Characterization Of The Role Of Two Sets Of DMSO Respiratory Systems In The Deep-sea Environmental Adaptation Of Shewanella Piezotolerans WP3

Dimethyl suifide(DMS)is a volatile anti-greenhouse sulfur compound in surface seawater.The removal of DMS from marine environment is mediated mainly by bio-oxidation to dimethyl sulfoxide(DMSO),effectively limiting the quantity of DMS available for releasing into the atmosphere.Although previous studies demonstrated that DMSO as a substantial sink of DMS is highly abundant in abyssal ocean water,few data are available on the turnover mechanisms of DMSO in this environment.As a genus the Shewanella are widely distributed in marine and freshwater environments,and they are also one of the more abundant ?-protobacteria population in deep sea.The hallmark of Shewanella is their ability to utilize a diverse array of terminal electron acceptors in the absence of oxygen and use a borad range of carbon substrates.Besides,they also have the capacity to thrive at low temperatures(~4°C).As a result,they show great potential for remediation of various environmental pollutants and in microbial fuel cells(MFCs),where their metabolism is harnessed to make electricity.Shewanella piezotolerans WP3 was isolated from a west Pacific deep-sea sediment at a depth of 1,914 m.Our previous study demonstrated that WP3 was able to utilize DMSO as a terminal electron acceptor for anaerobic growth.In this study,two putative dms gene clusters were identified in the WP3 genome,and they can be divided into type I and type VI by gene order.Genetic and physiological analyses demonstrated that both dms gene clusters were functional and the transcription of both gene clusters was affected by changes in pressure and temperature.Notably,the type I system is essential for WP3 to thrive under in situ conditions(4°C/20 MPa),whereas the type VI system is more important under high-pressure or low-temperature conditions(20°C/20 MPa,4°C/0.1 MPa).Additionally,DMSO-dependent growth conferred by the presence of both dms gene clusters was higher than growth conferred by either of the dms gene clusters alone.These data collectively suggest that the possession of two sets of DMSO respiratory systems is an adaptive strategy for WP3 survival in deep-sea environments.Two DMSO respiratory systems(type I and type VI)have been identified in the S.piezotolerans WP3 genome,in which type I is most common in Shewanella,whereas type VI is found only in WP3.Phylogenetic analyses were performed to examine the evolutionary relationships of DMSO reductases among Shewanella strains.Results demonstrated that type VI and type I DMSO reductases are closely evolutionarily related.Genetic and physiological analyses demonstrated that two DMSO respiratory systems in WP3 are functionally independent.In addition,type VI DMSO respiratory system don't rely on periplasmic DmsE protein for electron transfer,implying that two DMSO reductases in WP3 have a distinct subcellular localizaztion.Immunoelectron microscopy was used to observe the subcellular localizaztions of two DMSO reductases in WP3.Results demonstrated that type I DMSO reductase is located on the outer leaflet of the outer membrane,whereas the type VI DMSO reductase is located within the periplasmic space.Both type I and type VI DMSO reductases tend to accept electrons from CymA preferentially.Collectively,we proposed a core electron transport model of DMSO reduction in the deep-sea bacterium S.piezotolerans WP3.In view of fact that the presence of both dms gene clusters was essential for WP3 to achieve the maximum growth under in situ conditions,we concluded that the possession of two sets of DMSO reductases with distinct subcellular localizations might be an adaptive strategy for WP3 to achieve maximum DMSO utilization in deepsea environments.Finally,we investigated the roles of global regulator Crp on DMSOdependent anaerobic growth in WP3.Genetic and physiological analyses demonstrated that strains lacking crp gene failed to grow in DMSO medium,but no growth deficiency could be observed under aerobic conditions.The transcription assay revealed that,compared with WP3-WT,the expression of two dms gene clusters in ?crp was greatly down-regulated in the context of DMSO reduction.Furthermore,in vitro assay demonstrated that Crp binds directly to the promoter regions of both dms promoters,indicating that both dms gene clusters are under direct control of Crp protein.