Analysis Of Anaerobic DMSO Respiration System And Its Regulation Mechanism In Deep Sea Bacterium Shewanella Piezotolerans WP3

Bacterial respiration,the most basic and important biological metabolism pathways,is flexible and various.There is close relationship between one bacterial respiration pathway and others.Bacterial respiration with DMSO not only involves in the global sulfur cycle,iron cycle,the cooling effect of global climate and rainfall acidity,but also is closely related to changes in atmospheric ozone concentration. Microorganisms living in the extreme environment of deep-sea must have special physiological metabolic pathways to adapt to extreme environments.They can play an important role in "the global ecosystem".The deep sea bacteria used in this study was isolated from deep-sea sediments of 1914m depth in west Pacific.The phylogenetic analysis and molecular study shows that the bacterium is a psychrophilic and piezotolerant microbe with the term as Shewanella piezotolerans WP3,which will be an ideal material to study the adaptation mechanism of extremophiles to the extreme environments.In this experiment,combined with bioinformatics and molecular biology technique,we analysed the composition,expression and regulation of anaerobic DMSO respiration system in WP3.We have a deeper understanding of the unique genes,special metabolic pathway and the role of deep-sea microbes in environment.Shewanella species have been isolated from many aquatic environments.They can produce DMS through respiration of DMSO under anaerobic condition.Before the investigation of WP3,five dimethyl sulphoxide(DMSO) family subsystems were found.TypeI dmsEFABGH is present in 10 of the 20 sequenced Shewanella species. Deletion of typeâ… components in Shewanella oneidensis MR-1 leads to a severe defect in DMSO reduction.Compared with the typeâ… subsystem,the typeâ…¡andâ…¢subsystems contain an additional gene that is predicted to encode an outer-membrane lipoprotein,whereas the typeâ…£subsystem has an added gene that encodes an endonuclease-â…¢related protein.The typeâ…£subsystem is more characteristic of Escherichia coli,as it lacks genes that encode the MtrAB-like module(DmsEF),it is predicted to encode a periplasmic localized DmsAB reductase complex and it encodes the inner-membrane anchoring protein DmsC.Two dms gene clusters exist in WP3,including typeâ… subsystem and a new type subsystem without dmsF.There are two regulator genes before dmsA-2.This new dms gene cluster is typeâ…¥subsystem.The function of two regulator genes is unknown.In contrast with MR-1,deletion of typeâ… components in WP3 leads to no defect in DMSO.Gene expression was greatly induced under anaerobic growth conditions for genes tested in the typeâ…¥dms gene cluster(32 to 45fold) compared with a slight induction for genes tested in the typeâ… dms gene cluster(3 to 7fold). Typeâ… dms gene cluster is not necessary for anaerobic respiration with DMSO in WP3.We tested phenotype growth of some single dins gene deletion strains and double dmsB genes deletion strain compared with wildtype WP3 at 4℃and 20℃.The result shows that WP3 has two types of functional dms gene clusters.When one gene cluster is destroyed,the other one also can make mutant have the grestest ability of reducing DMSO as the same as the wildtype WP3 by increasing the expression of dms genes.In WP3,DmsA-1 is 90%similar to DmsA in Shewanella sediminis HAW-EB3 and 89%similar to DmsA in Shewanella woodyi ATCC 51980;DmsA-2 is 61% similar toDmsA in Shewanella putrefaciens CN32 and 60%similar toDmsA in Shewanella oneidensis MR-1.DmsA-1 is 53%similar to DmsA-2 in the sequence similarity of the primary structure.It is relatively low,compared to other strains,but they have same function.By contrast,variability in the use of DMSO can be provided by the presence of multiple copies of entire DMSO-reductase-containing subsystems in a single organism,differences in the types of electron-transfer components in these subsystems and the high degree of variability within the protein sequence of the DMSO reductase components. Escherichia coli can respire anaerobically using dimethyl sulfoxide(DMSO) or trimethylamine-N-oxide(TMAO) as the terminal electron acceptor for anaerobic energy generation.Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.In S.oneidensis,however,CRP has also been linked to the regulation of anaerobic respiration,whereas in other bacteria,anaerobic growth is governed by the redox-sensing protein FNR(fumarate nitrate-reduction regulator). Interestingly,the S.oneidensis electron-transport regulatorA(EtrA),which shares a high degree of amino acid sequence identity with the E.coli FNR protein(73.6%),is not essential for anaerobic growth and reduction of electron acceptors by MR-1.By contrast,S.oneidensis MR-1 crp mutants are deficient in the anaerobic reduction of Fe(â…¢),Mn(â…£),fumarate,nitrate and dimethyl sulphoxide(DMSO).The mechanism of CRP activation under anaerobic conditions is unknown,as CRP lacks obvious redox-sensing domains and therefore is not expected to respond to redox changes.WP3 crp gene deletion mutant is under construction.The relationship between the crp gene and WP3 anaerobic respiration is uncertain.WP3 etrA gene(swp2580) is 88%similar to the MR-1 etrA gene.We have been successfully constructed WP3 etrA gene mutant and tested its phenotype growth.WP3 etrA gene deletion mutant also can reduce N a₂S₂O₃ under anaerobic condition.The etrA gene is not essential for anaerobic growth and reduction of DMSO or N a₂S₂O₃ by WP3.In the Sunping' study of WP3 fur gene(ferric uptake regulator),WP3 fur gene deletion mutants are deficient in the anaerobic reduction of some electron acceptors including DMSO.There is no DNA-binding box of Fnr near the dms gene cluster.It is predicted that Fur indirectly impact the anaerobic reduction of DMSO by affectting the maturation of cytochrome C system for the anaerobic respiration of the electron transport chain.This suggests that Fnr dose not directly activate the transcriptional expression of dms gene clusters.This also reflects the regulation of cellular respiration is complex and required multiple genes through multiple pathways.