Screening And Identification Of Bacteria From Hydrothermal Vents And Their Responsive Mechanism To The Deep-sea Environment

Deep-sea hydrothermal vents are special regions in the bottom of oceans which are characterized by their extreme environmental conditions , including high pressure, high gradient of temperatures and high concentrations in toxic elements (sulfides,heavy metals). These ecosystems are some of the most productive ecosystems on our planet,even though they depend on microbial chemosynthesis instead of solar energy and photosynthesis. With the sharp thermal and chemical gradients,there are abundant diversity of microorganism including thermophile,psychrophile,acidophile, alkaliphile,barophilie and halophiles. It has opened a window into a largely unknown and unexplored biosphere that changed our fundamental views about the extent of habitable microbial environments on earth and the possible origins of life itself. Microorganisms present in extreme environments generally utilize different CO2 fixation pathways and one of them is the reductive tricarboxylic acid (rTCA) cycle. rTCA cycle is a carbon dioxide (CO2) oxidation pathway distinct from the well-known reductive pentose phosphate cycle (Calvin Benson cycle) in plants,algae,and various bacteria. In this pathway,four molecules of CO2 are oxidized to produce one molecule of oxaloacetate in one cycle. The key enzymes of rTCA cycle are ATP-dependent citrate lyase ,pyruvate:ferredoxin oxidoreductase,and 2-oxoglutarate:ferredoxin oxidoreductase.In this study,the molecular identification and phylogenetic analysis was conducted on oceanbic cultivatable microorganisms. A total of 16 bacteria strains were isolated from sediments and sulfide of India Ocean Hydrothermal Vents by using MMJHS oligo-medium. The identification and phylogenetic analysis of these bacteria were carried out based on the comparison of 16S rDNA gene sequences. The results showed that 12 bacteria strains belong toγ-proteobacteria,of which 6 strains are the members of genus Halomonas,4 strains belong to the genus Psychrobacte and 2 belong to genus Alcanivorax; the rest 4 strains are the members of Bacillus,of which 1 is Bacillus licheniformis. Among these 16 strains,all of them are gram-negative. Indole test,H2S test,MR test and VP test were negative for all strains. Our study lays a foundation for further study and development of microbial resources in deep-sea Hydrothermal Vents.Several recent molecular studies have demonstrated the presence and dominance of bacteria belonging to the epsilon subdivision of Proteobacteria (ε-Proteobacteria) that are both free-living and being found in association with metazoans at deep-sea hydrothermal vents.Caminibacter profundus strain DSMZ 15016 , a thermophile and chemolithoautotrophic sulfur oxidizers of theε-subdivision of proteobacteria,was first examined for activities of the key enzymes of the rTCA and CBB CO2 fixation pathways. All three key reductive tricarboxylic acid (rTCA) cycle genes aclB(βsubunit for ACL),porA(αsubunit for POR),and oorA(αsubunit for OOR) while failed at amplifying the form I and II RubisCO -the key enzyme in the Calvin-Benson cycle genes,cbbL and cbbM from genome DNA. Furthermore,the genes aclB and oorA were also cloned from RNA and the cbbL and cbbM were failed tobe cloned. These results paralleled with the tests on rTCA enzymatic activities ----ATP-dependent citrate lyase,pyruvate:ferredoxin oxidoreductase,and 2-oxoglutarate:ferredoxin oxidoreductase,while no activities was detected on ribulose 1,5-bisphosphate carboxylase (RubisCO) activity. The combination of enzymatic and genetic analysis demonstrated that the Epsilonproteobacteria Caminibacter profundus strain DSMZ 15016 use the rTCA cycle for carbon assimilation.Enzymatic analysis showed that there were optimal ACL expression at 45℃,especially on the 6th day,while both of the OOR and POR with the best expression on the 4th day. By designing primers and using SYBR GreenⅠreal-time quantitative PCR,the effects of environment factors to aclB and oorA genes activities were tested. The results revealed that aclB and oorA genes had the optimal expression on day 2 under the salt 30 g/L and pH7.0,at 40℃and 45℃,respectively. Furthermore,there were optimal expression of aclB under the stress of anoxygen whereas oorA at 3% oxygen. However,there were differences when using various concentration of oxygen. Both of them were acclimated better under the stress of anoxygen. This study revealed the response of energy metabolism of Epsilonproteobacteria to the changes of environment. It. has significance to deeper understand the energy metabolism of Epsilonproteobacteria and may learn the significantly contribution of rTCA to the global autotrophic processes.