| Hydrothermal vent in deap sea is an unique environment on the earth which is characterized with high pressure, oligo nutrients, darkness, and wide fluctuation of temperature, hydrogen content, oxgen content, salinity and pH. Hydrogen bacteria are widely distributed in deep-sea hydrothermal vents utilizing dissolved H2 in hydrothermal flow as energy source .With this energy, CO2 is converted into simple organic materials. They are the primary producers in the deep-sea hydrothermal ecosystem, and play an important role in the carbon and energy cycle. Hydrogenase is a class of redox protein, divided into NiFe-hydrogenases and Fe-hydrogenases and the iron-sulfur-free hydrogenases. The type I of NiFe-hydrogenases usually anchores on the plasmic membrane with two catalytic subunits hynS and hynL, and hynL containing active center Ni atoms.In this paper, deep-sea hydrothermal chemoautotrophic Caminibacter profundus strain DSMZ 15016 is taken as the research object. Primers for the membrane-bound type I NiFe Hydrogenase large subunit hynL gene were designed based on alignments of five phylogenetically distinct bacterial hydrogenase. These included (with accession numbers in parentheses): Caminibacter mediatlanticus strain TB-2(AY691430), Caminibacter hydrogeniphilus strain AM1116 (AJ309655), Nautilia lithotrophica strain 525 (AJ404370), Nautilia sp. strain Am-H (AF357197), Lebetimonas acidiphila strain Pd55 (AB167820). The hynL gene sequence was cloned and bioinformatic analysis was conducted.Scanning electron microscopy and EDS was used to study on the strain. The hynL relative expression, methyl amethyst (MV) reduction hydrogenase activity, and growth of bacteria strain in response to different environmental factors, such as incubation time, temperature, hydrogen content, oxygen content, salinity and pH were studied.The results showed that hynL gene was cloned, and 864 bp of hynL sequence segment was obtained. The deduced amino acid sequence of hynL had a highest similarity of 99% with that of Lebetimonas acidiphila, and it belongs to the same branch of Epsilonproteobacteria D groups of the type I NiFe hydrogenase large subunit.Scanning electron microscopy analysis showed that the surface of bacteria has S and Fe deposit, suggesting that the hydrogenease may involve in the transfer of production of hydrogen H+, H2S, and then the formation of Fe2+, FeS precipitation. In growth assay, the doubling time of strain was 6-48 h, 12 h into early exponential growth phase and 48-120 h into stationary phase. The growth reached the maximum amount to 4.73×107 cell/mL and it declined after 120 h. Bacteria can grow in the range of 25-65℃(optimum 55℃), 0.5%-5.0% sea salt (optimum 3.0%), pH2.0-7.1 (optimum pH5.5), O2 content 0%-1% (optimum0.3%) and H2 content 0%-80% (optimum 60%).MV (methyl amethyst) reduction hydrogenase activity increased rapidly with the incubation time and reached the maximum in the exponential growth phase (24 h), followed by a quick decline. At 24 h it reached the maximum at temperature of 55℃, 60% H2 content, 0.3% O2 concentration, salt concentration of 30 gL-1 and pH5.5. HynL gene expression was determined by the relative quantitative method to quantify and 16S rDNA as an internal reference. hynL gene expression increased rapidly with the incubation time and reached the maximum in the exponential growth phase (12 h), followed by a quick decline. Measured in the same range, hynL relative expression showed a same trend as MV reduction hydrogenase activity and growth of the strain.Our results suggested that C. profundus regulates the expression of hydrogenase gene in response to the changes of different environmental factors to complete the catalytic process of energy metabolism and provide energy for growth and propagation of bacteria in hydrothermal environment. This study provides a theoretical basis to reveal the deep-sea hydrothermal geochemical cycle and explore the molecular mechanisms of the origin and evolution of life.
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