| Dimethylsulfoniopropionate(DMSP)is an organic sulfur molecule that is mainly produced by phytoplankton and macroalgae,and it is prevalent in the marine environment.One of its biological metabolites is volatile gas dimethyl sulfide(DMS)which is the largest natural source of sulfur in the atmosphere.DMSP and DMS are critical in the global sulfur cycle and affect the global climate.The Antarctic region has become a hotspot in DMS studies because of the high spatial and temporal variability in DMS(P)concentration,but the level of bacterial-mediated DMS production remains unclear.In this study,Antarctic Rhodococcus sp.NJ-530 capable of degrading DMSP into DMS was obtained,and then analysis of complete genomic data and bioinformatics identified candidate genes involved in DMSP metabolism,especially DMSP lyase gene ddd D-Rh,constructing the pathway of DMSP degradation in Antarctic Rhodococcus sp.NJ-530.Heterologous expression of ddd D-Rh through DNA recombination technique was performed to verify its DMSP cleavage activity.Transcriptome sequencing was used to obtain a global transcriptional characterization of genes in Antarctic Rhodococcus sp.NJ-530 in response to DMSP,and quantitative real-time PCR(q RTPCR)was applied to reveal the effect of temperature and salinity on the transcription level of ddd D-Rh.The specific results are as follows: 1)A strain of Gram-positive Actinobacteria Antarctic Rhodococcus sp.NJ-530 was isolated successfully with DMSP as the sole carbon source,and its rate of DMS production from DMSP was measured as 3.96 pmol/mg pr/h through the gas chromatograph.Results of carbon source test showed that Antarctic Rhodococcus sp.NJ-530 could grow with DMSP as the sole carbon source,but compared with conventional carbon source,DMSP did not support as much growth of Antarctic Rhodococcus sp.NJ-530.2)Complete genome sequencing results of Antarctic Rhodococcus sp.NJ-530 revealed its genome size is 7,324,898 bp,including a chromosome and four plasmids.A total of 7371 protein-coding sequences(CDSs)were predicted,which were then annotated against several databases such as GO,KEGG,etc.Candidate genes involved in DMSP metabolism in Antarctic Rhodococcus sp.NJ-530 were identified and named ddd D-Rh,ddd B-Rh,ddd C-Rh,respectively.Thus,putative DMSP metabolic pathway in Antarctic Rhodococcus sp.NJ-530 would be: Ddd D-Rh cleaves DMSP into DMS and 3-hydroxypropionate(3-HP),and 3-HP is subsequently converted to malonate semialdehyde(MAL-SA)by Ddd B-Rh.Finally,MAL-SA is metabolized by Ddd C-Rh to generate acetyl-Co A and CO2.3)The Ddd D-Rh encoded by the candidate DMSP lyase gene ddd D-Rh contains two Cai B domains,which is likely to belong to the Co Atransferase III superfamily.The predicted molecular weight of Ddd D-Rh is 73.21 k Da,which is quite different from previously characterized Ddd D in sequence and evolution.Recombinant of Ddd D-Rh was mainly expressed as inclusion bodies in the BL21(DE3),and then low temperature dialysis was performed to refold Ddd D-Rh.In vitro assays showed that refolded Ddd D-Rh could cleave DMSP into DMS in the presence of acetylCo A,suggesting it was functional,and this was the first functionally Ddd D from Grampositive Actinobacteria.4)Transcriptome analysis showed that there were 2906 differentially expressed genes between Antarctic Rhodococcus sp.NJ-530 with and without DMSP induction,of which 1349 were up-regulated and 1557 were downregulated.Moreover,candidate ddd B-Rh and ddd C-Rh genes that might be involved in downstream steps of DMSP degradation were significantly up-regulated in this analysis.q RT-PCR revealed that high temperature(20 ?)facilitated its expression,while salinity had little effect.Overall,the molecular mechanism of DMSP metabolism in Antarctic Rhodococcus sp.NJ-530 was explored in this study,which provided understanding of the bacterial metabolism of DMSP and DMS emission in the Antarctic area. |
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