A Fis Family Regulator (FisR) Controls The Sulfide Oxidation Pathway In Cupriavidus Pinatubonensis JMP134

Sulfide and its derivatives are common pollutants in the air and water. Under natural conditions, sulfide is mainly generated from the volcanic activity and sulfur reduction by microorganisms under anaerobic conditions. Eukaryotes can also produce sulfide through normal intracellular metabolism. Previous studies of sulfide oxidation were mostly focused on the autotrophic microbes, and heterotrophic microbes were often overlooked. Therefore, the sulfide oxidation mechanism of heterotrophic microbe has more gaps to be filled.There are two enzymes involved in heterotrophic microbe sulfide oxidization: sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). In Cupriavidus pinatubonensis JMP134, there is a potential regulatory gene next to sulfide oxidation genes pdo and sqr, the corresponding protein is annotated as an σ54-dependent, Fis family transcriptional regulator (FisR). The transcriptional regulator usually contains three domain:the N-terminal regulatory (R) domain, the central AAA+ domain (C), and the C-terminal DNA binding domain (D). Unlike the σ70-dependent transcription, the σ54-dependent transcription absolutely requires the presence of an activator that couples the ATP hydrolysis with the transcription initiation process. Two regulators for sulfide oxidation have been reported, the biofilm growth-associated repressor (BigR) in plant pathogen Agrobacterium tumefaciens and the CsoR-like sulfurtransferase repressor (CstR) in Staphylococcus aureus, and they.are all repressors.In this work, fisR disrupted- and complemented-strains were constructed. Like the pdo & sqr double disrupted-strain, the fisR disrupted-strain also accumulated hydrogen sulfide and showed a similar growth phenotype under sulfide stress. Transcriptional analysis of the gene cluster shows that the expression level of pdo and sqr in the wild type is much higher than that in the fisR disrupted-strain. All above results confirmed that FisR is the activator necessary for the expression of pdo and sqr. Further, the addition of sulfide caused increased expression of pdo and sqr, indicting that sulfide can induce their expression.The regulatory (R) domain-deficient FisR (FisR-CD) was heterologous expressed and purified. FisR-CD bound at the upstream region of the pdo promoter, and the binding site was identified. In addition, a GFP promoter reporter system was constructed in the wild type strain, and there was a positive correlation between the concentration of sulfide and promoter strength, suggesting that FisR is activated by the sulfide addition.Through a series of experiments, the regulation mechanism of FisR on sulfide oxidation in the heterotrophic microbe Cupriavidus pinatubonensis JMP134 was preliminarily revealed. This study provides a new direction to further explore the sulfide oxidation pathways in heterotrophic microbes.