The Diversity Of Methylated Amines-metabolizing Bacteria In The Ocean And The Metabolic Mechanisms Of Methylated Amines

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.