威海近海沉積物細(xì)菌富集分離、新菌鑒定及B210捕食現(xiàn)象探究
發(fā)布時(shí)間：2018-07-15 13:14
【摘要】：近海沉積物中微生物多樣性豐富,蘊(yùn)藏大量的潛在新菌資源。本研究通過(guò)細(xì)菌富集分離手段,對(duì)不同富集時(shí)期的近海沉積物樣品進(jìn)行新菌資源挖掘,并通過(guò)454FLX+平臺(tái)宏基因組測(cè)序探究了不同富集時(shí)期富集培養(yǎng)液中細(xì)菌區(qū)系演替規(guī)律。本研究發(fā)現(xiàn)了大量潛在新菌,并對(duì)7株海洋新菌進(jìn)行了多相分類。本研究發(fā)現(xiàn)慢生單胞菌新菌B210具有捕食現(xiàn)象,并對(duì)其進(jìn)行了初步探究。通過(guò)富集分離,獲得326株細(xì)菌,包括49株潛在新菌。通過(guò)宏基因組測(cè)序分析得出:不同富集時(shí)期,富集培養(yǎng)液中有其獨(dú)特的細(xì)菌區(qū)系,為以后有目的性地挖掘海洋新菌提供指導(dǎo);物種豐富度會(huì)隨著富集時(shí)間的增加而降低,而至第30天時(shí),呈上升趨勢(shì);某些豐富度極低的細(xì)菌門(mén)類通過(guò)富集,數(shù)量得到了劇增,如酸桿菌門(mén)細(xì)菌。菌株Z1~T和JL1分別分離自石花菜和真江蘺,可降解瓊膠。呼吸醌為Q-8。菌株 Z1~T 的主要脂肪酸是 C_(18:1)ωa7c、C_(16:0) 和 summed feature 3(C_(16:1)ω7c and/or iso-C_(15:0)2-OH)。主要極性脂是磷脂酰乙醇胺、磷脂酰甘油和1種未知的氨基脂。DNA G+C含量是45.1 mol%。與最相近物種的16S rDNA序列相似性低于92.3%。根據(jù)多相分類,菌株Z1~T和JL1代表Gammaproteobacteriaa的一個(gè)新屬新種,命名為 Marinagarivorans algicola gen.nov.,sp.nov。模式菌株為 Z1~T(=ATCC BAA-2617~T=CICC 10859~T)。菌株NC2-42~T分離自威海近海沉積物,革蘭氏染色可變。呼吸醌是MK-7。細(xì)胞壁含二氨基庚二酸。極性脂組分包括磷脂酰甘油、磷脂酰乙醇胺、2種未知的糖脂、1種未知的磷脂、1種未知的氨基磷脂、1種未知的脂質(zhì)。主要脂肪酸是anteiso-C_(15:0)、iso-C_(16:0)和C_(16:0)。基因組DNAG+C含量58.1mol%。菌株NC2-42~T與Paenibacillus profundus有最高16SrDNA序列相似性(92.32%)。通過(guò)多相分類,NC2-42~T為 Paenibacillaceae 的一個(gè)新屬新種,命名為 Marinicrinis sediminis gen.nov.,sp.nov。模式菌株為 NC2-42~T(=KCTC 33676~T =MCCC 1K01238~T)。菌株XDB06~T分離于威海近海沉積物,革蘭氏染色陰性。呼吸醌是Q-8。主要脂肪酸是iso-C_(15:0)和C_(16:0)。極性脂成分為雙磷脂酰甘油、磷脂酰甘油、2種糖脂和 4 種磷脂�；蚪M DNA G+C 含量 65.0 mol%。與Wenzhouxiangella marina的16SrDNA相似性最高,為96.5%。通過(guò)多相分類,菌株XDB06~T是WenzhouxiangeHa 的一個(gè)新物種,命名為Wenzhouxiangella sediminis sp.nov。模式菌株為 XDB06~T(=KCTC 52041~T= MCCC 1K02285~T)。菌株0W14~T分離于文登海洋鹽田,革蘭氏染色陽(yáng)性。MK-7是唯一呼吸醌。肽聚糖類型是A4βL-Orn-D-Glu。主要脂肪酸組分是anteiso-C_(15:0)。極性脂組分是雙磷脂酰甘油、磷脂酰甘油、2種未知的糖脂和4種未知的磷脂�；蚪MDNA G+C 含量 44.6mol%。菌株 0W14T與Virgibacillus litoralis具有最高 16SrDNA 相似性(96.0%)。根據(jù)多相分類,菌株0W14T代表Bacillaceae的一個(gè)新屬新種,命名為 Marinisalinussediminis gen.nov.,sp.nov。模式菌株為 0W14T(=KCTC 33835~T=MCCC 1H00171~T)。菌株NC2-31~T分離于威海近海沉積物,革蘭氏染色陽(yáng)性。呼吸醌為MK-7,肽聚糖中含二氨基庚二酸。主要極性脂是雙磷脂酰甘油、磷脂酰甘油和磷脂酰乙醇胺。基因組DNA G+C含量46.3mol%。主要脂肪酸為iso-C_(15:0)、anteiso-C_(15:0)、iso-C_(16:0)和 SummedFeature3(C_(16:1)ω7cand/or iso-C_(15:0)2-OH)�；� 16SrDNA 序列的系統(tǒng)發(fā)育分析表明菌株NC2-31T在Bacillus內(nèi)形成獨(dú)立分支。根據(jù)多相分類,菌株NC2-31~T代表Baacillus的一個(gè)新物種,命名為Bacillusmarinisedimentorum sp.nov。模式菌株為 NC2-31~T(=KCTC33721~T = MCCC1K01239~T)。菌株WDN1C137~T分離于文登海洋鹽田,革蘭氏染色陰性。Q-10是唯一醌型。主要脂肪酸成分為C_(18:1)ω7c、cycloC_(19:0)ω8c、C_(16:0)。主要極性脂為磷脂酰甘油、磷酸糖脂、1種未知的糖酯、1種未知的脂質(zhì)、2種未知的氨基脂和3種未知的磷脂�；蚪MDNA G+C含量71.4 mol%。菌株WDN1C137T與Roseivivax jejudonensi.有最高16S rDNA相似性(94.5%)。根據(jù)多相分類,菌株WDN1C137T代表Rhodobacteracea 的一個(gè)新屬新種,Rhodosalinus sediminis gen.nov.,sp.nov。模式菌株為 WDN1C137~T(=KCTC52478~T = MCCC 1H00170~T)。菌株B210T分離于威海小石島近海沉積物,革蘭氏染色陰性。呼吸醌是MK-7。極性脂組分是雙磷脂酰甘油、磷脂酰甘油、磷脂酰乙醇胺、1種未知的磷脂、1種未知的脂質(zhì)。主要脂肪酸是iso-C_(15:0)。基因組DNA G+C含量是64.7 mol%。系菌株 B210T 與 Bradymonas sediminis FA350~T有最高 16S rDNA 序列相似性(89.2%)。通過(guò)多相分類,B210~T為Bradymonadaless的一個(gè)新屬新種。模式菌株為 B210~T(=KCTC42951~T)。對(duì)鑒定的慢生單胞菌新菌B210捕食現(xiàn)象進(jìn)行了初步探究。其捕食譜廣泛,能捕食革蘭氏染色陽(yáng)性和陰性細(xì)菌,這同黃色黏球菌的捕食譜相似,而不同于只能捕食革蘭氏陰性菌的蛭弧菌;通過(guò)掃描電子顯微鏡觀察、捕食接觸性試驗(yàn)推斷其捕食需要細(xì)胞間接觸。通過(guò)對(duì)菌株B210發(fā)酵液不同組分的抑菌作用的探究,發(fā)現(xiàn)其發(fā)酵上清液和胞內(nèi)產(chǎn)物都不具有抑菌作用,這進(jìn)一步佐證了其捕食行為需借助與獵物的細(xì)胞接觸。
[Abstract]:In the offshore sediments, the microbial diversity is abundant and contains a large number of potential new bacteria resources. Through the method of bacterial enrichment and separation, the new bacteria resources are excavated in the sediment samples of different enrichment period, and the succession law of the bacterial flora in the enrichment culture medium of different enrichment period is explored through the 454FLX+ platform macrogenome sequencing. This study found a large number of potential new bacteria and a multiphase classification of 7 new marine bacteria. This study found that B210 has a predatory phenomenon and has carried out preliminary exploration. Through enrichment and separation, 326 strains of bacteria, including 49 potential new bacteria, were obtained by Acer sequence analysis: enrichment culture and enrichment culture. There is a unique bacterial flora in the nutrient solution, which provides guidance for the future purpose of mining the new marine bacteria. The species richness will decrease with the increase of the enrichment time, and up to thirtieth days. Some kinds of bacteria with extremely low richness have been enriched, such as bacilli bacteria, bacteria Z1~T and JL1, respectively. The main fatty acids of the Q-8. strain Z1~T are C_ (18:1) Omega a7c, C_ (16:0) and summed feature 3 (C_ (16:1) Omega 7C). The main polar lipids are phosphatidylethanolamine, phosphatidyl glycerol and 1 kinds of unknown amino groups, which are 45.1 and the closest species The similarity of 16S rDNA sequence is lower than 92.3%. based on Polyphasic classification. Strain Z1~T and JL1 represent a new genus and new species of Gammaproteobacteriaa, named Marinagarivorans algicola gen.nov., sp.nov. model strain is Z1~T (=ATCC). Quinone is MK-7. cell wall containing two amino heptane diacid. Polar lipid components include phosphatidyl glycerol, phosphatidyl ethanolamine, 2 kinds of unknown glycolipids, 1 unknown phospholipids, 1 unknown amino phospholipids, 1 kinds of unknown lipids. The main fatty acids are anteiso-C_ (15:0), iso-C_ (16:0) and C_ (16:0). 58.1mol%. strains NC2-42~T and Paenibaci in genomic DNAG+C Llus profundus has the highest 16SrDNA sequence similarity (92.32%). Through multiphase classification, NC2-42~T is a new genus and new species of Paenibacillaceae, named Marinicrinis sediminis gen.nov., sp.nov. mode strain is NC2-42~T (=KCTC 33676~T). The strain is separated from the sediments of Weihai and gram-negative. The main fatty acids of Q-8. are iso-C_ (15:0) and C_ (16:0). The polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, 2 kinds of glycolipids and 4 kinds of phospholipids. The genomic DNA G+C content is the highest similarity between 65 mol%. and Wenzhouxiangella marina. The strain of Wenzhouxiangella sediminis sp.nov. model is XDB06~T (=KCTC 52041~T= MCCC 1K02285~T). Strain 0W14~T is separated from the marine salt field in Wendeng, and gram-positive.MK-7 is the only respiratory quinone. Lipoyl glycerol, 2 unknown glycols and 4 kinds of unknown phospholipids. Genomic DNA G+C content 44.6mol%. strain 0W14T and Virgibacillus litoralis have the highest 16SrDNA similarity (96%). According to multiphase classification, strain 0W14T represents a new species of Bacillaceae, named Marinisalinussediminis gen.nov., sp.nov. mode strain is 0 W14T (=KCTC 33835~T=MCCC 1H00171~T). Strain NC2-31~T was isolated from Weihai offshore sediments, gram-positive. Respiratory quinone was MK-7, and peptidoglycan contained two amino heptanic acid. The main polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol and phosphatidyl ethanolamine. Genomic DNA G+C content 46.3mol%. main fatty acids were iso-C_ (15:0), anteiso-C_. (15:0), iso-C_ (16:0) and SummedFeature3 (C_ (16:1) Omega 7cand/or iso-C_ (15:0) 2-OH). Phylogenetic analysis based on 16SrDNA sequence shows that strain NC2-31T forms an independent branch. 31~T (=KCTC33721~T = MCCC1K01239~T). Strain WDN1C137~T was isolated from the Wendeng ocean salt field. Gram-negative.Q-10 was the only quinone. The main fatty acids were C_ (18:1) Omega 7C, cycloC_ (19:0) Omega 8C, C_. 1 kinds of unknown sugar esters, 1 unknown lipids, 2 unknown amino fat and 3 were the main polar lipids. Genomic DNA G+C content 71.4 mol%. strain WDN1C137T and Roseivivax jejudonensi. have the highest 16S rDNA similarity (94.5%). According to the multiphase classification, the strain WDN1C137T represents a new species of Rhodobacteracea, Rhodosalinus sediminis. Strain B210T is separated from the sediments of Weihai small Shidao offshore, gram-negative. Respiratory quinone is a MK-7. polar lipid component which is diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, 1 kinds of unknown phospholipids, 1 kinds of unknown lipids. The main fatty acids are iso-C_ (15:0). The DNA G+C content of the group is 64.7 mol%. strain B210T and Bradymona S sediminis FA350~T has the highest 16S rDNA sequence similarity (89.2%). Through multiphase classification, B210~T is a new genus and new species of Bradymonadaless. The pattern strain is B210~T (=KCTC42951~T). The preliminary exploration of the identification of the new bacteria B210 of the new bacteria of the bacteria is preliminarily explored. It is widely used to capture Gram-positive and negative bacteria. This was similar to that of the Staphylococcus aureus, but different from the vermiculorvibrio that only preyed on the Gram-negative bacteria. Through the scanning electron microscope, the predatory contact test concluded that the predation needed intercellular contact. By investigating the Bacteriostasis of different components of the strain B210, the fermentation supernatant and the intracellular products were not found. It has bacteriostatic effect, which further confirms its predatory behavior by means of contact with prey cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93
本文編號(hào)：2124190
[Abstract]:In the offshore sediments, the microbial diversity is abundant and contains a large number of potential new bacteria resources. Through the method of bacterial enrichment and separation, the new bacteria resources are excavated in the sediment samples of different enrichment period, and the succession law of the bacterial flora in the enrichment culture medium of different enrichment period is explored through the 454FLX+ platform macrogenome sequencing. This study found a large number of potential new bacteria and a multiphase classification of 7 new marine bacteria. This study found that B210 has a predatory phenomenon and has carried out preliminary exploration. Through enrichment and separation, 326 strains of bacteria, including 49 potential new bacteria, were obtained by Acer sequence analysis: enrichment culture and enrichment culture. There is a unique bacterial flora in the nutrient solution, which provides guidance for the future purpose of mining the new marine bacteria. The species richness will decrease with the increase of the enrichment time, and up to thirtieth days. Some kinds of bacteria with extremely low richness have been enriched, such as bacilli bacteria, bacteria Z1~T and JL1, respectively. The main fatty acids of the Q-8. strain Z1~T are C_ (18:1) Omega a7c, C_ (16:0) and summed feature 3 (C_ (16:1) Omega 7C). The main polar lipids are phosphatidylethanolamine, phosphatidyl glycerol and 1 kinds of unknown amino groups, which are 45.1 and the closest species The similarity of 16S rDNA sequence is lower than 92.3%. based on Polyphasic classification. Strain Z1~T and JL1 represent a new genus and new species of Gammaproteobacteriaa, named Marinagarivorans algicola gen.nov., sp.nov. model strain is Z1~T (=ATCC). Quinone is MK-7. cell wall containing two amino heptane diacid. Polar lipid components include phosphatidyl glycerol, phosphatidyl ethanolamine, 2 kinds of unknown glycolipids, 1 unknown phospholipids, 1 unknown amino phospholipids, 1 kinds of unknown lipids. The main fatty acids are anteiso-C_ (15:0), iso-C_ (16:0) and C_ (16:0). 58.1mol%. strains NC2-42~T and Paenibaci in genomic DNAG+C Llus profundus has the highest 16SrDNA sequence similarity (92.32%). Through multiphase classification, NC2-42~T is a new genus and new species of Paenibacillaceae, named Marinicrinis sediminis gen.nov., sp.nov. mode strain is NC2-42~T (=KCTC 33676~T). The strain is separated from the sediments of Weihai and gram-negative. The main fatty acids of Q-8. are iso-C_ (15:0) and C_ (16:0). The polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, 2 kinds of glycolipids and 4 kinds of phospholipids. The genomic DNA G+C content is the highest similarity between 65 mol%. and Wenzhouxiangella marina. The strain of Wenzhouxiangella sediminis sp.nov. model is XDB06~T (=KCTC 52041~T= MCCC 1K02285~T). Strain 0W14~T is separated from the marine salt field in Wendeng, and gram-positive.MK-7 is the only respiratory quinone. Lipoyl glycerol, 2 unknown glycols and 4 kinds of unknown phospholipids. Genomic DNA G+C content 44.6mol%. strain 0W14T and Virgibacillus litoralis have the highest 16SrDNA similarity (96%). According to multiphase classification, strain 0W14T represents a new species of Bacillaceae, named Marinisalinussediminis gen.nov., sp.nov. mode strain is 0 W14T (=KCTC 33835~T=MCCC 1H00171~T). Strain NC2-31~T was isolated from Weihai offshore sediments, gram-positive. Respiratory quinone was MK-7, and peptidoglycan contained two amino heptanic acid. The main polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol and phosphatidyl ethanolamine. Genomic DNA G+C content 46.3mol%. main fatty acids were iso-C_ (15:0), anteiso-C_. (15:0), iso-C_ (16:0) and SummedFeature3 (C_ (16:1) Omega 7cand/or iso-C_ (15:0) 2-OH). Phylogenetic analysis based on 16SrDNA sequence shows that strain NC2-31T forms an independent branch. 31~T (=KCTC33721~T = MCCC1K01239~T). Strain WDN1C137~T was isolated from the Wendeng ocean salt field. Gram-negative.Q-10 was the only quinone. The main fatty acids were C_ (18:1) Omega 7C, cycloC_ (19:0) Omega 8C, C_. 1 kinds of unknown sugar esters, 1 unknown lipids, 2 unknown amino fat and 3 were the main polar lipids. Genomic DNA G+C content 71.4 mol%. strain WDN1C137T and Roseivivax jejudonensi. have the highest 16S rDNA similarity (94.5%). According to the multiphase classification, the strain WDN1C137T represents a new species of Rhodobacteracea, Rhodosalinus sediminis. Strain B210T is separated from the sediments of Weihai small Shidao offshore, gram-negative. Respiratory quinone is a MK-7. polar lipid component which is diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, 1 kinds of unknown phospholipids, 1 kinds of unknown lipids. The main fatty acids are iso-C_ (15:0). The DNA G+C content of the group is 64.7 mol%. strain B210T and Bradymona S sediminis FA350~T has the highest 16S rDNA sequence similarity (89.2%). Through multiphase classification, B210~T is a new genus and new species of Bradymonadaless. The pattern strain is B210~T (=KCTC42951~T). The preliminary exploration of the identification of the new bacteria B210 of the new bacteria of the bacteria is preliminarily explored. It is widely used to capture Gram-positive and negative bacteria. This was similar to that of the Staphylococcus aureus, but different from the vermiculorvibrio that only preyed on the Gram-negative bacteria. Through the scanning electron microscope, the predatory contact test concluded that the predation needed intercellular contact. By investigating the Bacteriostasis of different components of the strain B210, the fermentation supernatant and the intracellular products were not found. It has bacteriostatic effect, which further confirms its predatory behavior by means of contact with prey cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q93
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