The Mechanism Of A Sulfide Oxidation Pathway In Bacillus Licheniformis MW3 For Its Cascade Regulation

Hydrogen sulfide and its derivatives,an important part of biogeochemical cycle.widely exist in nature.Hydrogen sulfide is a kind of toxic gas,which can affect the activity of central nervous system and respiratory system by inhibiting respiratory chain.With the deepening of relevant studies,H2S is also considered as the third gas signal molecules after CO and NO,which can not only play an important regulatory role on cell physiology,but also resist oxidative pressure by directly participated in the process of cell scavenging free radicals as a reducing substance.Heterotrophic bacteria can produce H2S by oxidized organic matter in aerobic environment.The SQR-PDO pathway is important for the H2S oxidation of heterotrophic bacteria.which can oxidize sulfide into sulfite and thiosulfate through polysulfide.Sulfates are the salts with the highest degree of oxidation among sulfur compounds,they are also the major sulfur source for cysteine synthesis in plants and most microorganisms Industrial waste water with high concentration of sulfur also seriously affect the national ecological and environmental.Therefore,the research about sulfur oxidation play an important role in biogeochemical cycles.BigR.CstR and SqrR are reported as negative regulators from three different bacteria respectively,which negatively regulate the transcription of H2S oxidation-related genes.In addition.FisR,a new type of σ54-dependent regulator in Cupriavidus pinatubonensis JMP134.positively regulates the expression of sqr and pdo.Bioinformatics analysis showed that a gene cluster was located in upstream of sqr and pdo genes in Bacillus licheniformis MW3,which contained the two-component system NreBC and a series of sulfur transport-related genes.However,the mechanism of the regulatory factor yrkD and the two-component system NreBC regulating sqr and pdo is not clearIn this paper.the ability to oxidize H2S after induction was lost after deleted nreB and nreC genes,indicating that NreBC is an activated transcription factor.However,SQR could transcribe without induction when only yrkD was knocked out.SQR could not normally transcribe after the knockout of yrkD and nreBC strains.The comparison showed that YrkD could inhibit the expression of nreBC.After the establishment of the green fluorescent protein reporting system in MW3,it was found that the generation of green fluorescent protein could be induced by polysulfide,while the generation of green fluorescent protein could be induced by hydrogen sulfide instead of polysulfide on the basis of the knockout of yrkD strain.The results showed that the induction signals of YrkD and NreB are different.The induction of YrkD is sulfane sulfide,while the induction signal of NreB is HS-,which is a new type of induction signal and plays an important role in the sulfide metabolism.The RNA of the wild-type strain was extracted and analyzed for co-transcription.it was found that the co-transcription text existed in yrkD-yrkE-yrkF-yrkk-yrkI-yrkJ-ydfq-nreB-nreC-sqr-pdo.The sqr-pdo reversed strain was constructed,it was found that the SQR could still be expressed after the induction of sulfide when the sqr and pdo were reversed as a whole,which indicated that there were independent promoters in front of sqr-pdo.Transcription initiation sites were identified by the 5’ RACE analysis,the results confirmed that transcription initiation sites were located in front of yrkD and sqr.In B.licheniformis,a novel H2S oxidation pathway was found in this paper.This total pathway includes the known SQR-PDO pathway,which was cascade regulated by YrkD and NreBC.Meanwhile.NreBC,as a two-component system,has been found to be involved in the regulation of H2S oxidation pathway for the first time.In addition,known regulatory signals are polysulfide,and regulatory factors that can sense HS-signal has been found for the first time.The regulation mechanism of sulfide oxidation pathway revealed in this paper would enrich our cognition of the diversity of sulfur oxidation in heterotrophic bacteria,provide certain basis for the application of biological processing of H2S,and have certain ecological significance.