Metabolism And Function Of H₂S In Different Heterotrophic Bacteria

Hydrogen sulfide(H2S)is a toxic gas with a smell of rotten eggs.It exists in a variety of production processes and in nature.H2S can inhibit the respiratory chain and affect the activities of the central nervous system and respiratory system.It is the second killer after CO in industrial production.In the 1990s,researchers found that the mammalian cells can endogenously produce H2S,suggesting that H2S may have a certain physiological role.With the advancing of related research in recent years,H2S has been considered as the third gas signal molecule after the CO and NO.In mammalian cells,H2S is mainly produced from sulfur-containing amino acids,including cysteine,methionine and cystine.H2S production involves three enzymes:cystathionine ?-synthetase(CBS),cystathionine ?-lyase(CSE)and 3-mercaptopropionate sulfur transferase(3-MST).The excessive production and accumulation of H2S in mammalian cells may inhibit respiration and produce toxic effects.To avoid excessive accumulation of H2S,the endogenously produced H2S should be reoxidized to thiosulfate by enzymatic oxidation in mammalian cells.The H2S oxidation process mainly involves three enzymes:thioquinone oxidoreductase(SQR),sulfide dioxygenase(PDO)and sulfur transferase(RHOD).The pathway of H2S oxidation in autotrophic bacteria and its role in energy conservation have extensively been studied.SQR is reported mainly in the autotrophic bacteria and sulfur-dependent photosynthetic bacteria.SQR can be divided into six categories.Two types of PDO were found in gram-negative bacteria.They were able to catalyze the same substrate,and had high sequence similarity.Another type of PDO was found in Gram-positive autotrophic bacteria.The sequence similarity between this type of PDO and PDOs in the gram-negative autotrophic bacteria is low and their substrates are different.Previous studies have confirmed that heterotrophic bacteria also contain SQR,PDO and RHOD coding genes,and the above-mentioned proteins also play a role in the metabolism of sulfide.For example,we previously reported that SQR in Cupriavidus pinatubonensis JMP134 first oxidizes the sulfide to produce polysulfide.The polysulfide reacts spontaneously with GSH to fonn GSSH,and then PDO oxidizes GSSH to form sulfite.Sulfite can also spontaneously react with poly sulfide to generate thiosulfate.RHOD can accelerate the reaction of poly sulfide with GSH.Based on the above studies,it is speculated that similar to the situation in mammalian cells,H2S production and metabolism may be widely present in bacteria and have a certain physiological efect In this work,SQR and PDO were screened in all of the sequenced bacteria and archaea via bioinformatics.The SQR and PDO coding genes were found to be abundant in different bacteria and archaea.Taking into account the autotrophic bacteria in the metabolism of H2S has been studied more in depth,this work mainly studied the H2S production and metabolism in heterotrophic bacteria using C.pinatubonensis JMP134,Pseudomonas aeruginosa PAO1 and Escherichia coli BL21(DE3)as the research targets.E.coli BL21(DE3)genome contains no SQR and PDO coding genes.C.pinatubonensis JMP134 genome contains SQR and PDO coding genes that are located in the same operon.P.aeruginosa PAOl genome contains SQR and PDO coding genes,but SQR and PDO coding genes are far away in the genome.The deletion,complementation,overexpression of the genes were conducted with the analsyis of sulfide oxidation.SQR and PDO in heterotrophic bacteria were able to metabolize H2S.When the strains containing SQR and PDO were cultured,H2S was not accumulated and released.The strains containing SQR and PDO were able to metabolize H2S released from other strains in mixed culture.At the same time,P.aeruginosa PAO1 was used as the representative strain to study the H2S production pathway in heterotrophic bacteria.It was confirmed that P.aeruginosa PAO1 contained three enzymes CBS,CSE and 3-MST,which catalyzed the formation of H2S from organic sulfide,and sulfite reductase which catalyzed formation of H2S from inorganic sulfite.After deletion of the H2S synthesis-related genes and H2S metabolism-related genes in P.aeruginosa PAO1,three mutants were obtained.Pa?H2S(Pa?cbs?cse?mst?cysI)contains no H2S synthesis-related genes Pa3 K(Pa?sqr1?sqr2?pd)contains no H2 S metabolism-related genes Pa7K(?cbs?cse?mst?cysI?sqr1?sqr2?pdo)contains no H2S metabolism-related genes and H2S synthesis-related genes.Pa7K contains neither H2S-producing genes nor H2S metabolic genes,but its growth in LB medium is not affected and does not release H2S.If excess cysteine was added to LB medium,Pa7K will produce H2S in large quantities,indicating that P.aeruginosa PAO1 has other ways to produce H2S from cycteine.In the inorganic salt medium with excess inorganic sulfur,Pa7K no longer produces and accumulates H2S,which indicates that the key enzyme in inorganic sulfur pathway of P.aeruginosa PAO1 to produces H2S is via sulfite reductase.A sqr gene is not present in the genome of Cupriavidus necator H16,but C necator H16 did not release H2S during growth in LB.Further analysis of the genome of C.necator H16 revealed that the strain contained the fccA and fccB gene encoding the flavin cytochrome sulfur dehydrogenase(FCSD).FCSD has been reported in photosynthetic bacteria and contains a large subunit of flavin-binding protein(FccB)and a small subunit of cytochrome c(FccA).FCSD and SQR have similar function in vitro,capable of oxidizing sulfide to produce zero-valent sulfur,but the electrons generated during the oxidation of the sulfide by FCSD are passed to the cytochrome c.Whether FCSD can catalyze sulfide oxidation similar to SQR in vivo has not been reported.In this work,the FCSD coding genes of C necator H16 were knocked out,complemented,overexpressed;H2S oxidative capacity of FCSD was studied.The results showed that FCSD could metabolize 12S produced by C.necator H16.The mutant strain Cn?fccAB lost H2S-oxidizing ability and released H2S.The was expressed in the Pa3K(Pa?sqrl?sqr2?pdo),in which the H2S metabolic-related genes were knockout.It was found that FCSD also had the activity of oxidizing sulfide in Pa3K.Similar to SQR,the polysulfide produced by FCSD oxidation of H2S also contained disulfide and trisulfide.Furthermore,fccAB and pdo were co-expressed in Pa3K,and FCSD and PDO could synergistically metabolize H2S.FCSD oxidizes H2S to generate polysulfide,polysulfide and GSH spontaneous react to generate GSSH,and then PDO oxidizes GSSH to form sulfite.The sulfite can also spontaneously react with polysulfide to generate thiosulfate.This route is similar to the route of SQR/PDO in C.pinatuhonensis JMP134.The only difference is that C necator H16 does not contain RHOD that accelerates the reaction of polysulfide with GSH.Bioinformatics analysis showed that 190 of the 4999 strains with the sequenced genome had fccB,and most of the fccB were adjacent to fccA in the genome.FccBs can be divided into three classes,and each class is relatively conservative during evolution.P.aeruginosa is a gram-negative conditional pathogen that is widely distributed in different habitats.In order to further understand the physiological functions of H2S production and metabolism in heterotrophic bacteria,the physiological phenotype of P.aeruginosa PAO1 and its mutant strains including Pa3K,PaAH2S and Pa7K were studied.Several representative phenotypes of aeruginosa PAO1 including the production of pyocyanin,the formation of biofilm,the movement,the colony morphology on the blood plate,the production of rhamnolipid and the pathogenic ability were studied.The results showed that P.aeruginosa PAO1 was similar to that of Pa3K,while Pa?H2S was similar to that of Pa7K.In particular,the deletion of H2S synthesis related genes in P.aeruginosa PAO1 seriously affected the phenotype of pyocyanin production,the movement,the production of rhamnolipid,indicating that H2S synthesis rather than H2S metabolism of has an impact on various phenotypes of P.aeruginosa PAO1.The ability of mutans of P.aeruginosa PAO1 without the H2S synthesis-related genes to infect lettuce petiole is also significantly decreased.Transcriptome analysis based on the P.aeruginosa PAO1 and Pa?H2S showed that the deletion of H2S synthesis-related genes affected the quorum sensing system.Considering that the phenotypes of P.aeruginosa PAO1 are regulated by quorum sensing system,it is speculated that the deletion of H2S synthesis gene leads to the loss of H2S synthesis ability of P.aeruginosa PAO1,which in turn affects the quorum sensing system and leads to the change of the expression of phenotypic related genes regulated by the quorum sensing system,and finally affects the pathogenic ability of P.aeruginosa PAO1.The above studies showed that the physiological function of H2S may be related to the interaction of H2S with the quorum sensing system.The specific physiological functions of H2S still needs further study.