3株新菌的分类鉴定及新型耐热琼胶酶基因的克隆表达
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摘要
随着人类社会和经济的不断发展,陆地资源已经日益匮乏,为寻找支持人类进一步生存和发展的不竭资源,人们把注意力转向了占地球总面积71 %的海洋,对海洋资源的开发利用已成为各国战略性发展的主旋律。海洋是地球上最大的生物栖息地,其中生活的海洋微生物,种类众多且在特殊环境下形成了独特的代谢方式,成为海洋资源开发与利用的新宠。因此,对海洋微生物的生态、生理、遗传特征和生物活性物质等的研究都具有重大的理论意义和实际价值。本文在前期进行的微生物资源多样性研究的基础上,对几株新发现的新菌进行了分类鉴定并对其中一株新菌产生的琼胶酶进行了研究。
本文对分离自青岛近海的一株革兰氏阴性、严格需氧的琼脂降解菌YM01~T进行了分类学鉴定。结果表明,该菌细胞呈杆状,具有周生鞭毛,细胞首尾相连形成长链。YM01~T生长需要Na~+,不需要海水。能够水解羧甲基纤维素、淀粉、七叶灵和吐温80,不能水解卵磷脂、明胶、尿素以及酪蛋白。甲基萘醌-7(MK-7)为该菌主要的呼吸醌。其主要的脂肪酸组分为C_(16 : 0)(38.3 %), C_(16 : 1)ω7c和/或iso-C_(15 : 0) 2-OH(29.0 %), C_(18 : 1)ω7c(9.3 %)和C_(10 : 0) 3-OH(8.2 %)。主要的极性脂为磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)和氨基磷酸酯(PN)。该菌DNA的G+C含量为44.8 %。YM0~T与所有已经报道的细菌16S rDNA序列相似性都低于91 %,构建的系统进化树显示,YM01~T属γ-变形菌纲的交替单胞菌科,并形成了一个单独的分支类群。根据该菌在表型特征、化学分类特征和系统发生分析上所表现出来的特殊性,认定该菌为γ-变形菌纲中的一个新属新种,命名为嗜琼胶卵链菌(Catenovulum agarivorans gen. nov. sp. nov.)。
从中国南极中山站沿岸潮间带中分离到一株耐低温(0-30℃)、中度喜盐(7-8 % w/v, NaCl)的专性异养细菌,实验室编号为ZS2-28~T。基于16S rDNA的系统发生分析表明,ZS2-28~T属于α-变形菌纲Roseibaca, Roseinatronobacter和Rhodobaca属细菌分类群中一个单独的进化支,与其他已知种属细菌的相似度均低于97 %,为一株潜在的海洋新菌。该菌能够产生菌绿素-a,胞内含有聚β-羟基丁酸脂颗粒,胞外附着有分泌物。主要的细胞壁脂肪酸组分为C_(18 : 1)ω7c。主要的呼吸醌为泛醌10。主要的极性脂包括磷脂酰甘油、磷脂酰乙醇胺、磷脂酰胆碱和一种无法鉴定的氨基脂。基因组DNA G+C含量为63.3 mol%。根据表型、化学分类特征以及系统发生分析的结果,确定ZS2-28~T代表了一个新属新种。命名为南极红柠檬菌(Roseicitreum antarcticum gen. nov. sp. nov)。
此外,从人类唾液样品中分离出一株产可溶性棕色色素的细菌,实验室编号为MY15~T。基于16S rDNA序列的系统发生分析表明,该菌株与海洋类香味菌(Myroides marinus JS-08~T)、M. odoratimimus LMG 4092~T和深海类香味菌(M. profundi D25T)亲缘关系最近,序列相似性分别为96.5 %, 96.3 %和96.1 %。该菌革兰氏染色呈阴性,细胞杆状,不具有鞭毛,不能运动。不能产生Flexirubin类色素。MY15~T以甲基萘醌-6为主要的呼吸醌,C_(15:0) iso (51.2%), C_(17:0) iso 3-OH (12.9%)和C_(13:0) iso (10.5%)为主要的细胞壁脂肪酸。DNA G+C含量为34.3 mol%。根据该菌在遗传进化与表型特征上与类香味菌属其他细菌所表现出来的差异性,认定MY15T为类香味菌属的一个新种,命名为棕色类香味菌(Myroides phaeus gen. nov. sp. nov)。
本文在对嗜琼胶卵链菌YM01~T分类学研究的基础上,对其琼胶水解酶进行了进一步研究,发现YM01~T能够产生耐高温、耐蛋白酶K的琼胶酶,在100℃和90℃分别孵育120 min和240 min后仍能保持33.3 %和44.4 %的活性。此外,利用简并PCR和hiTail-PCR法克隆了一个琼胶酶基因。该基因编码781个氨基酸,理论分子量为89 kDa, SingalP分析其N-端51个氨基酸为信号肽序列,结构域分析表明该酶属于糖苷水解酶42家族。
As the development of society and economy, terrestrial resources have been more and more exhausted. People turned their attention to the oceans which accounted for 70 percent of the total area of the earth for the further development of human beings. The exploration and utilization of marine resources become a theme of the national strategic development. The oceans are the biggest habitat for various organisms, especially the marine microorganisms which contains a great number of species.They play an important role in the ocean exploration and utilization for its unique metabolic mechanism formed under extreme environments. Therefore, it is valuable both theoretically and practically to analyse the ecological, physiological and genetic characteristics of marine microbes, as well as the bioactive substances produced by them. On the basis of the previous research about the diversity of bacteria, we studied the marine microbial resources from two aspects, the taxonomy of novel bacteria and the analysis of their bioactive products.
A novel Gram-negative, strictly aerobic, agar-hydrolyzing bacterium designated YM01T isolated from the Yellow Sea of coastal region of Qingdao was studied using polyphasic approach. Cells were rod-shaped, peritrichously flagellated and formed long chains by end-to-end. This isolate absolutely required Na~+ ions but not sea water for growth. YM01~T was able to hydrolyze cellulose, starch, aesculin and Tween 80, but not egg yolk, gelatin, urea and casein. It contained menaquinone 7 as the predominant isoprenoid quinone and C_(16 : 0) (38.3 %), C_(16 : 1)ω7c and/or iso-C_(15 : 0) 2-OH (29.0 %), C_(18 : 1)ω7c (9.3 %) and C_(10 : 0) 3-OH (8.2 %) as the major cellular fatty acids. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and aminophospholipid (PN) were the major constituents of the phospholipids. The DNA G+C content was 44.8 mol%. 16S rDNA sequence analysis demonstrated that this isolate was unique, showing only <91.0 % sequence similarities to all published species and formed a distinct monophyletic clade closely related to the species of the family Alteromonadaceae within the group of Alteromonas-like Gammaproteobacteria. So, strain YM01T was considered to represent a novel genus and species in the Gammaproteobacteria, for which the name Catenovulum agarivorans gen. nov. sp. nov. is proposed.
Another psychrotolerant (growth occurs at 0–33℃) , moderately halophilic (optimal NaCl concentration of 7–8 %) and obligately heterotrophic marine bacterium, designated ZS2-25 28~T, was isolated from intertidal sediment samples collected from the coastal regions of the Chinese Antarctic Zhongshan Station on the Larsemann Hills. phylogenetic analysis based on 16S rDNA sequences indicated that strain ZS2-28~T formed a distinct evolutionary lineage within the clade of genera Roseibaca, Roseinatronobacter and Rhodobaca of the Alphaproteobacteria with only <91.0 % sequence similarities to those neighbors. This isolate contained bacteriochlorophyll a. Poly-β-hydroxy butyrate accumulation and slime production can be observed. It contained Q-10 as the predominant isoprenoid quinone and C_(18 : 1)ω7c as the major cellular fatty acids. The main polar lipids were phosphatidylglycerol, phospatidylethanolamine, phosphatidylcholine and an unidentified aminolipid. The G+C content of genomic DNA was 63.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain ZS2-28~T was considered to represent a novel genus and species, for which the name Roseicitreum antarcticum gen. nov. sp. nov. is proposed.
A novel bacterial strain, designated MY15~T, was isolated from human saliva. Phylogenetic analyses based on 16S rDNA gene sequences showed that the novel strain was most closely related to Myroides marinus JS-08~T, Myroides odoratimimus LMG 4092~T and Myroides profundi D25~T with 96.5 %, 96.3 % and 96.1 % sequence similarities, respectively. Cells were Gram-straining-negative, rod-shaped, lack flagella and non-motile. Flexirubin-type pigment is absent. This isolate contained menaquinone 6 as the major respiratory quinone, and C_(15:0) iso (51.2 %), C_(17:0) iso 3-OH (12.9 %) and C_(13:0) iso (10.5 %) as the dominant fatty acids. The G+C content of the DNA was 34.3 mol%. On the basis of this polyphasic study, strain MY15~T represents a novel species of the genus Myroides, for which the name Myroides phaeus sp. nov. is proposed.
On the basis of taxonomic study of Catenovulum agarivorans YM01~T, further research about the agar-hydrolyzing enzymes was performed. The results showed that this isolate produced a high temperature- and proteinase K- resistant agarase. After treatment at 100 and 90℃for 120 and 240 min respectively, the agarase remained the agar-hydrolyzing capacity for 33.3 and 44.4 %. In addition, one of the genes for the expression of agarase, aga A, was cloned by using degenerate PCR and hiTail-PCR. Aga A contained 781 amino acids and the theoretical molecular weight was 89 kDa. According to the result of SignalP analysis, aga A contained a 51 aa signal peptide. The conserved domain analysis revealed that aga A is a novel agarase belonging to the family glycoside hydrolase-42.
本文对分离自青岛近海的一株革兰氏阴性、严格需氧的琼脂降解菌YM01~T进行了分类学鉴定。结果表明,该菌细胞呈杆状,具有周生鞭毛,细胞首尾相连形成长链。YM01~T生长需要Na~+,不需要海水。能够水解羧甲基纤维素、淀粉、七叶灵和吐温80,不能水解卵磷脂、明胶、尿素以及酪蛋白。甲基萘醌-7(MK-7)为该菌主要的呼吸醌。其主要的脂肪酸组分为C_(16 : 0)(38.3 %), C_(16 : 1)ω7c和/或iso-C_(15 : 0) 2-OH(29.0 %), C_(18 : 1)ω7c(9.3 %)和C_(10 : 0) 3-OH(8.2 %)。主要的极性脂为磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)和氨基磷酸酯(PN)。该菌DNA的G+C含量为44.8 %。YM0~T与所有已经报道的细菌16S rDNA序列相似性都低于91 %,构建的系统进化树显示,YM01~T属γ-变形菌纲的交替单胞菌科,并形成了一个单独的分支类群。根据该菌在表型特征、化学分类特征和系统发生分析上所表现出来的特殊性,认定该菌为γ-变形菌纲中的一个新属新种,命名为嗜琼胶卵链菌(Catenovulum agarivorans gen. nov. sp. nov.)。
从中国南极中山站沿岸潮间带中分离到一株耐低温(0-30℃)、中度喜盐(7-8 % w/v, NaCl)的专性异养细菌,实验室编号为ZS2-28~T。基于16S rDNA的系统发生分析表明,ZS2-28~T属于α-变形菌纲Roseibaca, Roseinatronobacter和Rhodobaca属细菌分类群中一个单独的进化支,与其他已知种属细菌的相似度均低于97 %,为一株潜在的海洋新菌。该菌能够产生菌绿素-a,胞内含有聚β-羟基丁酸脂颗粒,胞外附着有分泌物。主要的细胞壁脂肪酸组分为C_(18 : 1)ω7c。主要的呼吸醌为泛醌10。主要的极性脂包括磷脂酰甘油、磷脂酰乙醇胺、磷脂酰胆碱和一种无法鉴定的氨基脂。基因组DNA G+C含量为63.3 mol%。根据表型、化学分类特征以及系统发生分析的结果,确定ZS2-28~T代表了一个新属新种。命名为南极红柠檬菌(Roseicitreum antarcticum gen. nov. sp. nov)。
此外,从人类唾液样品中分离出一株产可溶性棕色色素的细菌,实验室编号为MY15~T。基于16S rDNA序列的系统发生分析表明,该菌株与海洋类香味菌(Myroides marinus JS-08~T)、M. odoratimimus LMG 4092~T和深海类香味菌(M. profundi D25T)亲缘关系最近,序列相似性分别为96.5 %, 96.3 %和96.1 %。该菌革兰氏染色呈阴性,细胞杆状,不具有鞭毛,不能运动。不能产生Flexirubin类色素。MY15~T以甲基萘醌-6为主要的呼吸醌,C_(15:0) iso (51.2%), C_(17:0) iso 3-OH (12.9%)和C_(13:0) iso (10.5%)为主要的细胞壁脂肪酸。DNA G+C含量为34.3 mol%。根据该菌在遗传进化与表型特征上与类香味菌属其他细菌所表现出来的差异性,认定MY15T为类香味菌属的一个新种,命名为棕色类香味菌(Myroides phaeus gen. nov. sp. nov)。
本文在对嗜琼胶卵链菌YM01~T分类学研究的基础上,对其琼胶水解酶进行了进一步研究,发现YM01~T能够产生耐高温、耐蛋白酶K的琼胶酶,在100℃和90℃分别孵育120 min和240 min后仍能保持33.3 %和44.4 %的活性。此外,利用简并PCR和hiTail-PCR法克隆了一个琼胶酶基因。该基因编码781个氨基酸,理论分子量为89 kDa, SingalP分析其N-端51个氨基酸为信号肽序列,结构域分析表明该酶属于糖苷水解酶42家族。
As the development of society and economy, terrestrial resources have been more and more exhausted. People turned their attention to the oceans which accounted for 70 percent of the total area of the earth for the further development of human beings. The exploration and utilization of marine resources become a theme of the national strategic development. The oceans are the biggest habitat for various organisms, especially the marine microorganisms which contains a great number of species.They play an important role in the ocean exploration and utilization for its unique metabolic mechanism formed under extreme environments. Therefore, it is valuable both theoretically and practically to analyse the ecological, physiological and genetic characteristics of marine microbes, as well as the bioactive substances produced by them. On the basis of the previous research about the diversity of bacteria, we studied the marine microbial resources from two aspects, the taxonomy of novel bacteria and the analysis of their bioactive products.
A novel Gram-negative, strictly aerobic, agar-hydrolyzing bacterium designated YM01T isolated from the Yellow Sea of coastal region of Qingdao was studied using polyphasic approach. Cells were rod-shaped, peritrichously flagellated and formed long chains by end-to-end. This isolate absolutely required Na~+ ions but not sea water for growth. YM01~T was able to hydrolyze cellulose, starch, aesculin and Tween 80, but not egg yolk, gelatin, urea and casein. It contained menaquinone 7 as the predominant isoprenoid quinone and C_(16 : 0) (38.3 %), C_(16 : 1)ω7c and/or iso-C_(15 : 0) 2-OH (29.0 %), C_(18 : 1)ω7c (9.3 %) and C_(10 : 0) 3-OH (8.2 %) as the major cellular fatty acids. Phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and aminophospholipid (PN) were the major constituents of the phospholipids. The DNA G+C content was 44.8 mol%. 16S rDNA sequence analysis demonstrated that this isolate was unique, showing only <91.0 % sequence similarities to all published species and formed a distinct monophyletic clade closely related to the species of the family Alteromonadaceae within the group of Alteromonas-like Gammaproteobacteria. So, strain YM01T was considered to represent a novel genus and species in the Gammaproteobacteria, for which the name Catenovulum agarivorans gen. nov. sp. nov. is proposed.
Another psychrotolerant (growth occurs at 0–33℃) , moderately halophilic (optimal NaCl concentration of 7–8 %) and obligately heterotrophic marine bacterium, designated ZS2-25 28~T, was isolated from intertidal sediment samples collected from the coastal regions of the Chinese Antarctic Zhongshan Station on the Larsemann Hills. phylogenetic analysis based on 16S rDNA sequences indicated that strain ZS2-28~T formed a distinct evolutionary lineage within the clade of genera Roseibaca, Roseinatronobacter and Rhodobaca of the Alphaproteobacteria with only <91.0 % sequence similarities to those neighbors. This isolate contained bacteriochlorophyll a. Poly-β-hydroxy butyrate accumulation and slime production can be observed. It contained Q-10 as the predominant isoprenoid quinone and C_(18 : 1)ω7c as the major cellular fatty acids. The main polar lipids were phosphatidylglycerol, phospatidylethanolamine, phosphatidylcholine and an unidentified aminolipid. The G+C content of genomic DNA was 63.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain ZS2-28~T was considered to represent a novel genus and species, for which the name Roseicitreum antarcticum gen. nov. sp. nov. is proposed.
A novel bacterial strain, designated MY15~T, was isolated from human saliva. Phylogenetic analyses based on 16S rDNA gene sequences showed that the novel strain was most closely related to Myroides marinus JS-08~T, Myroides odoratimimus LMG 4092~T and Myroides profundi D25~T with 96.5 %, 96.3 % and 96.1 % sequence similarities, respectively. Cells were Gram-straining-negative, rod-shaped, lack flagella and non-motile. Flexirubin-type pigment is absent. This isolate contained menaquinone 6 as the major respiratory quinone, and C_(15:0) iso (51.2 %), C_(17:0) iso 3-OH (12.9 %) and C_(13:0) iso (10.5 %) as the dominant fatty acids. The G+C content of the DNA was 34.3 mol%. On the basis of this polyphasic study, strain MY15~T represents a novel species of the genus Myroides, for which the name Myroides phaeus sp. nov. is proposed.
On the basis of taxonomic study of Catenovulum agarivorans YM01~T, further research about the agar-hydrolyzing enzymes was performed. The results showed that this isolate produced a high temperature- and proteinase K- resistant agarase. After treatment at 100 and 90℃for 120 and 240 min respectively, the agarase remained the agar-hydrolyzing capacity for 33.3 and 44.4 %. In addition, one of the genes for the expression of agarase, aga A, was cloned by using degenerate PCR and hiTail-PCR. Aga A contained 781 amino acids and the theoretical molecular weight was 89 kDa. According to the result of SignalP analysis, aga A contained a 51 aa signal peptide. The conserved domain analysis revealed that aga A is a novel agarase belonging to the family glycoside hydrolase-42.
引文
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