Cloning And Analysis Of The Vibrio Harveyi Dam Gene

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.