Isolation And Cultivation Of Marine Oligotrophic SAR92 Clade Bacteria And Their Catabolic Pathways To Degrade Dimethylsulfoniopropionate

The SAR92 clade,a member of the Oligotrophic Marine Gammaproteobacteria Group(OMG),is widely distributed in the global marine environments.The SAR92 clade is a typical marine oligotrophic group showing no growth under high nutrient conditions and hence represents a hard-to-culture group.The SAR92 clade is found to be one of the most abundant and metabolically active bacterial groups in marine waters containing planktonic algal blooms,implying that this group has the ability to specifically degrade and utilize organic matter produced and released by algae.Studies on the SAR92 clade cultivable strain HTCC2207,have confirmed that it can degrade polysaccharides prominently produced by phytoplankton.However,there is no direct experimental evidence so far that the SAR92 clade is capable of degrading other algae-derived organic matter.Dimethylsulfoniopropionate(DMSP)is one of the most abundant organic sulfur compounds in marine environments and a major participant in the global sulfur cycle,which is produced mainly by marine phytoplankton.DMSP is an important carbon source and a major sulfur source of marine microorganisms,and its metabolism is mainly completed by marine bacteria.The marine Roseobacter and SAR11 clades of Alphaproteobacteria are the major DMSP-catabolizing bacterial groups in marine environments.Moreover,many copiotrophic groups of the Gammaproteobacteria class have been reported to catabolize DMSP.However,it still remains unclear whether and how oligotrophic groups of the Gammaproteobacteria class participate in the metabolism of DMSP in marine environments,which is in large part due to the lack of culturable strains and the difficulty to cultivate them.In this dissertation,a high-throughput dilution-to-extinction cultivation method was used to isolate bacteria from offshore surface seawater samples,through which the diversity of the isolated strains was analyzed.The phylogenetic,physiological and metabolic characteristics of the isolated SAR92 clade strains were investigated.The ability and catabolic pathways of the isolated SAR92 clade strains to catabolize DMSP,were then further systematically studied with the SAR92 clade’s contribution to global oceanic DMSP metabolism being also analyzed,through which the important role of SAR92 clade bacteria in the global sulfur cycle was confirmed.In addition,three marine bacterial strains were polyphasically taxonomically identified and their taxonomic status was established.1.Applying the high-throughput dilution-to-extinction method to isolate bacterial strains from offshore surface seawater samples and diversity analysis of the isolated strains.The high-throughput dilution-to-extinction cultivation method was used to isolate and culture bacteria from offshore surface seawater samples collected from three different sites of Yellow sea.China.A total of 205 bacterial isolates were obtained and most isolates belonged to different genera(clades)of the classes Alphaproteobacteria and Gammaproteobacteria.Among them,Sulfitobacter,Planktomarina and SAR116 clade of Alphaproteobacteria and Glaciecola,Colwellia,SAR92 clade,Psychromonas of Gammaproteobacteria were the dominant groups.Moreover,metagenomic analysis for one of the surface seawater samples showed that these dominant groups were also abundant in their in-situ environments.Meanwhile,culturable strains of three hard-to-culture oligotrophic clades including the SAR92,OM43 and SARI 16 clades were obtained,providing a foundation for further study on the physiological and metabolic characteristics and environmental adaptation mechanisms of these clades.2.Phylogeny,physiological and metabolic characteristics of the marine oligotrophic SAR92 clade bacteria.Three of the isolates belonged to the marine oligotrophic SAR92 clade obtained using the high-throughput dilution-to-extinction cultivation method,named strains H921,H231 and H455.were selected to be the representatives for further analysis of phylogeny,physiological and metabolic characteristics of the SAR92 clade.Phylogenetic analysis based on 16S rRNA genes and genome sequences showed that the SAR92 clade can be divided into three distinct subclusters,and strains H921,H231 and H455 belong to two of the three subclusters.respectively.The growth temperature range of the three isolated strains was 4-30℃.Cellular morphology observation showed that cells of all three isolated SAR92 clade strains possessed flagella.Incubation experiments showed typical oligotrophic characteristics for the growth of the there strains,including high concentration of glucose significantly inhibiting their growth and the inability to form visible colonies on solid media.Substrate utilization experiments showed that the three SAR92 clade strains can utilize certain kinds of algal polysaccharides and long-chain unsaturated fatty acids.These results enrich the understanding of the intra-clade diversity,physiological and metabolic characteristics of the oligotrophic SAR92 clade.3.The DMSP catabolic pathways of the marine oligotrophic SAR92 clade bacteria.Through the examination of the DMSP utilization ability and DMSP metabolic products,all four SAR92 clade strains including strains H231.H921 and H455 isolated in this dissertation and the SAR92 clade representative strain HTCC2207 were found to be able to utilize DMSP as a carbon source to grow and to produce dimethyl sulfide(DMS),thus experimentally confirming the ability of this clade to catabolize DMSP at strain level for the first time.Genome analysis showed that there was a gene cluster potentially encoding the DMSP lyase DddD-mediated DMSP cleavage pathway presenting in all the four SAR92 clade strains’ genomes,and a gene cluster potentially encoding the DmdA-mediated DMSP demethylation pathway in genomes of three strains.RT-qPCR results showed that the predicted DddD and DmdA genes in the four SAR92 clade strains could be upregulated by DMSP substrate.In vitro enzyme activity testing showed that the candidate DddD proteins in the four SAR92 clade strains all had the ability to cleave DMSP and produce DMS.The results of the dmdA gene complementation and functional testing experiments showed that the candidate dmdA gene products in strains H921,H231 and HTCC2207 all had DMSP demethylase activity.Based on the above experimental results,the DMSP catabolic pathways of these four SAR92 clade strains were proposed:strains H921,H231 and HTCC2207 have both a DddD-mediated DMSP cleavage pathway and a demethylation pathway,while strain H455 only has a DddD-mediated DMSP cleavage pathway.These results extend the understanding of the oligotrophic DMSP-catabolizing bacterial groups and their catabolic pathways.4.The role of SAR92 clade bacteria in global oceanic DMSP catabolism.Analysis of 120 SAR92 clade bacteria’s metagenome-assembled genomes revealed that 80%of these genomes carried homologous genes of the DMSP catabolism key enzymes DddD and/or DmdA,indicating that DMSP catabolism is prevalent in the SAR92 clade.Analysis of global oceanic metagenomes showed that SAR92 clade bacteria are widely distributed in marine environments,but their relative abundance is very uneven among different sites with the abundance in polar and surface seawater significantly higher than those in non-polar and deeper layer seawater.Analysis of global oceanic metagenomes and metatranscriptomes showed that,consistent with the distribution pattern of the SAR92 clade bacteria,homologous genes of the DMSP catabolism key enzymes DddD and DmdA of the SAR92 clade are widely distributed in global oceans’ surface seawater and have higher abundance in polar oceans’.These results indicated that the SAR92 clade is an important DMSP-catabolizing group,whose members play important roles in DMSP catabolism and sulfur cycle in global oceans.5.Polyphasic taxonomic analysis of three bacterial strains isolated from the Antarctic and Indian Ocean.Two strains,SM1352T and A20T,isolated from the intertidal sediments of the Antarctic and one strain,SM1810T,from the deep-sea sediments of the Southwest Indian Ocean were taxonomically identified using a polyphasic approach.Based on the analysis of their phenotypic,cytochemical and molecular genetic characteristics,the three marine bacterial strains were considered to represent three novel species in different genera in the phylum Bacteroidota,for which the names Aureibaculum luteum sp.nov.,Aureibaculum flavum sp.nov.and Pedobacter indicus sp.nov.are proposed.In summary,the results of this dissertation provide better understanding of the DMSP-catabolizing oligotrophic bacterial groups and their ecological function and lay a foundation for marine microbial resource utilization.