HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes.

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role.

Effect of ciprofloxacin concentration on the frequency and nature of resistant mutants selected from Pseudomonas aeruginosa mutS and mutT hypermutators.

The rapid emergence of drug resistance upon treatment of Pseudomonas aeruginosa infections with fluoroquinolones is a serious concern. In this study, we report the effect of hypermutability on the mutant selection window for ciprofloxacin (CIP) by comparing the hypermutator MPAO1 mutS and mutT strains with the wild-type strain. The mutant selection window was shifted to higher CIP concentrations for both hypermutators, presenting the mutS strain with a broader selection window in comparison to the wild-type strain. The mutation prevention concentrations (MPC) determined for mutT and mutS strains were increased 2- and 4-fold over the wild-type level, respectively. In addition, we analyzed the molecular bases for resistance in the bacterial subpopulations selected at different points in the window. At the top of the window, the resistant clones isolated were mainly mutated in GyrA and ParC topoisomerase subunits, while at the bottom of the window, resistance was associated with the overexpression of MexCD-OprJ and MexAB-OprM efflux pumps. Accordingly, a greater proportion of multidrug-resistant clones were found among the subpopulations isolated at the lower CIP concentrations. Furthermore, we found that the exposure to CIP subinhibitory concentrations favors the accumulation of cells overexpressing MexCD-OprJ (due to mutations in the transcriptional repressor NfxB) and MexAB-OprM efflux pumps. We discuss these results in the context of the possible participation of this antibiotic in a mutagenic process.

nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa.

nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved.

Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin.

The 8-oxodeoxyguanine (8-oxodG) repair system participates in the prevention and correction of mutations generated by oxidative DNA damage in prokaryotes and eukaryotes. In this study, we report that Pseudomonas aeruginosa strains deficient in this repair mechanism by inactivation of the mutT, mutM and mutY genes generate a high frequency of cells resistant to the antibiotic ciprofloxacin. In the mutT strain, the increase in ciprofloxacin resistance achieved at threefold minimal inhibitory concentration was about 1600-fold over the wild-type (WT) level, similar to the frequency achieved by the mismatch repair-deficient mutS strain. Molecular analysis of WT, mutT and mutY clones resistant to ciprofloxacin indicated that the nfxB gene was mutated in the majority of the cases, while mutS-derived resistant clones were mainly mutated in gyrA and parC genes. Cell viability analysis after treatment with paraquat or hydrogen peroxide indicated that 8-oxodG repair-deficient strains were considerably more susceptible to oxidative stress than the parental strain. Finally, it is shown that the ciprofloxacin resistance frequency of WT and repair-deficient strains increased significantly after cell exposure to paraquat. Thus, oxidative stress is strongly implicated in the emergence of ciprofloxacin-resistant mutants in P. aeruginosa, and the 8-oxodG repair pathway plays an important role in the prevention of these mutations.