Diversity Analyses Of Deep-sea Microorganisms And Study Of Enzyme Genes Within Ethanol Metabolism Of Halophilic Archaeon

Extremophiles live well in several extreme environments, where normal microorganisms cannot survive. Typical extreme habitats as Deep Ocean and Salt Lake give birth to many thermophilic, psychrophilic, piezophilic, halophilic and alkaliphilic microorganisms. Focus on the microbial life living in Deep Ocean and Salt Lake, three different studies were presented in this thesis.Firstly, a sediment sample collected from a deep-sea hydrothermal vent field located at a depth of 2,951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community and diversity based on the 16S rRNA and nifH genes libraries. Within the Archaea, the dominant clones belonged to the groups of MBGE and MGI within the phyla Euryarchaeota and Thaumarchaeota, respectively. About 60% of the bacterial clones belonged to the Proteobacteria. Additional bacterial phyla detected included the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3, and TM6. The detection of nifH genes suggests that biological nitrogen fixation occurred in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis shown that only cluster I and cluster III NifH are present, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.Secondly, a novel obligately piezophilic bacterium was isolated from seawater collected from Puerto Rico Trench at a depth of 6,000 m. The isolated strain YC-1 grew well with an optimal growth hydrostatic pressure of 50 MPa at 8℃, and no growth was observed at atmospheric pressure. The predominant cellular fatty acids are C16:1, C18:1 and C10:0 3-OH. The G+C content was 44.6 mol%. Strain YC-1 is Gram-negative, non-flagellum forming, and facultative anaerobic heterotroph. The 16S rRNA gene sequence of strain YC-1 was closely similar to the uncultured deep-sea bacterial clone Osedax_syml (97.2%), and was closely related to cultivated members of the genus Neptunomonas (92.7-93.2%). Based on taxonomic and phenotypic differences observed, the strain YC-1 appears to represent a new species within the family Oceanospirillaceae, which named Profundimonas piezophila gen. nov., sp. nov.Thirdly, we have studied the role of functional genes within ethanol metabolism of haloalkaliphilic Archaea Natronomonas pharaonis. It indicated that ethanol oxidation by N. pharaonis may be one of the mechanisms to degrade toxic compounds found in its habitat. In N. pharaonis, the adh gene and aldH2 gene encode alcohol dehydrogenase and aldehyde dehydrogenase, respectively, they are key enzymes involved in alcohol metabolism. Reverse transcription-PCR analysis showed that the aldH2 gene was inducible by ethanol, but the adh gene transcription was not affected by ethanol. It indicated that aldehyde dehydrogenase of N. pharaonis possibly contribute to the alcohol resistance, and alcohol dehydrogenase may play other physiological roles in addition to ethanol degradation. The recombinant alcohol dehydrogenase was haloalkaliphilic and thermophilic, and the recombinant aldehyde dehydrogenase was thermophilic, alkaliphilic and salt independent.