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Research On Diversity Of Halophile From Three Different Salterns Of Shandong Province And Taxonomy Analysis Of Six Novel Halophilic Strains

Posted on:2017-03-27Degree:MasterType:Thesis
Country:ChinaCandidate:J XiaFull Text:PDF
GTID:2180330485981138Subject:Microbiology
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Since the invention of the microscope, microorganisms, living under extreme conditions caused people’s attention. There may be many potential new species resources in extreme environments, it is a huge space for research and exploiting. This article investigated the distribution of halophiles from high-salt environment, collected from three different salterns for the study, while the application of traditional bacterial culture methods and pure high-throughput Illumina MiSeq sequencing, respectively. In addition, during the study of the halophiles’diversity survey from different salt samples, six halophilic new bacteria were separated and completed multiphase Identifications were performed, enriching the halophiles’repository.In this study, water samples and sediment samples were collected from Shandong Province, Feicheng city, Jingzhi saltern (36°8’24.45"N,116°49’22.46" E), Weifang city, Weifang saltern (36°59’20.82"N,119°9’56.89" E) and Weihai city, Wendeng saltern (36°57’56.49"N,122°0’38.85" E). After dilution spread plate method, number of different samples of culturable microorganisms were counted. More than 1000 halophiles were selected after the experiment. Followed by the PCR method,764 bacterial 16S rRNA gene were measured, in which 88 from Feicheng,134 from Weifang, remaining 542 from Wendeng.16S rRNA gene similarity lowered than 97%, six halophiles (SYD6T, XJ16T,2W06T,2W32T, JZ3C29T, WDS2C18T) were conducted multiphase classification. Cultured bacterial community from Feicheng saltern comprises Proteobacteria (78,88%, α-proteobacteria 3,3%; γ-proteobacteria 75, 85%)、Firmicutes (5,6%)、Bacteroidetes (5,6%); Cultured bacterial community from Weifang saltern comprises Firmicutes (82,61%)、Proteobacteria (51,38%, α-proteobacteria 5,4%; γ-proteobacteria 46,34%)、Bacteroidetes (1,1%); Cultured bacterial community from Wendeng saltern comprises Proteobacteria (405, 75%, α-proteobacteria 52,10%; β-proteobacteria 1;γ-proteobacteria 352,65%)、 Firmicutes (128,24%)、Bacteroidetes (7,1%). Except that,123 potential novel strains was discovered, representing 51 novel species, there are even 14 novel genera (16S rDNA similarity<= 94%).Through the taxonomic research, we propose a novel order including family Balneolaceae、Rhodothemaceae、Chitinophagaceae and Saprospiraceae within class Cytophagia. Besides that we also propose a novel family(Balneolaceae), which comprises Fodinibius、Aliifodinibius、Gracilimonas and Balneola。16S rRNA gene similarity lowered than 97%, six halophiles (SYD6T, XJ16T,2W06T,2W32T, JZ3C29T, WDS2C18T) were conducted multiphase classification.A bacterial strain, designated SYD6T, was isolated from a marine solar saltern on the coast of Weihai, Shandong Province, PR China. Cells of strain SYD6T were rod-shaped, red, and approximately 5.0-9.0 mm in length and 0.4-0.6 mm in width. The strain was Gram-stain negative, facultatively anaerobic, heterotrophic, catalase-positive and oxidase-negative. Growth occurred in 4-25%(w/v) NaCl [with 2-15%(w/v) MgCl2.6H2O also present], at 20-50℃ and pH 6.5-8.5. Optimal growth was observed at 37-42℃, pH 7.5-8.0, with 6-8%(w/v) NaCl [with 2-4%(w/v) MgCl2.6H2O]. Nitrate was not reduced. Glucose, sucrose, maltose, fructose and ribose stimulated growth, but not glycerol, xylose or mannitol. The G+C content of the genomic DNA was 61.5 mol%(HPLC). The sole methyl naphthoquinone was MK-7 and the predominant cellular fatty acids (> 10%) were iso-C15:0 2-OH/C16:1 ω7c, iso-C16:0, C18:1 ω7c/C18:1 co6c and C18:1 ω7c. The predominant polar lipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and an unknown lipid. Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain SYD6T was affiliated with the phylum Bacteroidetes. The most closely related neighbours were species of the genus Salisaeta and strain SYD6T had a 16S rRNA gene sequence similarity of 91.97% with Salisaeta longa DSM 21114T. On the basis of these phenotypic and phylogenetic data, strain SYD6T represents a novel species of a new genus of the family Rhodothermaceae, for which the name Longimonas halophila gen. nov., sp. nov. is proposed. The type strain of the type species is SYD6T (=CICC 10838T =KCTC 42399T).A Gram-stain-negative, oxidase-negative, catalase-positive and facultatively anaerobic strain designated as XJ16T was isolated from a marine solar saltern on the coast of Weihai, Shandong Province, China. Cells of strain XJ16T were long rod-shaped, approximately 2-10 μm in length and 0.4-0.6 μm in width. The colonies were ochre. Nitrate was reduced to nitrite. Growth occurred at an optimal temperature of 33-37℃ (range 20-45℃) and in the presence of 8-10%(w/v) NaC1 (range 2-20%, w/v). The pH range for growth was 6.5-9.5, with an optimum at 7.5-8.0. A phylogenetic analysis based on the 16S rRNA gene sequence demonstrated that strain XJ16T was affiliated with the phylum Proteobacteria. The most closely related neighbours were species of the genus Thioalkalivibrio and strain XJ16T had a 16S rRNA gene sequence similarity of 93.1% with Thioalkalivibrio sulfidiphilus HL-EbGr7T and of 93.0% with Thioalkalivibrio denitrificans ALJDT. The G+C content of the genomic DNA was 65.9 mol%(HPLC). The sole respiratory quinone was Q8 and the predominant cellular fatty acids (>10%) were iso-C15:0 2-OH/C16:1 ω7c, C18:0 and C16:0 10-CH3. The predominant polar lipids in strain XJ16T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. On the basis of these phylogenetic, physiological and biochemical characteristics, strain XJ16T should be classified as a novel genus and species within the family Ectothiorhodospiraceae and the name Halofilum ochraceum gen. nov., sp. nov. is proposed. The type strain is XJ16T (=KCTC 42259T=MCCC 1H00120T=CICC 23817T).A novel Gram-stain-negative, rods or bent rods, facultatively anaerobic, oxidase and catalase positive bacterium, designated 2W06T, was isolated from a marine solar saltern in the coast of Weihai, Shandong Province, PR China. Strain 2W06T required NaCl for growth. Optimal growth occurred at 33-37℃ (range 20-42℃) and pH 7.5-8.0 (range 6.5-8.5) with 8-10%(w/v) NaCl (range 4-18%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2W06T was most closely related to the type strain of Spiribacter salinus with 96.4% sequence similarity, followed by Alkalilimnicola ehrlichii (95.3%) and Arhodomonas recens (95.1%). Similarity to other species was less than 95.0%. The draft genome sequence of strain 2W06T consists of 188 contigs (> 200 bp) of 2,146,154 bp. The genomes of 2W06T and Spiribacter salinus M19-40T have an ANI value of 73.0%, and the POCP value between the two genomes is 80.9%. The genomic DNA G+C content of strain 2W06T is 65.0 mol% as calculated from the draft genome sequence. The predominant polar lipid profile of the novel isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylcholine. Besides, some other unknown phospholipids and glycolipids were also detected. Major cellular fatty acids were C16:0 and C18:1 ω7c and the main respiratory lipoquinones were MK-7 and Q8. Based on data from a chemotaxonomic, physiological and biochemical study,2W06T may represent a novel species of the genus Spiribacter in the family Ectothiorhodospiraceae. Here we propose the name Spiribacter halophilus sp. nov. to accommodate strain 2W06T. The type strain of Spiribacter halophilus is 2W06T(= KCTC 42499T= MCCC 1H00126T= CICC23868T). In addition, the emended description of the genus Spiribacter was also proposed.A novel Gram-stain-negative, rods, facultatively anaerobic, oxidase-negative and catalase-positive bacterium, designated 2W32T, was isolated from a marine solar saltern on the coast of Weihai, Shandong Province, PR China. Strain 2W32T was tolerant to moderate salt conditions. Optimal growth occurred at 33-37℃ (range 20-45℃) and pH 7.5-8.0 (range pH 7.0-8.5) with 6-10%(w/v) NaCl (range 2-18%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2W32T shared highest 16S rRNA gene sequence similarities to Aliifodinibius sediminis YIM J21T (94.6%), Aliifodinibius roseus YIM D15T (94.4%), Fodinibius salinus YIM C003T (93.6%), Gracilimonas tropica CL-CB462T (88.6%) and Balneola vulgaris 13IX/A01/164T (86.4%) and less than 83.0% to other species of the phylum Bacteroidetes. The isolate and closely related species formed a novel family level clade in the phylum Bacteroidetes. The polar lipid profile of the novel isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid, an unidentified glycolipid and an unidentified lipid. Dominant cellular fatty acids (> 10%) were iso-C15:0, iso-C17:1 ω9c and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) and the sole respiratory quinone was MK-7. The DNA G+C content of strain 2W32T was 47.5 mol%. Comparative analysis of 16S rRNA gene sequences and characterization indicated that strain 2W32T represents a novel species within the genus Aliifodinibius, for which the name Aliifodinibius halophilus sp. nov. is proposed. The type strain of Aliifodinibius halophilus is 2W32T (=KCTC 42497T =CICC 23869T). In addition, a novel family Balneolaceae fam. nov., is proposed to accommodate the genera Fodinibius, Aliifodinibius, Gracilimonas and Balneola.A novel Gram-stain-negative, rods, facultatively anaerobic, oxidase-negative and catalase-positive bacterium, designated JZ3C29T, was isolated from a saltern of city Feicheng, China. Strain JZ3C29T was tolerant to moderate salt conditions. Optimal growth occurred at 40℃ (range 20-50℃) and pH 7.5-8.5 (range pH 7.0-9.0) with 8-10% (w/v) NaCl (range 2-16%). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JZ3C29T shared highest 16S rRNA gene sequence similarities to Gracilimonas tropica CL-CB4621 (90.5%), Gracilimonas mengyeensis YIM J14T (90.5%) and Gracilimonas rosea CL-KR2T (90.4%) and less than 90.0% to other species of the phylum Bacteroidetes. The polar lipid profile of the novel isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine,3 unidentified glycolipids,4 unidentified phospholipids and 2 unidentified lipids. Dominant cellular fatty acids (> 10%) were iso-C15:0, iso-C17:1 ω9c and summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) and the sole respiratory quinone was MK-7. The DNA G+C content of strain JZ3C29T was 44.4 mol%. On the basis of these phenotypic and phylogenetic data, strain JZ3C29T should be classified as a novel genus and species within the family Balneolaceae, for which the name Rhodohalobacter halophilus gen. nov., sp. nov. is proposed. The type strain is JZ3C29T (=MCCC 1H00131T=KCTC 52046T).A bacterial strain, designated WDS2C18T, was isolated from a marine solar saltern in the coast of Weihai, Shandong Province, PR China. Cells of strain WDS2C18T were long rod-shaped, red, and approximately 6.0-12.0 μm in length and 0.3-0.4 μm in width. The strain was Gram-stain-negative, facultatively anaerobic, heterotrophic, catalase-positive and oxidase-negative. Optimal growth was observed at 40℃, pH 7.5-8.0, with 8-12%(w/v) NaCl. Nitrate was not reduced. Glycerol, sucrose, starch and D-mannitol stimulated growth, but not glucose, D-fructose, D-galactose, D-lactose, D-maltose, D-mannose, D-xylose, D-ribose, D-arabinose, L-rhamnose or D-cellobiose. The G+C content of the genomic DNA was 58.1 mol%(HPLC). The sole methyl naphthoquinone was MK-7 and the predominant cellular fatty acids (>10%) were iso-C15:0 2-OH/C16:1 ω7c, iso-C17:1 ω9c, iso-C15:0 and iso-C17:0. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, glycolipid,3 unidentified phospholipids and 2 unidentified lipids. Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain WDS2C18T was affiliated with the phylum Bacteroidetes. The closest related neighbours were species of the genera Salisaeta and Longimonas, strain WDS2C18T shared highest 16S rRNA gene sequence similarities to Salisaeta longa DSM 21114T (91.7%) and Longimonas halophila SYD6T (90.8%) and less than 90.0% to other species of the family Rhodothermaceae. On the basis of these phenotypic and phylogenetic data, strain WDS2C18T should be classified as a novel genus and species within the family Rhodothermaceae, for which the name Longibacter salinarum gen. nov., sp. nov. is proposed. The type strain is WDS2C18T (=MCCC 1H00132T= KCTC 52045T).
Keywords/Search Tags:halophilic bacteria, Illumina MiSeq, polyphasic taxonomy, novel family
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