Microbial Diversity And Environmental Adaptation Mechanisms In Typical Marine Environments

Planktonic bacteria are the most abundant microbes in marine ecosystems.They have extremely high genetic and metabolic diversity and thus play a significant role in marine biogeochemical cycles.Here we attempted to shed new light on the diversity, environmental adaptation mechanisms,and co-evolution with environments of some typical bacterioplankton groups in China seas and global oceans.These bacterial groups play a special role in marine carbon cycling and light utilization but received little attention,including:Cytophaga-Flavobacteria(CF),which are proficient in degrading high molecular weight particulate organic carbon;planktonic Archaea,the metabolic potential and ecological functions of which are largely unknown;planktonic Proteobacteria with the ability of fixing CO₂;and mixtrophic aerobic anoxygenic photosynthetic bacteria(AAPB) and pigmented heterotrophic bacteria(PHB),which can facultatively utilize or absorb light.Firstly,we chose the Yangtze River estuary(YRE) in the East China Sea(ECS) as a typical eutrophic water and systematically investigated the diversity pattern of CF, planktonic archaea,and CO₂ fixation proteobacteria there.CF cluster is one of the most abundant bacterial groups in the ocean.A newly designed CF specific 16S rRNA gene primer pair was used to construct two clone libraries from YRE and ECS bacterioplankton samples.An abundant CF diversity and a distinct diversity distribution pattern were revealed.Seventy partial 16S rRNA gene sequences of CF were clustered into 26 subgroups,including 7 subgroups that distributed only in the ECS,17 subgroups that distributed only in the YRE,and 2 subgroups that were shared by both stations.In comparison,the CF species diversity was high in the ECS,but the CF subgroup diversity was high in the YRE.Cosmopolitan subgroups dominated both stations. Subgroups with the soil,coast,and freshwater origins were detected only in the YRE,in accordance with the geographical location and environmental features there.The adaptation of CF species to the two completely different stations with respect to hydrology and the development and succession of local CF populations resulted in these significant differences between the YRE and ECS CF communities. By the construction of two archaeal 16S rRNA gene clone libraries from YRE bacterioplankton samples,we also revealed the presence of planktonic archaea in typical Chinese marine waters,which has been found to be cosmopolitan in marine ecosystems. Sequencing of the clones in 21 unique RFLP patterns showed the two archaeal groups: Marine Groupâ… (MGâ… ) that was affiliated with Crenarchaeota and Marine Groupâ…¡(MGâ…¡) that was affiliated with Euryarchaeota.MGâ… dominated both libraries.A large part of sequences were most related to uncultured archaea,including 2 sequences that were in 98%similarity with a nitrification marine archeaon Nitrosopumilus maritimus, suggesting that archaea may play an important role in estuarine ecosystems.Proteobacteria with the potential of fixing CO₂ is another understudied group in bacterioplanton.Here we for the first time successfully designed two primer pairs targeting the Formâ… and Formâ…¡rbcL genes(encoding RubisCO large subunit), respectively,and applied them to the inshore and offshore samples from the ECS.Our results showed that the Formâ… rbcL diversity was high at the inshore station with high salinity and low productivity,while the Formâ…¡rbcL diversity was high at the offshore station with low salinity and high productivity.A part of sequences showed a low similarity(60～78%) with their nearest neighbors in the GenBank database.The results indicate the presence of carbon-fixing proteobacteria in the ECS and suggest that we should draw more attention to their potential contribution in marine carbon cycling.Secondly,we focused on a typical functional bacterial group in marine bacterioplankton - AAPB and systematically studied their diversity pattern in global oceans and co-evolution with environments.The diversity data of an AAPB marker gene,pufM,was collected from the surface seawater of the Pacific,Atlantic,and Indian oceans,and Chinese marginal seas as well.The results showed that AAPB populations were highly diverse in global oceans and,more importantly,their diversity decreased with the increased Chl.a concentrations.This finding indicated that AAPB diversity and abundance followed a reverse trend from oligotrophic to eutrophic oceans.Depth profile of AAPB diversity was also investigated in three stations in the surface and upper twilight zones of the central Pacific,Atlantic,and Indian oceans by constructing six pufM gene clone libraries.The high AAPB diversity in oligotrophic oceans was further confirmed.Furthermore,we for the first time obtained abundant pufM sequences(136 sequences within 37 OTU) from deep twilight zones(200 m). Phylogenetic analysis showed that deep AAPB populations were diverse too,covering all the subgroups that were found in surface stations.They had a lower diversity,GC and GC3 contents but a higher dominance than surface stations.These results support the hypothesis that AAPB can utilize dim light to acquire energy and thus distribute into the layers blow euphotic zones.To further probe the co-evolutionary relationship between anoxygenic photosynthetic bacteria and environment,we retrieved the pufM sequences and related source information of 89 anoxygenic photosynthetic bacterial cultures from public database.Phylogenetic analysis revealed that horizontal gene transfer(HGT) occurred in 11 of total 21 pufM phylogenetic subgroups.HGT occurred among species within not only the same class but also different phyla or subphyla.Source environmental features of anoxygenic photosynthetic bacteria were closely related to their phylogeny.Species from oxic and anoxic environments clustered into separate and distinct clades in the phyiogenetic tree.HGT between ancient anoxygenic photosynthetic bacteria and long-term adaptive evolution into different niches may result in such coevolutionary events.Then,in addition to the analysis of bacterial diversity by culture-independent molecular methods,we also employed culture-dependent methods to study the diversity, abundance,and light absorption features of pigmented heterotrophic bacteria(PHB), which is another interesting bacteria group with respect to light utilization.PHB cultures were isolated from the coast and shelf of China seas and adjacent Pacific and were identified by 16S rRNA gene analysis.The results showed that PHB abundance and their percentage in total cultivable bacterial amount decreased from coast to open ocean with the highest values(9.9×10³ cells mL^-1 and 39.6%,respectively) occurring at the YRE station.In the depth profiles,PHB were only distributed in the euphotic zones and the highest values of both abundance and percentage proportion occurred at the surface stations.Total 247 PHB isolates were screened with 16S rRNA gene PCR-RFLP and then sequenced.The PHB strains we obtained covered 6 major bacterial groups (α-Proteobacteria,γ-Proteobacteria,Actinobacteria,Bacilli,Flavobacteria,and Sphingobacteria) and 25 genera.Dominant group differed among different stations,and different genus showed different colors,including golden yellow,yellow,red,pink,and orange etc.Cellular pigments absorbed the light between 450～550 nm.These results indicated that PHB were widely distributed in marine environments with high diversity. They may play a unique and important role in marine ecosystems due to the absorption and utilization of light.Finally,we established a set of new criterion for clustering pufMgene sequence by distance methods and a new method for assessing 16S rRNA gene copy number in bacterial genomes by combining PFGE and PCR amplification.The cutoff for discriminating species,genus,and(sub-)phylum based on pufM gene sequences were 0.06,0.15,and 0.48,respectively.