【作者】 王宁；

【导师】 张玉忠； 陈秀兰；

【作者基本信息】 山东大学 ， 微生物学， 2021， 博士

【摘要】 甲基胺类物质,包括单甲胺(monomethylamine,MMA)、二甲胺(dimethylamine,DMA)、三甲胺(trimethylamine,TMA)和氧化三甲胺(trimethylamine N-oxide,TMAO),是全球重要的有机氮化合物,也是全球碳循环和氮循环的重要参与者。多种微生物可以利用甲基胺类物质作为碳源、氮源或能源。挥发性的甲基胺类物质(MMA、DMA和TMA)是海洋痕量气体的重要组成部分,可以参与海洋气溶胶的形成,从而在气候调节中发挥重要的作用。因此,研究甲基胺类物质的代谢具有重要的生态学意义。从表层海水到深海沉积物中,甲基胺类物质的浓度可以达到数百纳摩尔甚至微摩尔量级。在表层海水中,能够利用甲基胺类物质的细菌主要是α-变形菌纲(Alphaproteobacteria)的细菌,其中的海洋玫瑰杆菌类群(Marine clade,MRC)和SAR11类群是降解甲基胺类物质的最主要微生物类群。然而,在深海和沉积物中代谢甲基胺类物质的细菌类群尚未被报道。目前,已报道的细菌代谢甲基胺类物质的途径主要有三条:甲烷生成途径、厌氧TMA脱氢途径和好氧TMA氧化途径。其中,好氧TMA氧化途径是表层海水中甲基胺类物质代谢的主要途径,该途径中TMA首先被氧化为TMAO,TMAO被进一步分解为DMA、MMA和铵。海洋细菌氧化TMA生成TMAO的分子机制在结构解析的基础上得到了阐明,TMAO代谢为DMA的分子机制也已通过同源模建和生化分析等方法被提出,然而,到目前为止,DMA和MMA代谢的分子机制尚不清楚。本论文中,我们首先采集了马里亚纳海沟区域5个不同深度的海水样品,研究了其中微生物群落的多样性以及代谢甲基胺类物质细菌的多样性;随后以甲基胺类物质代谢细菌为研究对象,对好氧TMA氧化途径中MMA和DMA代谢关键酶的性质、结构及催化的分子机制进行了系统的研究。1.马里亚纳海沟不同深度海水中代谢甲基胺类物质细菌的多样性分析为分析马里亚纳海沟不同深度海水中微生物群落组成和多样性,我们对从马里亚纳海沟区域采集的不同深度的海水(水深:5 m、800 m、2500 m、5000 m和10905 m)滤膜样品进行了宏基因组测序。基于宏基因组数据的分析结果,我们在细菌中鉴定出148个门、89个纲和2431个属,还有很多细菌未被鉴定到相应的分类单元。其中,变形菌门(Proteobacteria)占绝对优势,主要包括α-变形菌纲和γ-变形菌纲(Gammaproteobacteria),是所有海水样品中的优势类群。在不同深度的海水中,微生物的群落组成存在较大差异,相比于表层海水,800 m及以下深度海水中的微生物多样性更高。为了分析马里亚纳海沟不同深度海水中代谢甲基胺类物质细菌的多样性,我们通过以MMA、DMA、TMA或TMAO为唯一氮源的固体平板和液体培养基的筛选,从马里亚纳海沟海水样品中分离出了 366株能够利用甲基胺类物质的细菌,这些细菌分属于2个门、3个纲、20个属和41个种。不同深度海水中利用甲基胺类物质的细菌种类存在明显差异。在能够利用甲基胺类物质的可培养细菌中,变形菌门在每个深度都占主导地位,这和宏基因组的测序结果相一致;此外,我们在800 m及以下深度海水中分离出很多放线菌门(Actinobacteria)的细菌,说明放线菌门在深海甲基胺类物质代谢过程中可能发挥重要的作用。我们进一步对γ-变形菌纲的细菌Marinobacter nauticus D-M4822利用四种甲基胺类物质的能力做了分析,发现它可以利用四种甲基胺类物质(MMA、DMA、TMA或TMAO)为唯一氮源生长。基因组分析表明,该菌株可能利用好氧TMA氧化途径进行甲基胺类物质的代谢,而且该菌株可能含有新的TMAO转运蛋白。本研究揭示了马里亚纳海沟不同深度海水中代谢甲基胺类物质细菌的多样性,为进一步研究海洋中甲基胺类物质的代谢过程奠定了基础。2.海洋细菌代谢MMA的分子机制研究MMA是一种重要的海洋痕量气体,在海洋环境中广泛存在。γ-谷氨酰甲胺合成酶(GmaS)催化 MMA 到 γ-谷氨酰甲胺(γ-glutamylmethylamide,GMA)的转化,这是许多海洋细菌进行MMA代谢的第一步。据估计海洋表层海水中约23%的细菌含有gmaS基因,它是检测MMA利用菌的有效生物标记基因。然而,到目前为止,仍缺乏其晶体结构,GmaS催化MMA生成GMA的分子机制也尚不清楚。为了研究GmaS的催化机制,我们以海洋细菌Rhodovulum sp.12E13来源的GmaS(RhGmaS)为研究对象,首先对其进行了异源表达与纯化,研究了RhGmaS的基本酶学性质。RhGmaS催化反应的最适温度为60℃,最适pH为8.0。RhGmaS 对 L-谷氨酸、ATP 和 MMA 的Km值分别为 67.18±4.98 mM、0.42±0.05 mM和26.94±1.73 μM。底物特异性分析表明,除了MMA之外,RhGmaS还可以将乙胺、羟胺、丙胺、氯化铵、DMA或TMA作为底物,表明其具有比较广泛的底物特异性。RhGmaS对MMA的Km值最低,表明MMA可能是RhGmaS的天然底物。我们进一步解析了 RhGmaS的晶体结构,在一个不对称单元里存在三个RhGmaS分子组成的三聚体,而凝胶过滤分析表明RhGmaS在溶液中是十二聚体,与电镜分析结果一致。因此,RhGmaS在溶液中以十二聚体形式存在,在不对称单元中的三聚体表明RhGmaS具有较高的对称性。为了阐明MMA代谢的分子机制,我们又解析了五个RhGmaS与底物或底物类似物的复合物在不同状态下的晶体结构。基于结构和关键氨基酸残基突变分析,我们提出了RhGmaS催化MMA生成GMA的分子机制。在RhGmaS催化过程中,氨基酸残基Arg312参与极化ATP的γ-磷酸,并稳定γ-谷氨酰磷酸中间体;Asp177参与MMA的脱质子化,协助MMA攻击γ-谷氨酰磷酸生成四面体中间体;而Glu186作为催化碱,吸引四面体中间体的质子,最终生成γ-谷氨酰甲胺。序列分析表明,我们提出的RhGmaS转化MMA到GMA的分子机制在含有GmaS的细菌中可能普遍存在。我们的结果对深入理解MMA的代谢过程以及全球碳、氮循环具有重要的意义。3.海洋细菌代谢DMA关键酶DmmABC的酶学特性初步研究DMA是重要的有机氮化合物,作为海洋微生物的氮源,广泛分布于海洋环境中。DMA单加氧酶(Dmm)在NADPH和四氢叶酸的参与下,催化DMA生成 MMA 和 5,10-亚甲基四氢叶酸(5,10-methylene tetrahydrofolate,CH2=THF),参与海洋中DMA的代谢过程。它由dmmD、dmmA、dmmB和dmmC四个基因编码,其中,DmmA、DmmB和DmmC三个亚基可以在体外单独形成一个有催化活性的复合体DmmABC,催化DMA生成MMA和甲醛,而亚基DmmD参与甲醛的释放过程。虽然DmmABC的功能已被报道,但到目前为止,关于其复合物的酶学特性尚未有报道,DmmABC的结构及其催化DMA生成MMA的分子机制也尚不清楚。本文中,我们选择了来自MRC类群典型菌株Ruegeria pomeroyi DSS-3的DmmABC为研究对象,研究了 DmmABC的酶学特性及其各亚基的功能。首先对DmmABC进行了异源表达和纯化,已成功表达组装了均一、稳定的DmmABC复合物蛋白。凝胶过滤层析结果显示,DmmABC是一个异源寡聚体,分子量大小介于158 kDa和440 kDa之间。动态光散射(Dynamic light scattering,DLS)结果显示,DmmABC在溶液中的分子量约为202.7 kDa。鉴于其三个亚基的总单体分子量约为100 kDa,我们推测,DmmABC复合物的构成形式是DmmA2B2C2。随后我们研究了 DmmABC的酶学特性,DmmABC的最适酶活温度为30℃,最适酶活pH为7.0。Fe3+对DmmABC酶活的促进作用最明显,而Zn2+和Cu2+则抑制了约90%的DmmABC酶活。进一步我们通过表达DmmABC的亚基缺失突变体研究了 DmmA、DmmB和DmmC三个亚基的功能,揭示了它们对全酶组装及全酶酶活的重要性。我们的结果为后续解析DmmABC复合物的结构并阐明其催化机制奠定了基础。4.南极沉积物菌株SM1355T的多相分类研究南极地区环境条件极端,存在大量尚未被开发利用的微生物资源。菌株SM1355T分离自中国南极长城站附近的潮间带沉积物样品,细胞呈棒状,为革兰氏阴性好氧菌,没有鞭毛,不能滑行。菌株SM1355T’生长温度范围为4-35℃,生长的NaCl浓度范围为0.5-7%,能够水解七叶苷、吐温20、吐温40和吐温60,但不能水解DNA、淀粉、酪氨酸和吐温80,也不能还原硝酸盐为亚硝酸盐。菌株SM1355T主要的细胞脂肪酸成分为anteiso-C15:0、iso-C15:0和iso-C15:1G,主要极性脂为磷脂酰乙醇胺(phosphatidylethanolamine,PE)和一种未鉴定结构的脂类,基因组DNA的G+C含量为36.2 mol%。16S rRNA基因序列分析表明该菌株与黄杆菌科(Flavobacteriaceae)内Flaviramulus ichthyoenteri Th78T 和Algibacte’r agarilyticus KYW563T具有最高序列相似性,分别是96.3%和96.0%。根据16S rRNA基因序列构建的系统进化树显示菌株SM1355T在黄杆菌科内部形成一个独立的进化分支。以上分类数据表明,菌株SM1355T’隶属于黄杆菌科内的一个新属,并代表该新属内的一个新种,命名为Changchengzhania lutea gen.nov.,sp.nov.。新菌株的鉴定丰富了人们对南极海洋微生物多样性的了解,为进一步开发和利用南极细菌资源奠定了基础。综上所述,本论文对海洋中代谢甲基胺类物质的细菌类群及其代谢分子机制进行了研究,研究结果对更好地理解海洋细菌代谢甲基胺类物质的生化过程及其参与的全球碳、氮循环具有重要意义。更多还原

【Abstract】 Methylated amines,including monomethylamine(MMA),dimethylamine(DMA),trimethylamine(TMA)and trimethylamine N-oxide(TMAO),are important organonitrogen molecules,and play important roles in the global carbon and nitrogen cycling.Many microorganisms use methylated amines as carbon,nitrogen and/or energy sources.As important oceanic trace gases,volatile methylated amines(MMA,DMA and TMA)may affect the global climate through participation of the formation of marine aerosols.Thus,researches on the metabolism of methylated amines have important ecological significance.The concentrations of methylated amines in marine environments can reach up to hundreds of nanomolar and even micromolar levels.Methylated amines-utilizing bacteria in the surface seawater are mainly Alphaproteobacteria,among which the marine Roseobacter clade(MRC)and SAR11 clade are of vital importance in methylated amines metabolism.However,it is unclear that which kinds of bacteria are capable to metabolize methylated amines in the deep sea and marine sediments.Till now,three different pathways for microbial metabolism of methylated amines have been reported:a methanogenesis pathway,an anaerobic TMA dehydrogenase pathway and an aerobic TMA oxidation pathway.The aerobic TMA oxidation pathway is the major pathway for methylated amines metabolism in the surface seawater.In this pathway,TMA is oxidized to TMAO,which is further catabolized to DMA,MMA,and ammonium.Recently,the molecular mechanism of TMA oxidization was elucidated based on structural analysis,and the catalytic mechanism of the conversion of TMAO to DMA was also proposed on the basis of homology modelling and biochemical analyses.However,the molecular mechanisms of MMA and DMA metabolism have not been reported.In this dissertation,we first investigated the diversities of microbial communities and methylated amines-degrading bacteria in the seawaters from five different water depth of Mariana Trench.Then,we focused on the key enzymes involved in MMA and DMA metabolism,and studied their catalytic mechanisms.1.Diversity of methylated amines-degrading bacteria isolated from the seawaters from five depths of Mariana Trench.We used metagenomics to investigate the composition and diversity of microbial communities in the seawater samples from five different depths in Mariana Trench.In total,148 bacterial phyla were identified,comprising 89 classes and 2431 genera.Among them,Proteobacteria,especially Alphaproteobacteria and Gammaproteobacteria,were dominant in all seawater samples.The composition of bacterial communities exhibited significant differences in different water depth,and the microbial diversities were higher in deep seawater depths(≥800 m)than that in the surface seawater.In order to analyze the diversity of bacteria capable to degrade methylated amines in Mariana Trench,we screened methylated amines-utilizing bacteria using both agar plates and liquid mediums with MMA,DMA,TMA or TMAO as the sole nitrogen source.A total 366 bacterial strains were isolated from seawater samples,which belonged to 2 phyla,comprising 3 classes,20 genera and 41 species.The diversities of methylated amines-utilizing bacteria isolated from five seawater depths from Mariana Trench were significantly different.Among them,Proteobacteria was dominant in all seawater samples,which is consistent with the result of metagenomic analysis.In addition,we also isolated many bacteria belonged to the phylum Actinobacteria from deep seawaters(≥800 m),indicating that the Actinobacteria may play important roles in the metabolism of methylated amines in deep sea.Furthermore,we selected one gammaproteobacterium,Marinobacter nauticus D-M4822,which could utilize all of the four methylated amines(MMA,DMA,TMA or TMAO)as the sole nitrogen source for growth,for further study.Genomic analysis indicated that strain D-M4822 adopts the aerobic oxidation pathway to metabolize methylated amines and may contain a new TMAO transporter.These results reveal the diversity of methylated amines-utilizing bacteria in deep sea,which lays a foundation for further study on the metabolism of methylated amines,and offers a better understanding of the global nitrogen and carbon cycles.2.The molecular mechanism of bacterial conversation of oceanic 1VIMA to GMAMMA is an important oceanic trace gas and widespread in the oceans.The y-glutamylmethylamide synthetase(GmaS)catalyzes the conversion of MMA to y-glutamylmethylamide(GMA),which is the first step in MMA metabolism in many marine bacteria.The gmaS gene occurs in-23%of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria.However,the molecular mechanism of GmaS catalyzing the conversion of MMA to GMA is still unclear because of the lack of structural information.Here,we studied the molecular mechanism of GmaS from Rhodovulum sp.12E13(RhGmaS).We expressed RhGmaS in Escherichia coli BL21(DE3)cells and purified it The enzymatic properties of RhGmaS were characterized.The optimal temperature for RhGmaS enzymatic activity was 60℃,and the optimal pH was 8.0.The Km values of RhGmaS to glutamate,ATP and MMA were 67.18 ± 4.98 mM,0.42±0.05 mM and 26.94± 1.73μM,respectively.Substrate specificity analysis suggested that,in addition to MMA,RhGmaS can also accept ethylamine,hydroxylamine,propylamine,ammonium chloride,DMA or TMA as a substrate.Thus,RhGmaS has a relatively broad substrate specificity.The Km of RhGmaS for MMA was the lowest among the tested ammonia analogs,indicating that MMA is likely the natural substrate of RhGmaS.We further determined the crystal structures of apo-RhGmaS,with three monomers arranged as a trimer in an asymmetric unit.Both gel filtration analysis and electron microscopic analysis demonstrated that RhGmaS is a dodecamer in solution.All the solved RhGmaS structures contain three molecules arranged as a trimer in an asymmetric unit,suggesting that the dodecameric RhGmaS has a relatively high symmetry.To gain insight into the catalytic mechanism of RhGmaS,RhGmaS with different ligands in five states were also determined.Based on structural and mutational analyses,we proposed the molecular mechanism of RhGmaS catalyzing the conversion of MMA to GMA.During the catalysis of RhGmaS,the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate;Asp 177 is responsible for the deprotonation of MMA,assisting the attack of MMA onγ-glutamyl phosphate to produce a tetrahedral intermediate;and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate GMA.Sequence alignment analysis suggested that the proposed catalytic mechanism of RhGmaS has universal significance among bacteria containing GmaS.These results provide novel insights into MMA metabolism and broaden our understanding of the biogeochemical cycles of carbon and nitrogen.3.Preliminary characterization of DmmABC,a key enzyme involved in DMA metabolism in marine bacteriaDMA is ubiquitous in the ocean,and represents an important nitrogen source for marine microorganisms.DMA monooxygenase(Dmm)catalyzes the conversion of DMA to MMA and 5,10-methylene tetrahydrofolate,with the participation of NADPH and tetrahydrofolate,which involves in DMA metabolism.Dmm is encoded by four genes dmmD,dmmA,dmmB and dmmC.Three subunits(DmmA,DmmB and DmmC)can form DmmABC complex,which presents in vitro catalytic activity,catalyzing the conversion of DMA to MMA and formaldehyde.DmmD plays a key role in releasing formaldehyde.Till now,the enzymatic properties have not been reported,and the structure and molecular mechanism of DmmABC catalyzing the conversion of DMA to MMA is still unclear.In this study,we characterized DmmABC originated from DSS-3,a type strain of MRC.We first expressed and purified DmmABC,then we obtained uniform and stable proteins.Gel filtration analysis demonstrated that DmmABC is a heterooligomer and its molecular mass is between 158 kDa and 440 kDa.Dynamic light scattering(DLS)analysis indicated that the molecular mass of DmmABC is～202.7 kDa in solution.Because the total molecular mass of the three subunits is～100 kDa,it is reasonable to surmise that the form of the DmmABC complex is DmmA2B2C2.Moreover,the enzymatic properties of DmmABC were characterized.The optimal temperature for DmmABC enzymatic activity was 30℃,and the optimal pH was 7.0.Fe3+ significantly activated the enzymatic activity of DmmABC,whereas Zn2+and Cu2+inhibited its activity by～90%.Further,we studied the function of each subunit of DmmABC,and revealed that all the subunits are essential for the assembly and the enzymatic activity of DmmABC complex.These results lay a foundation for futher research on the structure of DmmABC complex and the catalytic mechanism of DMA metabolism.4.Study on the taxonomic identification of strain SM1355T isolated from Antarctic intertidal sedimentThe environmental conditions in the Antarctic are extreme,and a large number of Antarctic microbial resources have not been exploited.Strain SM1355T isolated from the Antarctic intertidal sediment sample is a Gram-negative,aerobic,non-flagellated,non-gliding,rod-shaped bacterial strain.The strain could grow at 4-35℃ and with 0.5-7%(w/v)NaCl.It was positive for hydrolysis of aesculin and Tweens 20,40 and 60,but negative for hydrolysis of DNA,starch,L-tyrosine or Tween 80.And it did not reduce nitrate to nitrite.The predominant fatty acids of this strain were anteiso-C15:0,iso-C15:0 and iso-C15:1 G and the major polar lipids were phosphatidylethanolamine(PE)and one unidentified lipid.The genomic DNA G+C content of strain SM1355T was 36.2 mol%.Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1355T formed a distinct phylogenetic lineage within the family Flavobacteriaceae,sharing the highest 16S rRNA gene sequence similarity with Flaviramulus ichthyoenteri Th78T(96.3%)and Algibacter agarilyticus KYW563T(96.0%).Based on the results of the polyphasic characterization for strain SM1355T,it is identified as the representative of a novel species in a new genus of the family Flavobacteriaceae,for which the name Changchengzhania lutea gen.nov.,sp.nov.is proposed.The taxonomic identification of new strains increases our understanding of the diversity of marine microorganisms in Antarctic.In conclusion,our study reveals the methylated amines-metabolizing bacteria in the ocean and the metabolic mechanisms of methylated amines,which provide novel insights into marine methylated amines metabolism and broaden our understanding of the biogeochemical cycles of carbon and nitrogen.更多还原