海洋琼胶降解细菌的多样性研究与Agarivorans albus QM38 β-琼胶酶基因的克隆与表达
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摘要
对青岛近海海水中琼胶降解细菌进行了系统的分离,筛选得到87株海洋琼胶降解细菌。从中选出15株有代表性的菌株,克隆其16SrDNA序列,进行分子鉴定。结果表明,这些细菌主要分布在Cellulophaga, Cytophaga, Microbulbifer, Glaciecola, Pseudoalteromonas, Pseudomonas, Alteromonas和Agarivorans共8个属中。其中QM5, QM21, QM36鉴定为Cellulophaga lytica , QM15和QM28鉴定为Glaciecola mesophila, QM11, QM35, QM47, QM65分别鉴定为Cytophaga fucicola, Pseudoalteromonas atlantica, Pseudoalteromonas haloplanktis, Alteromonas addita。其余5株菌能够鉴定到属的水平。
对细菌QM42和DH166的鉴定结果表明,这两株细菌都是革兰氏阴性的杆状细菌,生长需要NaCl,说明它们都是海洋细菌。QM42细胞宽度为0.6-0.7μm,长度为1.5-2.5μm,不产生芽孢。它能够产生氨肽酶、氧化酶和接触酶,并且能够利用下列糖类产酸:葡萄糖、木糖、乳糖和纤维二糖。细菌DH166是从东海中部海区50米深度的水样中分离到的一株海洋细菌。其生长对于营养条件的要求比较苛刻,培养基中没有丰富的有机物,它不能够生长。在1%和6%的NaCl含量的条件下都不能生长。对这两株细菌进行了脂肪酸分析和16SrDNA序列分析。初步鉴定QM42属于一个潜在的新种,而且可能是一个新属,与Marinobacter,Microbulbifer和Alcanivorax等属相近。细菌DH166属于Pseudoalteromonas属的一个潜在的新种。
对细菌QM38进行了进一步的研究。通过形态观察,生理生化特征鉴定以及16SrDNA序列分析,鉴定细菌QM38为Agarivorans albus。初步研究了细菌QM38琼胶酶摇瓶发酵的条件,对其产生的琼胶酶粗酶液进行了分析,研究了底物浓度和pH对酶活力的影响并分析了其酶解产物。
根据已经发表的其他细菌的琼胶酶的基因序列设计引物,从Agarivorans albus QM38的基因组DNA中克隆得到了三条编码琼胶酶的基因agaD01、agaD02和agaD03,并进行了测序。其中两条基因,agaD02和agaD03在Agarivorans属的细菌中是首次被克隆和测序。agaD01和agaD02在GenBank中的收录号分别为EF051475和EF199908。序列分析的结果表明,在GenBank数据库中,共有三条序列与agaD01有相似性,分别是来自弧菌JT0107编码β-琼胶酶的agaA基因,序列号为D14721,相似性有97.8%;Agarivorans sp. JA-1编码β-琼胶酶的基因,序列号为EF100136,相似性有98.9%;Agarivorans sp. JAMB-A11编码琼胶酶agaA11的基因,序列号为EF100136,相似性有98.2%。和其他序列则基本上没有相似性。与agaD02有相似性的只有一条序列,就是弧菌JT0107编码β-琼胶酶的agaB基因,序列号为D21202。这两条基因相似性有98.8%。在GenBank数据库中,只有两条序列与agaD03有相似性,一条是弧菌PO-303编码β-琼胶酶agarase-c的基因,序列号为AB218419,这两条基因相似性有96.8%。另外一条是Pseudoalteromonas sp. CY24编码胞外琼胶酶的基因,序列号为AY293310,这两条基因相似性有96.5%。但是agaD03和数据库中的其他基因基本上没有任何的同源性。
对agaD01和agaD02编码的蛋白质产物进行了生物信息学分析,结果显示,对这两个蛋白及其同源蛋白的结构和功能的研究很少,如果能进行结构与功能的研究,得到其催化特性,将能够获得许多新的数据与材料。
设计带有酶切位点的引物扩增细菌QM38的agaD02基因,产物克隆到载体pMD19-T Simple,经过酶切及测序验证后,将此基因亚克隆到原核表达载体pET24a,构建表达载体pET24a- agaD02,转化大肠杆菌BL21,提取质粒,双酶切进行验证。利用IPTG诱导表达,通过对诱导表达后的菌落进行碘液染色,发现有透明区域,证明表达成功而且有琼胶酶分泌到细胞外。
利用硫酸铵沉淀、分子筛层析和离子交换层析等方法,从Agarivorans albus QM38的培养液上清中分离纯化出一个琼胶酶组分。经SDS-PAGE电泳显示为单一条带,测定其分子量为44.2kD。根据其分子量推断,此琼胶酶组分可能是agaD03基因的编码产物。
A systematic screening for agar-degrading bacteria from the seawater samples yielded 87 isolates with promising agarolytic activity, among which 15 strains were selected for further analysis. To investigate the phylogenetic position of these strains,the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. A molecular phylogenetic dendrogram was constructed based on genetic distance analysis.Sequence comparison allowed to classify the strains as members of genera Cellulophaga, Cytophaga, Microbulbifer, Glaciecola, Pseudoalteromonas, Pseudomonas, Alteromonas and Agarivorans. Strains QM5, QM21 and QM36 were identified to be Cellulophaga lytica. Strains QM15 and QM28 were identified to be Glaciecola mesophila. Strains QM11, QM35, QM47 and QM65 were identified to be Cytophaga fucicola, Pseudoalteromonas atlantica, Pseudoalteromonas haloplanktis and Alteromonas addita, respectively.
Two strains, QM42 and DH166, were isolated from coastal seawater of Qingdao and the East China Sea, respectively. Both of them are gram negative marine bacteria. They can’t be cultured without NaCl supplement. Strain QM42 was positive in tests of catalase, oxidase, aminopeptidase and urease, but negative in the test of Voges Proskauer. This organism could grow and ferment glucose, xylose, lactose, and cellobiose. Strain DH166 was positive in tests of catalase, oxidase, gelatinase and lecithinase, but negative in the test of Voges Proskauer. This organism could not be cultured with 1% and 6% NaCl supplement. The major fatty acids detected in strain QM42 were 16:1w7c/15 iso 2-OH (29.11 %), 16:0 (19.53%), 18:1 w7c (19.62%), 18:0(10.78%), 12:0(7.56%), 12:0 2OH(2.31%), 12:0 3OH(3.27%), 10:0 3OH(3.53%), 14:0(1.89%) and 17:0(1.09%). The major fatty acids detected in strain DH166 were 16:1w7c/15 iso 2-OH (11.57 %), 16:0 (9.26%), 18:1 w7c (4.27%), 15:0(10.77%), 12:0 3OH(2.26%), 11:0 3OH (6.60%), 15:1 w8c (13.69%), 17:1 w8c (21.19%), 17:1 w6c (1.94%) and 17:0 (8.61%). Comparative 16S rRNA gene sequence analysis revealed that strain DH166 shared approximately 95.8% sequence similarity with members of the genus Pseudoalteromonas. But the partial sequences of the 16SrDNA of QM42 only showed similarities of about 90% to different members of the genus Microbulbifer. Further phylogenetic analysis of the 16S rRNA gene sequence showed that strain DH166 formed a separate branch within the genera Pseudoalteromonas. Thus strain DH166 maybe represent a potential novel species in the genera.The strain QM42 could be differentiated from its closest phylogenetic neighbours on the basis of several phenotypic features. It probably be a member of a new species and may be a member of a new genus in theγ-proteobacteria.
The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. This strain was gram negative, obligately aerobic, curved rod and polarly flagellated. On the basis of several phenotypic characters, biochemical and morphological characters and a phylogenetic analysis of the gene coding for the 16S rRNA, this strain was identified as Agarivorans albus strain QM38. An extracellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40℃, pH 7.6. Its activity was greatly affected by different concentration of agarose. The hydrolysis products were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE).
Threeβ-agarase genes, agaD01, agaD02 and agaD03, were amplified by PCR using chromosomal DNA of strain QM38 as a template. Two of them, agaD02 and agaD03, were first described in genus Agarivorans. The accession numbers for agaD02 and agaD03 in GenBank were EF051475 and EF199908, respectively. Homology searches were carried out with a BLAST program at the National Center for Biotechnology Information (NCBI) website. The nucleotide sequences of the agaD01, agaD02 and agaD03 gene were compared with entries in the GenBank database. The agaD01 gene were 97.8% and 98.9% identical to the two agarase genes from vibrio sp. JT0107 and Agarivorans sp. JA-1, and 98.2% with agaA11 gene from agarivorans sp. JAMB-A11. The agaD02 gene showed 98.8% sequence identify to the agaB gene from vibrio sp. JT0107. The agaD03 gene had a identify of 96.8% and 96.5% with two agarase genes from vibrio sp. PO-303 and Pseudoalteromonas sp.
CY24. The deduced amino acid sequences of the agaD01 and agaD02 genes were compared with entries in the DDBJ database. The proteins sequences were analyzed by molecular software tools.
According to the sequence of agaD02 gene, a pair of primers were respectively designed and synthesized. After amplification with PCR, the product was cloned into pMD19-T simple vector using TA cloning. The recombinants were sequenced and identified by restrictive endonuclease digestion. The target sequences were then subcloned into a highly efficient eukaryotic expression vector pET24a. The expression vectors were identified by double restrictive endonuclease digestion and then were transformed into E. coli BL21(DE3).The recombinant E. coli BL21 (pET24a-agaD02) were induced by IPTG on Petri dish and were dyed with Lugol iodide solution after culture for 24h at 37℃. A clear Zone was observed around the colony of the E. coli. This is to certificate that the pET24a-agaD02 eukaryotic expression vectors are successfully constructed.
An extracellularβ-agarase was purified to electrophoretic homogeneity by ammonium sulfate precipitation and successive liquid chromatographies. The molecular mass of the enzyme was determined to be 44.2 kDa by SDS-PAGE. Thisβ-agarase might be encoded by the agaD03 gene.
对细菌QM42和DH166的鉴定结果表明,这两株细菌都是革兰氏阴性的杆状细菌,生长需要NaCl,说明它们都是海洋细菌。QM42细胞宽度为0.6-0.7μm,长度为1.5-2.5μm,不产生芽孢。它能够产生氨肽酶、氧化酶和接触酶,并且能够利用下列糖类产酸:葡萄糖、木糖、乳糖和纤维二糖。细菌DH166是从东海中部海区50米深度的水样中分离到的一株海洋细菌。其生长对于营养条件的要求比较苛刻,培养基中没有丰富的有机物,它不能够生长。在1%和6%的NaCl含量的条件下都不能生长。对这两株细菌进行了脂肪酸分析和16SrDNA序列分析。初步鉴定QM42属于一个潜在的新种,而且可能是一个新属,与Marinobacter,Microbulbifer和Alcanivorax等属相近。细菌DH166属于Pseudoalteromonas属的一个潜在的新种。
对细菌QM38进行了进一步的研究。通过形态观察,生理生化特征鉴定以及16SrDNA序列分析,鉴定细菌QM38为Agarivorans albus。初步研究了细菌QM38琼胶酶摇瓶发酵的条件,对其产生的琼胶酶粗酶液进行了分析,研究了底物浓度和pH对酶活力的影响并分析了其酶解产物。
根据已经发表的其他细菌的琼胶酶的基因序列设计引物,从Agarivorans albus QM38的基因组DNA中克隆得到了三条编码琼胶酶的基因agaD01、agaD02和agaD03,并进行了测序。其中两条基因,agaD02和agaD03在Agarivorans属的细菌中是首次被克隆和测序。agaD01和agaD02在GenBank中的收录号分别为EF051475和EF199908。序列分析的结果表明,在GenBank数据库中,共有三条序列与agaD01有相似性,分别是来自弧菌JT0107编码β-琼胶酶的agaA基因,序列号为D14721,相似性有97.8%;Agarivorans sp. JA-1编码β-琼胶酶的基因,序列号为EF100136,相似性有98.9%;Agarivorans sp. JAMB-A11编码琼胶酶agaA11的基因,序列号为EF100136,相似性有98.2%。和其他序列则基本上没有相似性。与agaD02有相似性的只有一条序列,就是弧菌JT0107编码β-琼胶酶的agaB基因,序列号为D21202。这两条基因相似性有98.8%。在GenBank数据库中,只有两条序列与agaD03有相似性,一条是弧菌PO-303编码β-琼胶酶agarase-c的基因,序列号为AB218419,这两条基因相似性有96.8%。另外一条是Pseudoalteromonas sp. CY24编码胞外琼胶酶的基因,序列号为AY293310,这两条基因相似性有96.5%。但是agaD03和数据库中的其他基因基本上没有任何的同源性。
对agaD01和agaD02编码的蛋白质产物进行了生物信息学分析,结果显示,对这两个蛋白及其同源蛋白的结构和功能的研究很少,如果能进行结构与功能的研究,得到其催化特性,将能够获得许多新的数据与材料。
设计带有酶切位点的引物扩增细菌QM38的agaD02基因,产物克隆到载体pMD19-T Simple,经过酶切及测序验证后,将此基因亚克隆到原核表达载体pET24a,构建表达载体pET24a- agaD02,转化大肠杆菌BL21,提取质粒,双酶切进行验证。利用IPTG诱导表达,通过对诱导表达后的菌落进行碘液染色,发现有透明区域,证明表达成功而且有琼胶酶分泌到细胞外。
利用硫酸铵沉淀、分子筛层析和离子交换层析等方法,从Agarivorans albus QM38的培养液上清中分离纯化出一个琼胶酶组分。经SDS-PAGE电泳显示为单一条带,测定其分子量为44.2kD。根据其分子量推断,此琼胶酶组分可能是agaD03基因的编码产物。
A systematic screening for agar-degrading bacteria from the seawater samples yielded 87 isolates with promising agarolytic activity, among which 15 strains were selected for further analysis. To investigate the phylogenetic position of these strains,the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. A molecular phylogenetic dendrogram was constructed based on genetic distance analysis.Sequence comparison allowed to classify the strains as members of genera Cellulophaga, Cytophaga, Microbulbifer, Glaciecola, Pseudoalteromonas, Pseudomonas, Alteromonas and Agarivorans. Strains QM5, QM21 and QM36 were identified to be Cellulophaga lytica. Strains QM15 and QM28 were identified to be Glaciecola mesophila. Strains QM11, QM35, QM47 and QM65 were identified to be Cytophaga fucicola, Pseudoalteromonas atlantica, Pseudoalteromonas haloplanktis and Alteromonas addita, respectively.
Two strains, QM42 and DH166, were isolated from coastal seawater of Qingdao and the East China Sea, respectively. Both of them are gram negative marine bacteria. They can’t be cultured without NaCl supplement. Strain QM42 was positive in tests of catalase, oxidase, aminopeptidase and urease, but negative in the test of Voges Proskauer. This organism could grow and ferment glucose, xylose, lactose, and cellobiose. Strain DH166 was positive in tests of catalase, oxidase, gelatinase and lecithinase, but negative in the test of Voges Proskauer. This organism could not be cultured with 1% and 6% NaCl supplement. The major fatty acids detected in strain QM42 were 16:1w7c/15 iso 2-OH (29.11 %), 16:0 (19.53%), 18:1 w7c (19.62%), 18:0(10.78%), 12:0(7.56%), 12:0 2OH(2.31%), 12:0 3OH(3.27%), 10:0 3OH(3.53%), 14:0(1.89%) and 17:0(1.09%). The major fatty acids detected in strain DH166 were 16:1w7c/15 iso 2-OH (11.57 %), 16:0 (9.26%), 18:1 w7c (4.27%), 15:0(10.77%), 12:0 3OH(2.26%), 11:0 3OH (6.60%), 15:1 w8c (13.69%), 17:1 w8c (21.19%), 17:1 w6c (1.94%) and 17:0 (8.61%). Comparative 16S rRNA gene sequence analysis revealed that strain DH166 shared approximately 95.8% sequence similarity with members of the genus Pseudoalteromonas. But the partial sequences of the 16SrDNA of QM42 only showed similarities of about 90% to different members of the genus Microbulbifer. Further phylogenetic analysis of the 16S rRNA gene sequence showed that strain DH166 formed a separate branch within the genera Pseudoalteromonas. Thus strain DH166 maybe represent a potential novel species in the genera.The strain QM42 could be differentiated from its closest phylogenetic neighbours on the basis of several phenotypic features. It probably be a member of a new species and may be a member of a new genus in theγ-proteobacteria.
The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. This strain was gram negative, obligately aerobic, curved rod and polarly flagellated. On the basis of several phenotypic characters, biochemical and morphological characters and a phylogenetic analysis of the gene coding for the 16S rRNA, this strain was identified as Agarivorans albus strain QM38. An extracellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40℃, pH 7.6. Its activity was greatly affected by different concentration of agarose. The hydrolysis products were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE).
Threeβ-agarase genes, agaD01, agaD02 and agaD03, were amplified by PCR using chromosomal DNA of strain QM38 as a template. Two of them, agaD02 and agaD03, were first described in genus Agarivorans. The accession numbers for agaD02 and agaD03 in GenBank were EF051475 and EF199908, respectively. Homology searches were carried out with a BLAST program at the National Center for Biotechnology Information (NCBI) website. The nucleotide sequences of the agaD01, agaD02 and agaD03 gene were compared with entries in the GenBank database. The agaD01 gene were 97.8% and 98.9% identical to the two agarase genes from vibrio sp. JT0107 and Agarivorans sp. JA-1, and 98.2% with agaA11 gene from agarivorans sp. JAMB-A11. The agaD02 gene showed 98.8% sequence identify to the agaB gene from vibrio sp. JT0107. The agaD03 gene had a identify of 96.8% and 96.5% with two agarase genes from vibrio sp. PO-303 and Pseudoalteromonas sp.
CY24. The deduced amino acid sequences of the agaD01 and agaD02 genes were compared with entries in the DDBJ database. The proteins sequences were analyzed by molecular software tools.
According to the sequence of agaD02 gene, a pair of primers were respectively designed and synthesized. After amplification with PCR, the product was cloned into pMD19-T simple vector using TA cloning. The recombinants were sequenced and identified by restrictive endonuclease digestion. The target sequences were then subcloned into a highly efficient eukaryotic expression vector pET24a. The expression vectors were identified by double restrictive endonuclease digestion and then were transformed into E. coli BL21(DE3).The recombinant E. coli BL21 (pET24a-agaD02) were induced by IPTG on Petri dish and were dyed with Lugol iodide solution after culture for 24h at 37℃. A clear Zone was observed around the colony of the E. coli. This is to certificate that the pET24a-agaD02 eukaryotic expression vectors are successfully constructed.
An extracellularβ-agarase was purified to electrophoretic homogeneity by ammonium sulfate precipitation and successive liquid chromatographies. The molecular mass of the enzyme was determined to be 44.2 kDa by SDS-PAGE. Thisβ-agarase might be encoded by the agaD03 gene.
引文
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