Genomic Studies Of Deep-Sea Sedimentary Bacteria And Filamentous Fungus

Marine is one of the most important ecosystems on the Earth. It harbors a variety of bacteria which are valuable resources for discovery of new drugs and enzymes. More than 60% of the Earth's surface is covered by sea water with depth more than 1000 m. The deep-sea floor is covered with fine-grained sediments. Deep-sea sediment is a dynamic geo- and biosphere that hosts rich microbial communities because of its great width and depth. The deep-sea is an extreme environment and harbors a variety of extremophiles adapted to low or high temperature, high pressure, high salty and low nutrient. These extremophiles will have great potential in industry, medicine and environment protection. Though deep-sea sediments contain huge microbial resources, it is still less exploited because of the sample and culture difficulty. So it is important to discover and exploit the bacterial resources in deep-sea sediments.In this dissertation, we identified a bacterium, SM-A87, isolated from southern Okinawa Trough. Then the complete genome of SM-A87 was sequenced and analyzed to find its role in deep-sea particulate organic material (POM) degradation. Pseudoalteromonas sp. SM9913 is a cold-adapted bacterium isolated from Okinawa Trough with water depth of 1855 m. It can secret a large quantity of exopolysaccharides and cold-adapted proteases. In order to find the special features of deep-sea species of Pseudoalteromonas, the complete genome of P. sp. SM9913 was sequenced and compared to Pseudoalteromonas haloplanktis TAC125 which is a cold-adapted bacterium isolated from the Antarctic surface water. Trichoderma pseudokoningii SMF2 was a filamentous fungus isolated from beach soil. It has nematicidal activity and can kill nematode Meloidogyne incognita. The secondary metabolite peptaibols T. pseudokoningii SMF2 secreted have antimicrobial activity and can induce programmed cell death of tumor. In this dissertation, we sequenced and analyzed the genome of SMF2 in order to find the pathway of peptaibols synthesis and provide foundation for further research.Identification of strain SM-A87:Strain SM-A87 was isolated from southern Okinawa Trough sediments at 2 meters below seafloor with water depth of 1245 m. It is a Gram-negative, non-motile bacterium. After 48 h cultivation at 28℃on marine agar, the colonies were yellow to orange and circular, about 1-3mm in diameter, and were adherent to the agar. Cells were rod-shaped and ranged from 0.3 to 0.6 mm in width and from 1.5 to 3.3 mm in length and were non-motile. Cells in old cultures might form coccoid bodies.Cells are strictly aerobic, oxidase-and catalase-positive. Flexirubin-type pigments are absent. MK-6 is the predominant respiratory quinone. SM-A87 synthesized mainly terminally branched iso-and anteiso-fatty acids. The strain can grow at 4-38℃(25-30℃optimum), at pH 5.0-8.5 and in the presence of 0-12% NaCl (3%, optimum).The DNA G+C content of SM-A87 is 35.8%. The phylogenetic tree revealed that strain SM-A87 can form a distinct lineage within the family Flavobacteriaceae. Strain SM-A87 had 92.9% 16S rRNA gene sequence similarity to its nearest neighbor Salegentibacter holothuriorum, and 91.8% and 91.5% to Mesonia algae and Gramella portivictoriae, respectively.According to the phenotypic characteristics, chemotaxonomy and phylogeny analysis, SM-A87 was classified as a new genus and species in the family Flavobacteriaceae for which the name Wangia profunda is proposed. The type strain is SM-A87T. In the International Journal of Systematic and Evolutionary Microbiology (IJSEM) Validation List no.116, it was renamed to Zunongwangia profunda.Genomic study of Zunongwangia profunda SM-A87:SM-A87 is a new genus and species in the family Flavobacteriaceae of the phylum Bacteroidetes that was isolated from deep-sea sediment. Its genome is composed of only one circular chromosome with 5128187 bp. It has 4653 predicted ORFs of which the average length is 960 bp. The genome contains 47 tRNA genes and 3 rRNA operons. This is the first sequenced genome of a deep-sea bacterium from the phylum Bacteroidetes. The genome harbors all the genes of glycolysis, the pentose phosphate pathway and the tricarboxylic/citric acid cycle as well as the key enzyme of ED metabolic pathway. All this reflects the metabolic versatility of SM-A87.SM-A87 can produce a large quantity of capsular polysaccharide, and the genome contains two gene clusters for polysaccharide synthesis and export. SM-A87 contains 130 predicted peptidases,61 of which have signal peptides. The extracellular peptidases are more halophilic than the intracellular peptidases. The halophilicity of the extracellular peptidases helps them function in saline environments and decompose extracellular organic nitrogen matter in the marine salty condition. The extracellular peptidases mainly belong to families of metallopeptidases and serine peptidases. SM-A87 has many genes related to carbohydrate transport and metabolism. This shows that the deep-sea bacterium of the phylum Bacteroidetes is also good at decomposing extracelluar materials.SM-A87 has two CRISPR loci. The first one is 5595 bp and the second one is 2115 bp. CRISPR is related to phage defense., But the spacers of these two CRISPR loci do not have similar sequences in the virus databases, this may because the updated virus databases just have a small section sequences of all marine virus.SM-A87 is a moderate halophile and can tolerate 0-12% NaCl. Halophilic proteins usually contain more acidic residues and have lower pIs (predicted isoelectric point) than nonhalophilic proteins. We predicted the pIs of all the proteins of SM-A87 and found that the pIs of extracelluar proteins are lower than that of intracellular proteins. This shows that its extracellular proteins are more halophilic than intracelluar proteins. This also indicated that SM-A87 may concentrate organic compatible solutes in the cell so the intracellular ion concentration is not high. The presence of glycine betaine transporter in SM-A87 confirms this conclusion.Genomic and comparative genomic study of Pseudoalteromonas sp. SM9913: P. sp. SM9913 (SM9913 for short) is a deep-sea cold adapted bacterium with the optimum growth temperature of 15℃. The genome of SM9913 is composed of two chromosomes. One is 3.3 Mb (chrⅠ) and the other is 700 kb (chrⅡ). The genome size and structure are all similar to the two chromosomes of P. haloplanktis TAC125 (TAC125 for short). The two chromosomes of SM9913 have 3711 ORFs in total, of which 66.9% can be annotated with known or predicted function. Sixty-two tRNA genes and eight rRNA operons as well as one extra 5S rRNA gene are all located in chr I.Phylogenetic tree based on the 16S rRNA gene indicates that SM9913 and TAC125 are with high similarity. The average nucleotide identity between SM9913 and TAC125 is 85%, which indicates that the two strains are not the same species, but have a high identity. The two strains share 2698 orthologous genes, accounting for 72.7%and 77.4%of all the genes of SM9913 and TAC125, respectively. Twelve genomic islands (GIs) larger than 15 kb can be identified. Eleven GIs are located in chr 1 and one GI is located in chr 2. Most genes in the GIs of SM9913 are specific genes that do not have orthologs in TAC125. These specific genes may confer some features of SM9913 that differentiate it from TAC125SM9913 has fewer dioxygenase genes than TAC125 and lacks the fatty acid metabolism gene cluster that is related to ROS (reactive oxygen species) resistance, indicating a possible sensitivity to reactive oxygen species. Accordingly, experimental results showed that SM9913 was less tolerant of H2O2 than TAC125. SM9913 was only able to grow at concentrations of up to 5 mM H2O2, while TAC125 grew well even at 10-15 mM H2O2. This may be because the oxygen concentration in deep sea sediment is very low, so the production of ROS in deep-sea sediment is less than that in surface sea water.GI-2 of SM9913 contains some genes that are related to drug resistance. SM9913 can resist some antibiotics, such as ampicillin, penicillin and amoxicillin, while TAC125 is susceptible to them. The Okinawa Trough is at the edge of the continental shelf of the East China Sea. Compared to Antarctic sea water, the water and sediments in the Okinawa Trough are likely to contain more materials from the continent, including antibiotics. So SM9913 can resist many antibiotics.There are some heavy metal resistance and efflux genes in GI-8 and GI-9 of SM9913. Experimental results proved that SM9913 is more resistant to zinc than TAC125. It is still unclear why deep-sea bacteria are more resistant to heavy metals than surface sea bacteria. However, SM9913 may be adapted to high metal concentrations because the negatively-charged EPS that is secreted by deep-sea bacteria sometimes can adsorb more cations around the cell than are neededWith the polar and lateral flagellar systems, SM9913 can swim in the sea water and swarm on the sediment particle surface, which is advantageous in the acquisition of nutrients from particle materials in deep-sea sediment. The polar flagellum can be seen under SEM. But the lateral flagella were not observed. This may be because lateral flagella biosynthesis genes are not expressed under normal pressure.The genome contains a glycogen production operon, SM9913 can accumulate glycogen with this operon when nutrients are widely available and use the stored glycogen when nutrients are absent from the environment, which would improve its ability to survive in the deep-sea environment.Genomic study of Trichoderma pseudokoningii SMF2:The genomic sequence of T. pseudokoningii SMF2 was sequenced and assembled by Solexa Genome Analyzer. The genome composed of 114 contigs and 93 scaffolds is about 31 Mb. Its extracelluar proteases are mainly metallo and serine peptidases. Genomic sequence has some similarity to other sequenced strains in the same genus but has differences in GO function categories. The genome has 22 gene clusters related to synthesis of secondary metabolite. These clusters may be responsible for the synthesis of peptabols, which needs further research. Our prelimitary study on the genome of SMF2 will be beneficial for further research on its genome, transcripsome and proteinsome.