Insights Into The Diversity And Catabolic Mechanisms Of Dimethylsulfoniopropionate-catabolizing Bacteria In The Antarctic Ocean

Dimethylsulfoniopropionate(DMSP)is an important organic sulfur compound in the ocean,which plays an important role in the global carbon and sulfur cycling.DMSP is produced up to 103 Tg each year by many marine phytoplankton,intertidal macroalgae,coastal angiosperms,coral and bacteria.Microbial degradation of DMSP is an important step in the global sulfur cycle.It is reported that marine bacteria metabolize DMSP via three different pathways,the lysis pathway,the demethylation pathway and the oxidation pathway.In the lysis pathway,microorganisms use diverse DMSP lyases to cleave DMSP to produce dimethylsulfide(DMS)and acrylate or 3-hydroxypropionate-CoA(3-HP-CoA).Eight different DMSP lyases have been reported,including DddP,DddL,DddQ,DddW,DddK,DddY,DddD and Almal.Volatile DMS is an important participant in the global sulfur cycle,which can enter the atmosphere from the oceans.In the atmosphere,DMS may form cloud condensation nuclei and further influence the global weather and climate.Microorganisms can use trimethylamine monooxygenase(Tmm)oxidize DMS to dimethylsulfoxide(DMSO).In marine organisms,DMSO is an effective cryoprotectant and free-radical scavenger.In this dissertation,DMSP-catabolizing bacteria were firstly screened from Antarctic samples,and a strain,Psychrobacter sp.D2,which grew on DMSP and produced DMS,was obtained;the pathway and metabolic mechanism of P.sp.D2 utilizing DMSP were further elucidated,and a novel ATP-dependent DMSP lyase termed DddX was identified,which belongs to the acyl-CoA synthetase superfamily and catalyses the conversion of DMSP to DMS and acryloyl-CoA;the molecular mechanism of bacterial oxidation of oceanic DMS into DMSO was also studied;in addition,a novel strain,SM1701T,isolated from the surface of Antarctic macroalgae,was taxonomically identified using a polyphasic approach.1.Diversity of DMSP-catabolizing bacteria in the Antarctic ocean and the discovery of DMSP catabolic gene cluster in an Antarctic bacterium Psychrobacter sp.D2The microbial cleavage of DMSP generates volatile DMS through the action of DMSP lyases,and about 300 million tons of DMS is produced annually.Recently,several bacteria were reported to produce DMS from DMSP but lack known DMSP lyases in their genomes,suggesting the presence of novel enzyme(s)for DMSP degradation in nature.In this dissertation,using DMSP as the sole carbon source,DMSP-catabolizing bacteria were screened from Antarctic marine samples including seawater and sediments.The diversity of the obtained bacteria was also analyzed.A total of 175 DMSP-catabolizing bacteria were obtained,and they belong to the genera Pseudoalteromonas,Psychrobacter,Arthrobacter,Arcobacter,Paraglaciecola and Polaribacter,with the genera Pseudoalteromonas(47%)and Psychrobacter(44%)being predominant Among these DMSP-catabolizing bacteria,Psychrobacter.sp.D2,which grew well on DMSP and produced DMS,was selected for further investigation with its genome and transcriptome sequenced.A gene cluster comprising four genes(1696,1697,1698,and 1699)was found to be likely involved in DMSP metabolism in P.sp.D2.RT-qPCR analysis showed that the transcripts of these four genes were all upregulated under the induction of DMSP.In the gene cluster,1696,1698 and 1699 are homologs of DddT,DddC and DddB,respectively,which have been reported to be involved in DMSP import and 3-HP-CoA catabolism.The results of physiological experiments and bioinformatics analyses suggested that 1697 likely encodes a novel DMSP lyase,which was termed as DddX.These results reveal the diversity of DMSP-catabolizing bacteria in the Antarctic ocean and identify a possible novel DMSP lyase gene,which are helpful for further study of the microbial catabolism of DMSP.2.The essential role of dddX in DMSP degradation in P.sp.D2To verify the function of dddX in DMSP catabolism,the genetic manipulation system of P.sp.D2 was constructed firstly.Genetic analyses suggested that dddX encodes a functional DMSP lyase enzyme degrading DMSP to DMS.Then DddX was overexpressed and purified,and the enzymatic activity and metabolites analyses of the recombinant DddX were performed.In the presence of CoA and ATP,the recombinant DddX catalyzes the conversion of DMSP to DMS and acryloyl-CoA,which is different from the other reported DMSP lyases.To the best of our knowledge,DddX represents the first DMSP lyase of the acyl-CoA synthetase(ACD)superfamily.Liquid chromatography-mass spectrometry(LC-MS)analysis found that DMSP-CoA is formed in the catalytic reaction of DddX,indicating that DddX catalyzes a two-step reaction:1)the ligation of DMSP with CoA to form the intermediate DMSP-CoA;2)the subsequent cleavage of DMSP-CoA to DMS and acryloyl-CoA.The enzymatic properties of DddX were further examined.The optimal temperature and pH for DddX enzymatic activity were 40? and 8.5,respectively.Together,these results demonstrate that DddX is a novel ATP-dependent DMSP lyase,which provides a better understanding of the DMSP metabolism.3.The catalytic mechanism of DMSP lyase DddXTo elucidate the structure basis for DddX catalysis,the crystal structure of DddX in complex with ATP was firstly solved.Analysis for the crystal structure of DddX revealed that each DddX monomer contains a CoA-binding domain and an ATP-grasp domain,with one loop(Gly280-Tyr300)of the CoA-binding domain inserting into the ATP-grasp domain.Sequence alignment and mutational analysis indicated that His292 of Gly280-Tyr300 is highly conserved in the ACD superfamily.Deduced from the reported molecular mechanisms of the ACD superfamily,residue His292 is likely phosphorylated in the catalysis of DddX on DMSP.Through the docking of DMSP-CoA into the structure of DddX,two aromatic residues(Trp391 and Phe435)were found to form cation-? interactions with DMSP-CoA.Mutational analysis showed that these two residues play important roles in the DddX catalysis.Structure analysis showed that Glu432 is close to the DMSP moiety of DMSP-CoA,and may function as the catalytic base.Mutational analysis showed that the mutation of Glu432 abolished the enzymatic activity of DddX,indicating that the key role of Glu432 during the DddX catalysis.Based on all of the results,the molecular mechanism of DddX catalysis on DMSP was proposed.DddX is found in diverse groups of bacteria,including Alphaproteobacteria,Gammaproteobacteria,and Firmicutes,suggesting that this new DMSP lyase may play an important role in DMSP metabolism and global sulfur cycle.These results offer new insights into how diverse bacteria cleave DMSP to generate the climatically important gas DMS,extending the understanding of the important biogeochemical process of microbial cleavage of DMSP producing DMS.4.Structural and mechanistic insights into dimethylsulfoxide formation through dimethylsulfide oxidation catalyzed by trimethylamine monooxygenase(Tmm)DMS and DMSO are widespread in marine environment,and are important participants in the global sulfur cycle.Microbiol oxidation of DMS to DMSO represents a major sink of DMS in marine surface waters.The SAR11 clade and the marine Roseobacter clade(MRC)are the most abundant heterotrophic bacteria in the ocean surface seawater,and are active participants in marine carbon,nitrogen and sulfur cycles.It has been reported that Tmm from both MRC and SAR11 bacteria can oxidize DMS to generate DMSO.However,the structural basis of DMS oxidation has not been explained.In this dissertation,a Tmm homolog from the SAR11 bacterium Pelagibacter sp.HTCC7211(Tmm7211)was characterized.Tmm7211 was found to have DMS oxidation activity in vitro.The crystal structures of Tmm7211/FAD/NADPH and Tmm7211/FAD/NADPH soaked with DMS were further solved.Based on structural analyses and related literatures,the catalytic mechanism of Tmm7211 was elucidated,which comprises a reductive half-reaction and an oxidative half-reaction.FAD and NADPH molecules were found essential for the catalysis of Tmm7211.In the reductive half-reaction,FAD reacts with NADPH and O2 to form the C4a-hydroperoxyflavin.In the oxidative half-reaction,the binding of DMS repels the nicotinamide ring of NADP+,and NADP+generates a conformational change to form a hydrogen bond with Asp314,shutting off the substrate entrance and exposing the active C4a-hydroperoxyflavin to DMS to complete the oxidation of DMS.Sequence analysis revealed that the proposed catalytic mechanism of Tmm7211 may be widely adopted by MRC and SAR11 bacteria.These results provide important insight into the conversion of DMS into DMSO in marine bacteria,leading to a better understanding of the global sulfur cycle.5.Polyphasic taxonomic analysis of strain SM1701TStrain SM1701T was isolated from a macroalgae sample collected from the intertidal zone of the King George Island,West Antarctic.In this dissertation,the taxonomic position of strain SM1701T was determined based on phenotypic(morphological,physiological,and biochemical traits),chemotaxonomic and genotypic characterization.Cells of strain SM1701T are Gram-stain-negative,non-flagellated rods,and aerobic.The strain grows at 4-30?(optimum 20?),pH 6.0-8.0(optimum at pH 7.0)and with 0.5-5%(w/v)NaCl(optimum 2%).It hydrolyses gelatin,tyrosine and Tweens(40,60 and 80).The major polar lipids of strain SM1701T are phosphatidylethanolamine(PE)and two unidentified glycolipids(GL).The major cellular fatty acids of strain SM1701T include iso-C15:0,iso-C15:1G,iso-C16:1G,C16:0 and iso-C17:0 3-OH.The major respiratory quinone of strain SM1701T is menaquinone-7(MK-7).The genomic DNA G+C content of strain SM1701T is 34.1 mol%.Strain SM1701T has high 16S rRNA gene sequence similarities(92.5-93.8%)to members of the genus Crocinitomix in the family Crocinitomicaceae.The phylogenetic trees based on 16S rRNA gene sequences showed that strain SM1701T formed a separate phylogenetic branch,adjacent to the genus Crocinitomix,within the cluster of the family Crocinitomicaceae,suggesting that it should belong to a new genus.On the basis of the polyphasic characterization for strain SM1701T in this dissertation,it is considered to represent a novel species in a new genus in the family Crocinitomicaceae,for which the name Putridiphycobacter roseus gen.nov.,sp.nov.is proposed.The type strain is SM1701T(=KCTC 62302T=NBRC 113201T=CGMCC 1.16510T).In conclusion,in this dissertation,the diversity of the culturable DMSP-catabolizing bacteria isolated from the Antarcitc ocean,the molecular mechanisms for DMSP cleavage of a novel ATP dependent DMSP lyase DddX and bacterial oxidation of DMS into DMSO,and the taxonomic identification of a novel Antarctic marine bacterium have been detailedly studied,whose results provide a better understanding of DMSP metabolism and the global sulfur cycle,and lay a foundation for better utilization of marine microbial resources.