Polyphasic Taxonomy Of Bacteria From Deep-sea And Polar Region And Genomic Analysis Of Rheinheimera Nanhaiensis E407-8from Deep-sea

More than70%of the Earth's surface is covered by marine sediments, which form the largest global reservoir of organic carbon. There are lots of microbes in marine sediments. According to the statistics, marine sediments contain over half of the microbes on the Earth. The deep-sea (except for the hydrothermal vents) and the polar region are extreme environments with high salinity and low temperature. Microbes in these extreme environments must have evolved unique mechanisms to survive and exhibit high diversity in species, genes and ecological functions. Their metabolic pathways and genetic backgrounds may be very distinctive, which are greatly valuable for both fundamental studies and biotechnological applications. Besides, they may be promising sources for new bioactive moleculars.To study these microorganisms, the first and important step is the determination of their taxonomic postions. At present, polyphasic taxonomy, which contains phenotypic, genotypic and phylogenetic analyses, is frequently used for taxonomic characterization of bacterial strains. In this dissertation, we identified four bacteria, which were isolated from different marine sediments, through polyphasic taxonomic studies. The results revealed that these four strains represented different new bacterial species. Furhter more, we sequenced the genome of a marine bacterium Rheinheimera nanhaiensis E407-8, which isolated from the sediment of the South China Sea, and conducted a prelimiary analysis on the complete genome sequence.1. Polyphasic taxonomy of strain CF12-14from the sediment of the South China SeaStrain CF12-14was isolated from the sediment of the South China Sea at a water depth of1153m. Cells are Gram-negative, straight or slightly curved rods. Cells are approximately0.3-0.6μm in width and0.8-2.4μm in length. Cells can produce bubs and prosthecae, and motile with one polar flagellum. Colonies are circular, slightly convex and white to light-yellow.Oxidase and catalase activities of strain CF12-14are positive. Growth occurs at4-42℃, good growth occurs at25-40℃, and the optimum temperature for growth is30-35℃. Growth occurs in the presence of0.1-15%(w/v) NaCl (optimum,2-3%) with no growth at0or18%NaCl. The pH range for growth is6.0-11.5with the optimum pH at8.0-9.5.The dominant cellular fatty acids of strain CF12-14are iso-C15:0, iso-C17:0and iso-C17:1ω9c. The major polar lipids are phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). Ubiquinone8(Q-8) is the major respiratory quinone.The DNA G+C content of strain CF12-14is50.4mol%. Strain CF12-14has the highest16S rRNA gene sequence similarities to type strains of Idiomarina salinarum DSM21900T (94.7%) and I. seosinensis DSM21922(94.6%), and91.9-94.6%sequence similarities to those of other recognized Idiomarina species.Results from the polyphasic taxonomy study support the conclusion that CF12-14represents a novel Idiomarina species, for which the name Idiomarina maris sp. nov. is proposed. The type strain of Idiomarina maris is CF12-14T (=CCTCC AB208166T=KACC13974T).2. Polyphasic taxonomy of strain D25from the deep-sea sediment of the southern Okinawa TroughStrain D25was isolated from the deep-sea sediment of the southern Okinawa Trough. Cells are spindle-shaped, Gram-negative rods (0.7-0.9μm in width and1.6-2.4μm in length) and aerobic. Cells lack flagellum and are nonmotile. Colonies are pale yellow, circular and convex with entire margins and smooth surfaces. The pigments are diffusible and nonflexirubin type.Oxidase and catalase activities of strain D25are positive. Growth occurs in the presence of0-8%NaCl (optimum,1%), at8-42℃(optimum,30-35℃) and at pH5.0-9.0(optimum, pH6-7). No growth was observed at4or45℃. Strain D25hydrolyses casein, gelatin and DNA, but not starch or carboxymethyl cellulose. Nitrite is reduced, and nitrate is not reduced. Result of the Voges-Proskauer test is positive. Citrate is not utilized. Indole or H2S are not produced.The main cellular fatty acids of strain D25are iso-C15:0, iso-C17:1ω9c, iso-C17:03-OH and Summed Feature3(comprising C16:1ω7c and/or iso-C15:02-OH). The major respiratory quinone is MK-6.The DNA G+C content of strain D25is33.0mol%. Phylogenetic analyses of16S rRNA gene sequences show that strain D25fell within the genus Myroides, with99.2%,96.0%and93.4%sequence similarities to the only three recognized species of Myroides. However, the DNA-DNA similarity value between strain D25and its nearest neighbor Myroides odoratimimus JCM7460T is only49.9%(<70%).The results of the polyphasic taxonomy analysis suggest that strain D25represents a novel species of the genus Myroides, for which the name Myroides profundi sp. nov. is proposed. The type strain is D25T (=CCTCC M208030T=DSM19823T).3. Polyphasic taxonomy of strain BSs20135from the Arctic marine sedimentStrain BSs20135was isolated from the Arctic marine sediment. Cells are Gram-negative, straight or slightly curved rods (0.4-0.8in width and1.3-4.8μm in length), and motile by means of a single polar flagellum. Buds and prosthecae can be formed. Colonies are pale-yellow, circular, convex and smooth with entire edges. In the broth medium, cell aggregates are yellowish-brown.Oxidase and catalase activities of strain BSs20135are positive. Growth occurs at4-28℃(optimum,25℃) and1-5%(w/v) NaCl (optimum,2%NaCl), and NaCl is required for growth. Strain BSs20135hydrolyses aesculin and DNA, but not casein, Tween80or starch (API20NE). This strain does not reduce nitrate to nitrite (API20NE), and not produce indole, acetoin (Voges-Proskauer reaction) or H2S (API20E).The major cellular fatty acids of strain BSs20135are Summed Feature3(comprising C16:1ω7c and/or iso-C15:02-OH), C16:0, C17:1ω8c and C18:1ω7c.The genomic DNA G+C content of strain BSs20135is40.3mol%. Phylogenetic analysis of16S rRNA gene sequences indicates that strain BSs20135belongs to the genus Glaciecola. The strain BSs20135shows the highest16S rRNA gene sequence similarities to G. psychrophila170T (97.7%), G. mesophila KMM241T (97.4%) and G. polaris LMG21857T (97.1%) and lower16S rRNA gene sequence similarities (93.6-96.5%) with the other members of the genus Glaciecola.Based on16S rRNA gene sequence analysis, phenotypic and chemotaxonomic characterization, strain BSs20135represents a novel species, for which the name Glaciecola arctica sp. nov. is proposed. The type strain is Glaciecola arctica BSs20135T (=CCTCC AB209161T=KACC14537T).4. Polyphasic taxonomy of strain S3-22from the Antarctic marine sedimentStrain S3-22was isolated from the marine sediment of the Nella Fjord, Antarctica. Cells are Gram-negative rods (0.6-1.0μm in width and1.6-1.8μm in length), and motile with single polar flagellum. Colonies are white, small, circular (0.5-1.2mm in diameter), convex and smooth.Strain S3-22is facultatively aerobic. Oxidase and catalase activities of strain S3-22are positive. Growth occurs at4-25℃(optimum,15℃) and pH6.0-8.0(optimum, pH6.5-7.0). Strain S3-22grows with0.5-5%NaCl (optimum,2-3%). The strain does not grow without NaCl. It does not hydrolyse casein, aesculin (API20NE) or starch and is negative for DNase activity. Nitrate is reduced to nitrite (API20NE). Indole, acetoin (Voges-Proskauer reaction) or H2S are not produced (API20E).Strain S3-22contains Q-8as the only respiratory quinone and Summed Feature3(comprising C16:1ω7c and/or iso-C15:02-OH), C16:0and C18:1ω7c as the major cellular fatty acids.The genomic DNA G+C content of strain S3-22is45.6mol%. Phylogenetic analyses of16S rRNA gene sequences show that strain S3-22is affiliated with the genus Neptunomonas, with97.1%sequence similarity to Neptunomonas japonica JAMM0745T and94.8%to Neptunomonas naphthovorans NAG-2N-1261, the type strains of the only two recognized Neptunomonas species. DNA-DNA relatedness between strain S3-22and N. japonica JCM14595T is20.4%.Based on the evidence from the polyphasic study, strain S3-22represents a novel Neptunomonas species, for which the name Neptunomonas antarctica sp. nov. is proposed. The type strain is S3-22T (=CCTCC AB209086T=KACC14056T).5. Polyphasic taxonomy and genomic analysis of strain E407-8from the deep-sea of the South China SeaThe strain E407-8was isolated from the sediment collected from a water depth of1800m at the station E407(18°29.810'N,112°0.017'E) of the South China Sea. Based on our polyphasic taxonomic study, strain E407-8represents a novel species in the genus Rheinheimera, for which the name Rheinheimera nanhaiensis sp. nov. is proposed. The type strain is Rheinheimera nanhaiensis E407-8T (=CCTCC AB209089T=KACC14030T).Description of Rheinheimera nanhaiensis E407-8is as follow:Cells are Gram-negative. Facultatively aerobic. Cells are straight or curved rods (1.0-2.0μm in length and0.3-0.5μm in width). Motile by a single polar flagellum. Able to form prosthecae. Colonies are slightly yellow, circular and convex with smooth surfaces. Grows at10-48℃(optimum,37℃), but not at50℃. Grows in0-8%(w/v) NaCl (optimum,0.5-2.5%) and at pH5.5-10.0(optimum, pH7.5-8.5). the following enzyme activities are detected:Oxidase, catalase, gelatinase, DNase, a-chymotrypsin, trypsin, esterase (C4), esterase lipase (C8), N-acetyl-(3-glucosaminidase, alkaline and acid phosphatases, leucine arylamidase, valine arylamidase and naphthol-AS-BI-phosphohydrolase. Hydrolyses starch, casein and aesculin. Reduces nitrate to nitrite; Able to utilize D-glucose, N-acetyl-D-glucosamine, sucrose, trehalose, cellobiose and maltose as sole carbon and energy sources. Negative for indole and H2S production; negative for utilization of citrate and the Voges-Proskauer test.The genomic sequence of the strain Rheinheimera nanhaiensis E407-8was determined and analyzed. The genome is about4.0Mb, and the G+C content is51.3mol%. Its genome encodes all kinds of proteins and enzymes required for glycolysis, the citric acid cycle, pentose phosphate pathway and oxidative phosphorylation. This reflects the metabolic versatility of E407-8.The strain E407-8hydrolyses casein, gelatin (collagen) and elastin based on our previous experimental data. Elastin and collagen are the common major constituents of marine animals, and they are high molecular protein polymers, which are recalcitrant to degradation. And they are the important source for the deep-sea particulate organic nitrogen (PON) and high molecular weight dissolved organic nitrogen (HMW DON). The genome sequence of strain E407-8habors a very large number of extracellular peptidases (82), and they belong to four different peptidase categories (51serine peptidases;25metal peptidases;4threonine peptidases, and2cysteine peptidases). Among these peptidases, their families have also been classified for each categories,(e.g.13families for the51serine peptidases and12families for the25metal peptidases), and such a wide range distribution of peptidase families indicates that strain E407-8may have the hydrolytic ability to many proteins. The synergistic effect of these peptidases can promote the degradation efficiency of extracellular proteins. Thus strain E407-8can play an important role in the cycle of deep sea organic nitrogen.The physiological and biochemical studies show that the strain E407-8has the extracellular enzymatic activities of esterase (C4), esterase/lipase (C8) and amylase. Correspondingly, the genes encoding eight esterases/lipases and six amylases that are all with signal peptides are present in its genome. The recent studies have found that about80%of the single-celled organism will be killed by the virus, and form enormous organic carbon, thus the bacterial cell wall polysaccharides (peptidoglycan) and the microbial cell membrane components (lipids) may serve as the important source for the organic carbon in the marine sediments. Strain E407-8encodes the proteins that can degrade the bacterial cell wall polysaccharides, the plant polysaccharides (starch), and the microbial cell membrane components (lipids). Therefore, this strain may play an important role in the degradation of the deep sea organic carbon.