Autoregulation of topoisomerase I expression by supercoiling sensitive transcription.

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Escherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobacterium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of sub-optimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

Extending the definition of the GyrB quinolone resistance-determining region in Mycobacterium tuberculosis DNA gyrase for assessing fluoroquinolone resistance in M. tuberculosis.

Fluoroquinolone (FQ) resistance is emerging in Mycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target in M. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified in M. tuberculosis clinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

Temporal interplay between efflux pumps and target mutations in development of antibiotic resistance in Escherichia coli.

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥ 64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop.

Relationship between the expression of ompF and quinolone resistance in Escherichia coli.

The outer membrane porin protein, OmpF, is widely found in gram-negative bacteria. It is known that the decreased expression of OmpF causes resistance to multiple antibiotics, including quinolones. In order to characterize the influence of decreased OmpF expression on bacterial growth, the fitness of the ompF and gyrA mutant strain of Escherichia coli selected experimentally with quinolone was compared with that of the parent strain. The expression levels of ompF in clinical isolates and the mutant selected with quinolone were determined by real-time PCR. The bacterial growth of the experimentally selected mutants was also measured both in vitro and in a urinary tract infection model in mice. Decreased ompF phenotypes were frequently found in clinical isolates that exhibited alteration of topoisomerases. The mutant experimentally obtained by the resistance selection process with quinolone showed no loss of fitness either in vitro or in vivo. These results suggest that the decreased expression of ompF and gyrA mutation do not affect the survival of the bacteria, and in fact may be responsible for the spread of high-level resistance to quinolones.

Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth.

By real-time quantitative PCR (RTQ-PCR), two different standardisation methods were used to quantify expression of three target genes (RNAII and RNAIII transcripts of agr locus and ica transcript of icaADBC locus): (i) a relative quantification, using a transcript of three housekeeping genes (gyrase A, gyrA; guanylate kinase, gmk and 16S rRNA, 16S) as internal standard, and (ii) an absolute quantification, using cloned sequences of the target genes in known concentrations as external standards. To determine the efficiency and reliability of these two methods, the gene expressions were studied during the growth of a clinical isolate of Staphylococcus aureus. Between 3 and 20 h after inoculation, target gene transcription was analysed using LightCycler Apparatus, LC Data Analysis software and RelQuant software for relative quantification (Roche). For all target genes, the expression profiles obtained with gyrA or gmk as internal standards remained almost identical. However, these profiles varied between each other depending on the standard gene. Due to their important expression variations during growth phases, these two housekeeping genes seem inappropriate to be used as internal standards. The absolute quantification of the three transcripts of interest gave results similar to their relative quantification expressed versus 16S rRNA. Therefore, our study suggests the suitable use of 16S rRNA as internal standard in RTQ-PCR quantification of staphylococcal gene expression during the stationary phase of growth.

Prevalence of plasmid-mediated AmpC ß-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum ß-lactamase-producing E. coli in Japan.

In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC ß-lactamase (pAmpC)-producers, extended-spectrum ß-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and ß-lactam/ß-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

In vivo sortase A and clumping factor A mRNA expression during Staphylococcus aureus infection.

The Staphylococcus aureus cell surface protein clumping factor A (ClfA) and the enzyme sortase A (SrtA), which attach surface proteins to the cell wall, have both been shown to be virulence factors in models of septic arthritis and sepsis. The mRNA levels of clfA, srtA and the putative housekeeping gene gyrase B (gyrB) in S. aureus were determined using real-time PCR during the course of sepsis/septic arthritis. Expression was measured in joints, being a target of localized infection, and in kidneys, representing a systemic compartment. In infected kidneys, the mRNA levels of clfA, srtA and gyrB were all decreasing over time, from day 3 of infection to day 14. The transcript numbers of clfA and srtA decreased faster in septic mice than in mice with a non-septic disease. The mRNA levels of clfA and gyrB in joints, though, were increasing during the course of infection. These differences suggest that the specific tissue environment is decisive for the differentiation of staphylococci. Also, there was a negative relationship between bacterial load in a tissue and the numbers of clfA, srtA and gyrB transcripts per colony-forming unit. Possibly enters the majority of bacteria a metabolically dormant steady state at high bacterial loads.

Molecular cloning of apicoplast-targeted Plasmodium falciparum DNA gyrase genes: unique intrinsic ATPase activity and ATP-independent dimerization of PfGyrB subunit.

DNA gyrase, a typical type II topoisomerase that can introduce negative supercoils in DNA, is essential for replication and transcription in prokaryotes. The apicomplexan parasite Plasmodium falciparum contains the genes for both gyrase A and gyrase B in its genome. Due to the large sizes of both proteins and the unusual codon usage of the highly AT-rich P. falciparum gyrA (PfgyrA) and PfgyrB genes, it has so far been impossible to characterize these proteins, which could be excellent drug targets. Here, we report the cloning, expression, and functional characterization of full-length PfGyrB and functional domains of PfGyrA. Unlike Escherichia coli GyrB, PfGyrB shows strong intrinsic ATPase activity and follows a linear pattern of ATP hydrolysis characteristic of dimer formation in the absence of ATP analogues. These unique features have not been reported for any known gyrase so far. The PfgyrB gene complemented the E. coli gyrase temperature-sensitive strain, and, together with the N-terminal domain of PfGyrA, it showed typical DNA cleavage activity. Furthermore, PfGyrA contains a unique leucine heptad repeat that might be responsible for dimerization. These results confirm the presence of DNA gyrase in eukaryotes and confer great potential for drug development and organelle DNA replication in the deadliest human malarial parasite, P. falciparum.

Quinolone-resistant Haemophilus influenzae in a long-term-care facility: nucleotide sequence characterization of alterations in the genes encoding DNA gyrase and DNA topoisomerase IV.

Fluoroquinolone-resistant isolates of Haemophilus influenzae, obtained from a long-term care facility, were examined for nucleotide sequence differences in the quinolone-resistance-determining regions of gyrA, gyrB, parC, and parE. Similarities among the resistant isolates, plus multiple differences with susceptible isolates, suggest clonal dissemination involving two resistant subclones.

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

Dual targeting of DNA gyrase and topoisomerase IV: target interactions of garenoxacin (BMS-284756, T-3811ME), a new desfluoroquinolone.

We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 x 10(-12) to 4.0 x 10(-11)) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 micro g/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5' terminus of grlB and the 3' terminus of gyrA.

Small-colony mutants of Staphylococcus aureus allow selection of gyrase-mediated resistance to dual-target fluoroquinolones.

Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of Staphylococcus aureus RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the gyrA, gyrB, grlA, or grlB gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of S. aureus that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had gyrA(S84L) alterations; those obtained with sparfloxacin carried a gyrA(D83A) mutation or a novel gyrB deletion (DeltaRKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of S. aureus GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type grlB-grlA gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant gyrA or gyrB alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.

Purification and characterization of recombinant Staphylococcus haemolyticus DNA gyrase and topoisomerase IV expressed in Escherichia coli.

The subunits of DNA gyrase and topoisomerase IV from Staphylococcus haemolyticus were expressed in Escherichia coli, purified to homogeneity, and used to reconstitute active enzymes that were sensitive to known topoisomerase inhibitors. This represents the first description of a method for isolating type II topoisomerases of a coagulase-negative staphylococcal species.

DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA, gyrB and parC, parE genes.

Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.