Endogenous generation of hydrogen sulfide and its regulation in Shewanella oneidensis

Abstract

Hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. In this study, mechanisms of endogenous H2S generation in Shewanella oneidensis were investigated. As a research model with highly diverse anaerobic respiratory pathways, the microorganism is able to produce H2S by respiring on a variety of sulfur-containing compounds with SirACD and PsrABC enzymatic complexes, as well as through cysteine degradation with three enzymes, MdeA, SO_1095, and SseA. We showed that the SirACD and PsrABC complexes, which are predominantly, if not exclusively, responsible for H2S generation via respiration of sulfur species, do not interplay with each other. Strikingly, a screen for regulators controlling endogenous H2S generation by transposon mutagenesis identified global regulator Crp to be essential to all H2S-generating processes. In contrast, Fnr and Arc, two other global regulators that have a role in respiration, are dispensable in regulating H2S generation via respiration of sulfur species. Interestingly, Arc is involved in the H2S generation through cysteine degradation by repressing expression of the mdeA gene. We further showed that expression of the sirA and psrABC operons is subjected to direct regulation of Crp, but the mechanisms underlying the requirement of Crp for H2S generation through cysteine degradation remain elusive.

Keywords: Crp; H2S; Shewanella; endogenous generation; regulation.

Figure 1

H₂S generation by S. oneidensis. (A) Effects of Asp (3 mM), AOAA (50 μM), and PAG (7.5 mM), inhibitors of 3MST, CBS, and CSE respectively, on H₂S generation by the S. oneidensis wild-type. (B) Effects of PAG, the CSE inhibitor, at varying concentrations on H₂S generation by the S. oneidensis wild-type. (C) H₂S generation by indicated S. oneidensis mutants. Mutants showing a significant defect in H₂S generation were subjected to genetic complementation. The double mutant (ΔmdeAΔSO_1095) was complemented by either gene separately. Lead acetate–soaked paper strips show a PbS brown or black stain as a result of reaction with H₂S. Strips were affixed to the inner wall of a culture tube, above the level of the liquid cultures for ~20 h. Levels of the wild-type were averaged and set to be 100%, and used to normalize averaged H₂S levels of all other samples. In (C), the methylene blue formation assay was used for quantification of H₂S. Data are presented as the mean ± SD from at least five independent experiments. Asterisks indicate statistically significant difference (*, p < 0.05).

Figure 2

Effect of cysteine on S. oneidensis strains. (A) Effect of 10 mM cysteine on H₂S generation by S. oneidensis strains under aerobic conditions. Averaged H₂S level of the wild-type in the absence of cysteine was set to 100% for subsequent normalization. (B) Effect of 10 mM cysteine on H₂S generation by S. oneidensis strains under anaerobic conditions. Fumarate was used as the sole EA. Averaged H₂S level of the wild-type in the presence of cysteine was set to 100% for subsequent normalization because H₂S levels were extremely low without induction. (C) Effect of 10 mM cysteine on activities of indicated promoters. For each test gene, the promoter-lacZ (E. coli) construct was introduced into the chromosome of the wild-type and the activities of the promoter as single copy were assessed by β-galactosidase assay. Data are presented as the mean ± SD from at least three independent experiments.

Figure 3

Characteristics of H₂S generation by S. oneidensis strains under anaerobic conditions. Fumarate of 5 mM was used as EA to support growth to ~0.2 of OD₆₀₀, which was then added with either SO²⁻₃ or S₂O²⁻₃. H₂S generation was measured 8 h after the addition. Averaged H₂S level of the wild-type in the presence of cysteine was set to 100% for subsequent normalization as stated in Figure 2. “EA + Cys” represents the indicated EA plus cysteine. Data are presented as the mean ± SD from at least four independent experiments.

Figure 4

Crp-cAMP is essential to H₂S generation by S. oneidensis. (A) H₂S levels in anaerobic cultures of indicated mutants with SO²⁻₃ and S₂O²⁻₃ at 2 mM each were measured and normalized to values of the wild-type. TMAO of 5 mM was used as EA to support growth to ~0.2 of OD₆₀₀, which was then added with SO²⁻₃ and S₂O²⁻₃. (B) H₂S levels in aerobic cultures of indicated mutants with cysteine at 2 mM were measured and normalized to values from the wild-type. In both (A,B) The double mutant (ΔcyaCΔcyaA) was complemented by the cyaA gene. Data are presented as the mean ± SD from at least five independent experiments.

Figure 5

Crp on activities of indicated promoters. For each test gene, the promoter-lacZ construct was introduced into the chromosome of the Δcrp strain and the activities of the promoter as single copy were assessed by β-galactosidase assay. The same assay was performed with the complemented mutant for comparison. Data are presented as the mean ± SD from at least four independent experiments.

Figure 6

Impact of loss of on H₂S generation in S. oneidensis. (A) ArcA and Fnr on H₂S generation. H₂S levels in cultures of indicated mutants grown aerobically or anaerobically with 10 mM SO²⁻₃ or S₂O²⁻₃ as the sole EA were measured and normalized to values from the wild-type. Complementation of the ΔarcA mutant (ΔarcA^C) was conducted with aerobic growth. (B) ArcA and Fnr on activities of indicated promoters. For each test gene, the promoter-lacZ construct was introduced into the chromosome of the Δcrp strain and the activities of the promoter as single copy were assessed by β-galactosidase assay. Data are presented as the mean ± SD from at least four independent experiments.