Genes involved in cysteine metabolism are important for tellurite resistance in bacteria (Chasteen et al., 2009). We then decided to compare the tellurite sensitivity of strains BSIP1215 and BSIP1793 (ΔcymR). On plates containing methionine, the ΔcymR mutant was less resistant to tellurite than the wild-type strain with a growth inhibition area diameter of 47.7 and 30.3 mm, respectively (Fig. 4b). In contrast, on plates containing cystine, the same growth inhibition area diameter was obtained for both strains (40.2 mm for BSIP1793 and 40.6 mm for BSIP1215) (Fig. 4b). In addition, the black deposits were much more prevalent for the ΔcymR mutant than for
the wild-type strain and the selleck inhibitor blackening mostly surrounded the paper disk for strain BSIP1793
(Fig. 4a, left PD332991 panel). Tellurite might be reduced by the H2S produced by bacteria. The significant amount of H2S produced in the ΔcymR mutant was probably responsible for the quantity of tellurium deposits observed with this mutant. The diffusion of H2S into the plate could also explain why tellurite reduction occurred even in the zone of growth inhibition. To confirm the possible role of H2S in this phenomenon, we repeated the same disk assay, but kept the lid of the plate open in a moisturized atmosphere, allowing H2S diffusion outside from the plate. The growth inhibition area diameter of the ΔcymR mutant then markedly increased in the open plates, reaching 52.7 mm instead of 38.1 mm for the wild-type strain. Simultaneously, the blackening around the paper disk disappeared
Chloroambucil (Fig. 4a, right panel). A similar result was obtained when 5 mL of alkaline agar enriched with zinc acetate was poured on the lid to absorb H2S (data not shown). This indicated that H2S obtained from cysteine degradation probably participated in tellurite reduction, protecting the ΔcymR mutant from its toxicity. When H2S escaped from the plate, we observed a drastic increase in tellurite sensitivity for the ΔcymR strain similar to that obtained in the presence of methionine under conditions producing less H2S (Fig. 3a). We then tested the effect of CymR inactivation on the susceptibility of B. subtilis to other stress stimuli. We compared the sensitivity of strains BSIP1215 and BSIP1793 (ΔcymR) to paraquat, H2O2 and diamide using disk diffusion assays. The ΔcymR mutant was significantly more sensitive than the wild-type strain to diamide, a specific thiol oxidant that causes disulfide stress. This effect was observed with plates containing cystine or methionine (Table 1, data not shown). We further tested the effect of H2O2 and paraquat. On plates with methionine, the growth inhibition area in the presence of 10 μL of 2 M paraquat was 58.8 mm for the ΔcymR mutant and 49.3 mm for the wild-type strain. Under the same conditions, the zone of growth inhibition in the presence of 10 μL of 10 M H2O2 was 52.1 mm for the ΔcymR mutant and 41.4 mm for the wild-type strain.