the essential E. coli topoisomerase topo IV, is lethal at all temperatures when PriA, the main replication restart protein, is absent. This lethality is suppressed by the activation of a PriA-independent replication restart pathway (dnaC809 mutation). This result suggests that topo IV acts prior to full-chromosome replication completion.

A physical and functional interaction between Escherichia coli FtsK and topoisomerase IV.

FtsK and topoisomerase (Topo) IV are both involved in chromosome segregation in Escherichia coli. The former protein resides at the septal ring and is required for resolution of chromosome dimers. The latter protein is the chromosomal decatenase. We have demonstrated recently that Topo IV activity is concentrated at the septal proximal regions of the nucleoids late in the cell cycle. Here we demonstrate that FtsK and Topo IV physically and functionally interact. Topo IV was recovered in immunoprecipitates of FtsK. Two-hybrid analysis and immunoblotting showed that this interaction was mediated by the ParC subunit of Topo IV. In addition, we show that the C-terminal motor domain of FtsK stimulates the decatenation activity of Topo IV but not that of DNA gyrase, the other type II topoisomerase in the cell. Topo IV and FtsK appear to cooperate in the cell as well. Rescue of a parE temperature-sensitive mutation by overproduction of DnaX, which leads to stabilization of the temperature-sensitive Topo IV, required both the C-terminal domain of FtsK and dif, whereas rescue by overproduction of Topo III, which bypasses Topo IV function, did not. The interaction between FtsK and Topo IV may provide a means for concentrating the latter enzyme at the cell center.

Molecular evaluation of antibiotic susceptibility: Tropheryma whipplei paradigm.

Tropheryma whipplei, the agent of Whipple's disease, grows fastidiously only in cell cultures without plaque production, and only three strains have been passaged. The formation of bacterial clumps in the supernatant precludes enumeration of viable bacteria and MIC determination. We evaluated the bacteriostatic effects of fluoroquinolones against two T. whipplei isolates by measuring the inhibition of the DNA copy number increase by real-time quantitative PCR. The analysis of the T. whipplei genome database allowed the identification not only of the gyrA gene but also the parC gene encoding the alpha subunit of the natural fluoroquinolone targets DNA gyrase (GyrA) and topoisomerase IV (ParC), respectively. The parC gene was detected in actinobacteria for the first time. High ciprofloxacin MICs (4 and 8 micro g/ml) were correlated with the presence in T. whipplei GyrA and ParC sequences with an alanine residue at positions 83 and 80 (Escherichia coli numbering), respectively. Alanines at these positions have previously been associated with increased fluoroquinolone resistance in E. coli and mycobacteria. However, the MIC of levofloxacin was low (0.25 micro g/ml). The same T. whipplei GyrA and ParC sequences were found in two other cultured strains and in nine uncultured tissue samples from Whipple's disease patients, allowing one to speculate that T. whipplei is naturally relatively resistant to fluoroquinolones.

Emerging roles for plant topoisomerase VI.

Topoisomerase VI is a unique type II topoisomerase originally identified in archaea. Although lacking in most eukaryotic phyla, topoisomerase VI homologs have been recently identified and characterized in the plant Arabidopsis thaliana. Three new studies of Arabidopsis topoisomerase VI show that this enzyme is important to several processes involving DNA replication and gene expression.

Pharmacodynamic characterization of efflux and topoisomerase IV-mediated fluoroquinolone resistance in Streptococcus pneumoniae.

OBJECTIVES: Most in vitro investigations of fluoroquinolone resistance involving Streptococcus pneumoniae have described genotypic changes in quinolone resistance-determining regions (QRDRs) that occur as the result of exposure to fluoroquinolones obtained with static antimicrobial concentrations. The objectives of this study were to determine whether differences exist between moxifloxacin, sparfloxacin and levofloxacin antimicrobial effect (AME) and their ability to select out stepwise mutations with wild-type, efflux-expressing and parC-mediated fluoroquinolone resistance while simulating the in vivo dosing and pharmacokinetics of the respective agents. METHODS: A one-compartment pharmacodynamic model simulated fluoroquinolone dosing regimens. Duplicate 24 h experiments were carried out in Mueller-Hinton broth with 3% horse blood at 1 x 10(8) cfu/mL. Reserpine (10 mg/L) was added to select experiments conducted with efflux-expressing strains. AME was expressed as the area under the time-concentration kill curve (AUEC24). Strains expressing increased MIC post-time-concentration kill curve (TCKC) were evaluated for changes in QRDR. RESULTS: Moxifloxacin exhibited a greater AME against all isolates. Efflux was generally associated with partial loss of AME for all fluoroquinolones, and levofloxacin retained no AME against parC-expressing S. pneumoniae. Increased fluoroquinolone MIC post-TCKC was more common with efflux expression. The addition of reserpine was associated with enhanced AME for levofloxacin and moxifloxacin, but was not associated with altered resistance selection. Isolates recovered post-TCKC from experiments involving efflux- or parC mutation-containing isolates generally exhibited a more than four-fold increase in MIC, which was associated with commonly reported substitutions in both parC and gyrA. CONCLUSION: The results of this study generally indicate that resistance selection under pharmacodynamic conditions is similar to results reported with static fluoroquinolone concentrations. While moxifloxacin AME was greater than levofloxacin and sparfloxacin, the overall selection of resistant isolates did not differ.

C-terminal domain of gyrase A is predicted to have a beta-propeller structure.

Two different type II topoisomerases are known in bacteria. DNA gyrase (Gyr) introduces negative supercoils into DNA. Topoisomerase IV (Par) relaxes DNA supercoils. GyrA and ParC subunits of bacterial type II topoisomerases are involved in breakage and reunion of DNA. The spatial structure of the C-terminal fragment in GyrA/ParC is not available. We infer homology between the C-terminal domain of GyrA/ParC and a regulator of chromosome condensation (RCC1), a eukaryotic protein that functions as a guanine-nucleotide-exchange factor for the nuclear G protein Ran. This homology, complemented by detection of 6 sequence repeats with 4 predicted beta-strands each in GyrA/ParC sequences, allows us to predict that the GyrA/ParC C-terminal domain folds into a 6-bladed beta-propeller. The prediction rationalizes available experimental data and sheds light on the spatial properties of the largest topoisomerase domain that lacks structural information.