哈氏弧菌dam基因的克隆与分析
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摘要
DNA甲基化在生物界是一种普遍机制,几乎存在于所有生命体中。目前已发现的甲基化皆由DNA甲基转移酶(DNA methyltransferase)所介导,该酶能够利用S-腺苷甲硫氨酸为底物,将其甲基转移至腺嘌呤(A)的N6或胞嘧啶(C)的C5或N4位置上。DNA甲基化最早是作为限制-修饰系统的一个组成部分而被发现的,该系统是生物在长期进化过程中形成的一种自我保护机制,其中甲基转移酶所起的作用是甲基修饰某些特定序列中的腺嘌呤或胞嘧啶,以此特征将自身DNA与异源DNA区别开来。随后的研究发现一些独立存在的甲基转移酶,其功能与限制-修饰无关。DNA腺嘌呤甲基化酶(DNA adenine methylase,Dam)即是这种类型的酶之一。Dam广泛存在于细菌中,如大肠杆菌,沙门氏杆菌,耶尔森氏菌,奈瑟菌,以及霍乱弧菌等。与限制-修饰系统中的甲基化酶不同,Dam参与细胞生理代谢过程中的许多重要方面,如DNA复制,基因表达,甲基化介导的错配修复,换位等。近年来研究表明在某些人类病原菌中Dam与细菌致病力密切相关,其基因敲除导致细菌毒力大幅下降。基于其在细胞生命过程中所起的多种作用,dam基因的研究将有助于加深我们对细菌生命机制的理解。
弧菌是海洋环境中最常见的细菌类群之一,且大量存在于各种水生环境中。弧菌科多种属是人类或养殖动物病原性细菌,其中哈氏弧菌(Vibrio harveyi)能够感染多种养殖水生生物(虾、鱼、贝等),是危害我国水产养殖业发展的重要病原菌之一。本实验所用哈氏弧菌T4为我们从患病大菱鲆肠道内分离出的一株病原菌。(1)T4基因组DNA经提取纯化后分别用DpnI、DpnII和Sau3AI酶切,酶切结果经0.8%琼胶糖凝胶电泳后发现T4 DNA对DpnI和Sau3AI敏感,而对DpnII则具有抗性,说明该菌中存在具功能活性的DNA腺嘌呤甲基化酶,在其作用下T4染色体DNA处于被甲基化状态。(2)利用兼并PCR,染色体步移法的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。(3)运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。(4)对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bp DNA片段皆具有启动子活性,但前者的活性为后二者的3.3倍。(5)通过多种手段构建T4 dam缺失菌株,来研究dam与T4致病性的关系,但没有成功,表明dam对T4的生存可能是必需的。
本文报道了哈氏弧菌dam基因的克隆、dam基因启动子活性的定位及分析,为深入了解Dam在哈氏弧菌中的作用奠定基础。
DNA methylation occurs throughout the living world, including bacteria, plants, and mammals. DNA methylation occurs at the C-5 or N-4 position of cytosine and at the N-6 position of adenine using S-adenosyl methionine as a methyl donor catalyzed by enzymes known as DNA methyltransferases (Mtases).DNA methylation has historically been associated with DNA restriction-modification systems thought to be an important mechanism of self- protection in the long process of evolution. Restriction-modification systems contain a DNA methylase that distinguishes host DNA from foreign DNA by methylating adenine or cytosine of specific sequences. Subsequent research found some MTases that are not associated with restriction-modification systems, such as DNA adenine methylase (Dam), which methylates N-6 of adenine in GATC sequences. DNA adenine methylase (Dam) is widespread in bacterias, including Escherichia coli, Salmonella spp., Yersinia spp., Neisseria spp. , and Vibrio cholerae。Dam participates in many important physiological processes such as DNA replication, gene expression, methyl-directed mismatch repair and transposition。More recently it has become evident that Dam plays an important role in the pathogenesis of several bacterial species, and deletion of the dam gene significantly attenuates the virulence of these pathogens. Because Dam plays muLtiple important roles in cellular physiology, the research on the dam gene will help us to understand the mechanism of bacterial life.
The vibrios are among the most common bacteria in marine environments and highly abundant in aquatic environments, including estuaries, marine coastal waters and sediments, and aquacuLture setting worldwide. Some species are serious pathogens for human or animals reared in aquacuLture. Of these, V.harveyi is an important bacterial pathogen of multiple maricultured animals ,such as shellfish, shrimp, fish,and seriously inhibits the development of maricuLture in our country.The Vibrio harveyi T4 we used in this report is a bacterial pathogen isolated from the intestine of the sicken turbot.
After extraction, T4 genomic DNA was digest with DpnI, DpnII, Sau3AI. The 0.8% electrophoreses result indicated that T4 genomic DNA is sensitive to DpnI and Sau3AI,but can not be cleaved by DpnII,and this demonstrates that there is a functional DNA adenine methylase (Dam) which methylates the chromosome DNA at GATC site in T4.
The dam gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V.parahaemolyticus (95% in identity). The V.harveyi dam gene was subcloned into plasmid pBR322 and introduced into the E.coli strain ER2925 in which the dam gene had been knocked out. DpnI, DpnII, and Sau3AI restriction enzyme analysis of the genomic DNA of ER2925 transformed with V.harveyi dam indicated that the cloned V.harveyi dam gene couLd functionally complement the E. coli dam and methylate E.coli chromosome at the GATC sites.
The 3251 bp upstream flanking region of V.harveyi dam was obtained by genome walking and analyzed at the DNA and amino acid sequence levels. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length (ORF1101) and the other was of 1503 bp in length (ORF1503). The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V.parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V.parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with V.parahaemolyticus shikimate kinase. These three genes, together with dam, were arranged in the order of shikimate kinase - 3-dehydroquinate synthase– damX–dam..
The immediate upstream region of the V.harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named as P78, P112,and P477, respectively) and tested for promoter activity which showed that , while all the three fragments had detectable promoter activities, there was a marked difference among them: the activity of P78 was more than 3 times higher than that of P112 and P477.
Different genetic strategies to obtain the dam knockout mutant of T4 were futile, indicating that the dam gene may be essential for the viability of the bacterium.
弧菌是海洋环境中最常见的细菌类群之一,且大量存在于各种水生环境中。弧菌科多种属是人类或养殖动物病原性细菌,其中哈氏弧菌(Vibrio harveyi)能够感染多种养殖水生生物(虾、鱼、贝等),是危害我国水产养殖业发展的重要病原菌之一。本实验所用哈氏弧菌T4为我们从患病大菱鲆肠道内分离出的一株病原菌。(1)T4基因组DNA经提取纯化后分别用DpnI、DpnII和Sau3AI酶切,酶切结果经0.8%琼胶糖凝胶电泳后发现T4 DNA对DpnI和Sau3AI敏感,而对DpnII则具有抗性,说明该菌中存在具功能活性的DNA腺嘌呤甲基化酶,在其作用下T4染色体DNA处于被甲基化状态。(2)利用兼并PCR,染色体步移法的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。(3)运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。(4)对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bp DNA片段皆具有启动子活性,但前者的活性为后二者的3.3倍。(5)通过多种手段构建T4 dam缺失菌株,来研究dam与T4致病性的关系,但没有成功,表明dam对T4的生存可能是必需的。
本文报道了哈氏弧菌dam基因的克隆、dam基因启动子活性的定位及分析,为深入了解Dam在哈氏弧菌中的作用奠定基础。
DNA methylation occurs throughout the living world, including bacteria, plants, and mammals. DNA methylation occurs at the C-5 or N-4 position of cytosine and at the N-6 position of adenine using S-adenosyl methionine as a methyl donor catalyzed by enzymes known as DNA methyltransferases (Mtases).DNA methylation has historically been associated with DNA restriction-modification systems thought to be an important mechanism of self- protection in the long process of evolution. Restriction-modification systems contain a DNA methylase that distinguishes host DNA from foreign DNA by methylating adenine or cytosine of specific sequences. Subsequent research found some MTases that are not associated with restriction-modification systems, such as DNA adenine methylase (Dam), which methylates N-6 of adenine in GATC sequences. DNA adenine methylase (Dam) is widespread in bacterias, including Escherichia coli, Salmonella spp., Yersinia spp., Neisseria spp. , and Vibrio cholerae。Dam participates in many important physiological processes such as DNA replication, gene expression, methyl-directed mismatch repair and transposition。More recently it has become evident that Dam plays an important role in the pathogenesis of several bacterial species, and deletion of the dam gene significantly attenuates the virulence of these pathogens. Because Dam plays muLtiple important roles in cellular physiology, the research on the dam gene will help us to understand the mechanism of bacterial life.
The vibrios are among the most common bacteria in marine environments and highly abundant in aquatic environments, including estuaries, marine coastal waters and sediments, and aquacuLture setting worldwide. Some species are serious pathogens for human or animals reared in aquacuLture. Of these, V.harveyi is an important bacterial pathogen of multiple maricultured animals ,such as shellfish, shrimp, fish,and seriously inhibits the development of maricuLture in our country.The Vibrio harveyi T4 we used in this report is a bacterial pathogen isolated from the intestine of the sicken turbot.
After extraction, T4 genomic DNA was digest with DpnI, DpnII, Sau3AI. The 0.8% electrophoreses result indicated that T4 genomic DNA is sensitive to DpnI and Sau3AI,but can not be cleaved by DpnII,and this demonstrates that there is a functional DNA adenine methylase (Dam) which methylates the chromosome DNA at GATC site in T4.
The dam gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V.parahaemolyticus (95% in identity). The V.harveyi dam gene was subcloned into plasmid pBR322 and introduced into the E.coli strain ER2925 in which the dam gene had been knocked out. DpnI, DpnII, and Sau3AI restriction enzyme analysis of the genomic DNA of ER2925 transformed with V.harveyi dam indicated that the cloned V.harveyi dam gene couLd functionally complement the E. coli dam and methylate E.coli chromosome at the GATC sites.
The 3251 bp upstream flanking region of V.harveyi dam was obtained by genome walking and analyzed at the DNA and amino acid sequence levels. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length (ORF1101) and the other was of 1503 bp in length (ORF1503). The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V.parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V.parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with V.parahaemolyticus shikimate kinase. These three genes, together with dam, were arranged in the order of shikimate kinase - 3-dehydroquinate synthase– damX–dam..
The immediate upstream region of the V.harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named as P78, P112,and P477, respectively) and tested for promoter activity which showed that , while all the three fragments had detectable promoter activities, there was a marked difference among them: the activity of P78 was more than 3 times higher than that of P112 and P477.
Different genetic strategies to obtain the dam knockout mutant of T4 were futile, indicating that the dam gene may be essential for the viability of the bacterium.
引文
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