Studies On DNA Methylation In Model Streptomycetes

5-methylcytosine is one of the most common DNA methylation.Dcm methylatransferase encoded by dcm gene in E. coli K12can convertinternal cytosine in the sequence of5′CCWGG3′to5-methylcytosine(W=A/T). Although Dcm modification was discovered37years ago, itsbiological role remains unclear. According to the recent study,5-methylation is related with cancer in eukaryptes; it has been postulatedto be a component of a type II restriction-modification system that canrestrict exogenous DNA in prokaryotes; one latest obervation with respectto Dcm role in prokaryotes is that the synthesis of ribosome andsecondary metabolites can be affected by its expression.Streptomyces coelicolor and Streptomyces lividans are two modelstreptomycestes strains whose genome sequence have over99%identitybut they differ strikingly in the restriction of alien DNA from Escherichiacoli. There is no clear evidence regarding the methylation patterns ofthese two closely related strains. Using the HPLC-MS, this study detectedboth methylcytosine and6-methyladenine in S. coelicolor but nonemethylation in the general host S. lividans (lacking the SLG genomicisland that encodes the DNA sulfur modification). According to the preicted DNA methyltransferases of S. coelicolor in the Rebase(http://tools.neb.com/~vincze/genomes/view.php?view_id=214), wecloned all six methyltansferase genes (sco3104, sco3510, sco5331,sco6627, sco6844and sco6885) individually in an integrative plasmidpIB139and expressed in S. lividans HXY16. LC-MS/MS analysisdetected that dmC in strain HXY16::sco6627, and6mA in strainHXY16::sco6885. This finding is contradicted with the prediction that S.coelicolor doesn’t bear5mC as it contains multiplemethylcytosine-dependent restriction systems. This contradiction impliescomplicated regulation of the methylation and restriction system in S.coelicolor.Although the genomic sequence of Streptomyces coelicolor has over99%identity to that of S. lividans, they are siginificantly different in thephysiological differentiation. Are these differences related to the DNAmethylation? Given the uncertainty of the Dcm role, we introduced dcminto S. lividans to see the epigentic effect of Dcm expression by takingfull advantage of its complicated morphologicaland physiologicaldifferentiation. We obeserved two types of exoconjugants with respect tothe colony size upon the introduction of the dcm gene. Restrictivedigestion revealed that Dcm recognition site in the small colony ismethylated and protected from digestion but not in the big colony. PCRand Southern Blot confirmed that dcm gene is deleted together with the vector pPM927in big colonies. On the other hand, the silent gene clusterof actionrhodin in both agar plates and liquid media has been activated insmall colonies. The combined results of morphological study andactionrhodin detection reveal that dcm gene has a pleiotropic effects onthe growth of Streptomyces lividans.