枯草菌制限修飾系遺伝子BsuＭの分子遺伝学的解析

Many microorganisms prevent phage or plasmid infection by digesting its unmethylated DNA with restriction endonuclease, which recognizes specific target sequence on incoming DNA. In order to maintain the integrity of the chromosome, they also possess modification methylase that recognizes and methylates chromosomal DNA at the same target sequence as restriction endonuclease recognizes. Methylated chromosomal DNA is no more susceptible to restriction endonuclease. A set of genes of restriction endonuclease and modification methylase is called restriction modification (RM) system genes.Since the early investigation of the RM system by Luria, Arber, Messelson et al, lots of restriction modification systems have been found in various kinds of microorganisms and they were studied mainly on their enzymatic mechanisms.The research on RM system in Bacillus subtilis was started by Shibata and Ando in 1970\u27s. They revealed that B. subtilis Marburg strain 168 has only one RM system genes, named BsuM. The chromosomal location of the BsuM was determined by Saito et al, and mutant strain RM125 was isolated by Uozumi et al, which lacks both restriction and modification activities. Further, The recognition sequence of BsuM was determined by Bron et al, as CTCGAG. This sequence is the same as the recognition sequence of Xhol restriction enzyme. However, the sequence and components of BsuM genes and their products were remained unknown.In the genome sequencing project, Kasahara et al, revealed that B. subtilis Marburg 168 has only one orf, YdiS, the predicted product of which is homologous to restriction enzyme, and two orfs, ydiO and ydiP whose presumed products are homologous to the cytocine-specific DNA methytransferases. They were found in the prophage 3 region between groESL operon and gut operon, where classical BsuM mutations were located.In this study, PCR analysis revealed that the classical RM mutant strain RM125 is lacking the prophage 3 region (13kbp) containing five genes, ydiO, ydiP, ydiR, ydiS and ydjA.Disruption of ydiO or ydiP genes required disrupted ydiR, ydiS or ydjA. Interestingly, two novel genes, ydiR and ydjA, that locate upstream and downstream of the ydiS gene had to be disrupted to disrupt ydiO or ydiP.Northern analysis showed that the ydiO, ydiP, ydiR, ydiS and ydjA genes constitute two operons, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon, expressing during logarithmic phase of growth. The ydiO-ydiP operon was also transcribed by a read through mechanism from the groESL operon, which is the upstream operon of the ydiO-ydiP operon.The ratio of the transformation frequency of pHV1401 with 3 Xhol sites to that of pHV33 without Xhol site was several ten hold higher in the ydiR, ydiS or ydjA disruptants.The degree of methylation of the BsuM target sequence (CTCGAG) on the chromosomal DNA was estimated indirectly by susceptibility to Xhol (an isoschizomer to BsuM) digestion of DNA extracted from these disruptant strains. Six Xhol sites on the chromosome were examined. Each Xhol site was susceptible to Xhol digestion when DNA was extracted from the strain disrupted for both operons, while it was not susceptible when extracted from the wild type strain or the strain disrupted for the ydiR, ydiS or ydjA gene only.His-tagged YdiO was purified in a denatured form in E. coli, but its renatured form did not show methyltransferase activity. His-tagged YdiR protein could be produced in B. subtilis cell, and Ni column purified YdiR showed a weak endonuclease activity. This activity may be due to the presumed YdiR/YdiS/YdjA protein complex.Although biochemical evidences are not enough, these results show that ydiO and ydiP make an operon for DNA methylation, and ydiR, ydiS and ydjA make the other operon for DNA restriction, and these genes are component genes of restriction -modification system BsuM in B. subtilis Marburg. YdiR, YdiS and YdjA may make a protein complex, where only YdiS has a similarity to endonuclease. YdiO and YdiP methylate BsuM target sequence and YdiR/YdiS/YdjA digests unmethylated DNA at BsuM site. Smaller orfs in the prophage 3 region seemed not to be involved restriction and modification of B. subtilis.Prophage 3 region contains pseudogenes of integrase and terminase of bacteriophage and phosphomannomutase. These suggest that the RM system in B. subtilis Marburg originated from integration of a bacteriophage in a relatively recent period during evolution, and many genes, except RM system genes, may have been lost by accumulation of mutations