【作者】 王秀娟；

【导师】 张熙颖； 张玉忠；

【作者基本信息】 山东大学 ， 微生物学， 2021， 博士

【摘要】 二甲基巯基丙酸内盐(Dimethylsulfoniopropionate,DMSP)是海洋中重要的有机硫化合物,在全球碳循环和硫循环过程中发挥重要作用。单细胞真核浮游藻类、潮间带大型藻类、部分海岸带被子植物、珊瑚虫和部分海洋细菌等是DMSP的生产者,每年全球约产生109吨DMSP。微生物参与的DMSP的分解代谢过程是全球硫循环的重要步骤之一。目前已发现海洋微生物可以通过三种不同的代谢途径降解DMSP:裂解代谢途径、脱甲基代谢途径和氧化代谢途径。在裂解代谢途径中,海洋细菌利用DMSP裂解酶裂解DMSP产生二甲基硫(Dimethylsulfide,DMS)和丙烯酸[或 3-羟基丙酰辅酶 A(3-Hydroxypropionate-CoA,3-HP-CoA)]。目前已报道的 DMSP 裂解酶有 8 种:DddP、DddL、DddQ、DddW、DddK、DddY、DddD和Almal。挥发性气体DMS是全球硫循环的重要载体物质,可以参与云凝结核的形成,进而影响全球气候。海洋细菌可以利用三甲胺(Trimethylamine,TMA)单加氧酶Tmm将DMS氧化为二甲基亚砜(Dimethylsulfoxide,DMSO)。DMSO具有重要的生理功能,它可以作为胞内的冷冻保护剂和自由基清除剂。在本论文中,我们首先以自南极样品中筛选的DMSP利用细菌菌株Psychrobacter sp.D2为研究对象,从中鉴定出了一种新型ATP依赖的DMSP裂解酶DddX,序列比对发现,DddX属于酰基辅酶A合成酶超家族,可以催化DMSP裂解产生DMS和丙烯酰辅酶A,我们进一步阐明了该菌株利用DMSP的途径和代谢机制;在此基础上,我们进一步研究了 Tmm氧化DMS生成DMSO的分子机制;此外,对从南极海域大型海藻样品表面分离得到的一株细菌进行了分类学鉴定,鉴定为一个细菌新属的新种。1.南极海域DMSP利用菌株的多样性分析与南极海洋细菌Psychrobacter sp.D2代谢DMSP途径中关键基因簇的发现微生物通过DMSP裂解酶裂解DMSP产生挥发性的DMS,每年通过微生物的裂解作用约产生300万吨的DMS。最近,多种细菌被报道能够降解DMSP产生DMS,但在其基因组中并未发现已知的DMSP裂解酶基因,这表明自然环境中可能蕴藏着新的DMSP裂解酶或代谢途径。在本论文中,我们以DMSP为唯一碳源,从采集于南极的海水和沉积物样品中筛选DMSP利用菌株,并对筛选菌株进行了多样性分析。筛选到的175株细菌分布于Pseudoalteromonas、Psychrobacter、Arthrobacter、Arcobacter、Paraglaciecola 和 Polaribacter六个属,其中Pseudoalteromonas(47%)和Psychrobacter(44%)是优势属。为了阐明筛选菌株利用DMSP的机制,我们选取能以DMSP为唯一碳源生长且降解DMSP产生DMS的菌株Psychrobacter.sp.D2进行了全基因组测序和转录组测序,发现了P.sp.D2代谢DMSP的关键基因簇:1696-1699基因簇。RT-qPCR分析表明该基因簇在DMSP诱导下转录上调明显,其中的1696、1698和1699分别是DddT、DddC和DddB的同源蛋白,它们与DMSP的转运和3-HP-CoA的代谢相关。生理实验结果及生物信息学分析表明1697可能是一种新型DMSP裂解酶,命名为DddX。本部分研究结果揭示了南极海域中DMSP利用菌株的多样性,发现了一个可能的新型DMSP裂解酶基因,为进一步研究细菌如何代谢DMSP奠定了基础。2.菌株P.sp.D2代谢DMSP途径中关键基因dddX的功能为了研究dddX的功能,本论文首先建立了 P.sp.D2的遗传操作体系,通过dddX基因敲除、回补和表型验证等手段,证明了dddX能够编码有功能的DMSP裂解酶。随后对DddX进行了异源表达与纯化,检测了 DddX的体外酶活和代谢产物。DddX 在三磷酸腺苷(Adenosine triphosphate,ATP)和辅酶 A(Coenzyme A,CoA)的参与下,降解DMSP产生DMS和丙烯酰辅酶A,这与目前已知的DMSP裂解酶的裂解产物均不同,也是酰基辅酶A合成酶(Acyl-CoA synthetase,ACD)超家族中首个被发现的DMSP裂解酶。本论文利用高效液相色谱-质谱联用(Liquid chromatography-mass spectrometry,LC-MS)方法发现在 DddX 的催化过程中会产生中间代谢物DMSP-CoA,推测DddX通过两步反应催化DMSP裂解:(1),将DMSP与CoA连接形成DMSP-CoA反应中间体;(2),将DMSP-CoA裂解为DMS和丙烯酰辅酶A。进一步检测DddX的基本酶学性质发现,其最适酶活温度为40℃,最适酶活pH为8.5。这些结果证明DddX是一种新型ATP依赖的DMSP裂解酶,增强了对DMSP代谢的认识。3.DMSP裂解酶DddX裂解DMSP的分子机制研究为了研究DMSP裂解酶DddX裂解DMSP的分子机制,本论文首先解析了DddX/ATP复合物的晶体结构。晶体结构分析表明,DddX单体有两个结构域:CoA-binding结构域和ATP-grasp结构域,CoA-binding结构域的一段卷曲(Gly280-Tyr300)插入到ATP-grasp结构域中。序列分析和定点突变对酶活性的影响结果表明,卷曲Gly280-Tyr300中的His292在ACD超家族中高度保守。根据已报道的ACD超家族相关分子机制推测,氨基酸残基His292在DddX催化DMSP的过程中很可能被ATP分子磷酸化。通过将DddX的晶体结构与DMSP-CoA进行分子对接模拟分析,发现Trp391和Phe435能与DMSP-CoA形成cation-π相互作用,而定点突变分析结果进一步证实了这两个氨基酸残基在DddX的催化过程中的确发挥着重要作用。结构分析表明Glu432靠近DMSP-CoA分子的DMSP部分,可能是DddX催化过程中的催化碱基,突变432位的谷氨酸残基会导致DddX酶活的丧失,验证了 Glu432在催化过程中的重要性。综合实验结果,本论文提出了 DddX裂解DMSP产生DMS和丙烯酰辅酶A的分子机制。进一步分析发现,DddX存在于不同种类的细菌类群中,包括属于革兰氏阴性菌的α-变形菌门和γ-变形菌门,以及属于革兰氏阳性菌的厚壁菌门,表明DddX在DMSP的代谢及全球硫循环中发挥着重要作用。新型DMSP裂解酶DddX是被鉴定的第9种DMSP裂解酶,本文研究结果拓展了对于微生物裂解DMSP产生DMS这一重要的生物地球化学反应的认知和理解。4.海洋细菌TMA单加氧酶Tmm氧化DMS产生DMSO的分子机制研究DMS和DMSO在海洋中广泛存在,是全球硫循环的重要参与者。微生物氧化DMS产生DMSO是海洋表层海水中DMS消耗的主要途径。SAR11类群和海洋玫瑰杆菌类群(Marine Roseobacterclade,MRC)是海洋表层海水中丰度最高的异养细菌类群,同时也是海洋碳循环和硫循环的重要参与者。据报道来源于MRC和SAR11类群的TMA单加氧酶Tmm能够氧化DMS产生DMSO。然而,Tmm氧化DMS的分子机制尚不清楚。本论文首先鉴定了一个来源于SAR11细菌Pelagibacter sp.HTCC7211的Tmm同源蛋白Tmm7211,发现其在体外具有DMS氧化活性,并进一步解析了 Tmm7211/FAD/NADPH复合物的晶体结构。结合晶体结构分析及文献报道发现,Tmm7211的催化过程包括两个半反应:还原半反应和氧化半反应,而FAD和NADPH分子是Tmm7211催化反应中必需的辅因子。在还原半反应中,FAD被NADPH还原,在氧化半反应中,还原的FAD与一个O2分子反应形成C4a-(hydro)peroxyflavin。DMS的结合使NADP+的构象发生变化,NADP+与Tmm7211的Asp314形成新的氢键,导致底物进入通道关闭,并将C4a-(hydro)peroxyflavin的活性位点暴露给DMS,从而完成DMS的氧化半反应。序列分析表明所提出的Tmm7211氧化DMS产生DMSO的催化机制在MRC和SAR11类群细菌中是普遍存在的。本部分研究为海洋细菌如何将DMS转化为DMSO提供了重要见解,有助于更好地理解全球硫循环过程。5.南极细菌菌株SM1701T的多相分类研究菌株SM1701T分离自南极长城站附近海域的大型海藻样品表面,本论文基于其形态特征、生理生化特征、细胞化学特征和分子遗传学特征确定了该菌株的分类学地位。菌株SM1701T为细胞呈短杆状,无鞭毛,好氧的革兰氏阴性细菌。该菌株生长的温度范围为4-30℃,最适生长温度为20℃;pH耐受范围为pH 6.0-8.0,最适生长pH为7.0;NaCl耐受浓度为0.5-5%(w/v),最适生长NaCl浓度为2%(w/v)。菌株SM1701T能够水解吐温40、吐温60、吐温80、明胶和酪氨酸。菌株 SM1701T 的主要极性脂包括磷脂酰乙醇胺(Phosphatidylethanolamine,PE)和两种尚未鉴定结构的糖脂(Unidentifed glycolipid,GL)。该菌株主要脂肪酸成分为 iso-C15:0、iso-C15:1G、iso-C16:1G、C16:0和iso-C17:0 3-OH,主要呼吸醌为甲基萘醌7(Menaquinone 7,MK-7)。其基因组DNA G+C含量为34.1 mol%。16S rRNA基因序列分析表明菌株SM1701T与Crocinitomicaceae科Crocinitomix属已知种的序列相似性最高(92.5-93.8%)。基于16S rRNA基因序列构建的系统进化树显示,菌株SM1701T在Crocinitomicaceae科内形成邻近Crocinitomix属的独立进化分支。上述研究结果表明菌株SM1701T隶属于Crocinitomicaceae科内的一个新属并代表该新属内的一个新种,将该新属命名为Putridiphycobacter gen.nov.,新种命名为Putridiphycobacter roseus sp.nov.,菌株 SM1701T(=KCTC 62302T=NBRC 113201T=CGMCC 1.16510T)是该新种的模式菌株。综上所述,本论文对南极海域中DMSP利用菌株的多样性、新型ATP依赖的DMSP裂解酶DddX裂解DMSP的代谢产物和分子机制、DMS氧化产生DMSO的分子机制和新型细菌的分类鉴定进行了详细的研究,研究结果为更全面认知DMSP的代谢过程和全球硫循环过程,并为开发利用海洋微生物资源奠定了基础。更多还原

【Abstract】 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-π 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.更多还原