The methyl-specific restriction system of Streptomyces coelicolor A3(2) was analyzed by carrying out transformations with unmethylated and methylated pSET152 DNA. Streptomyces coelicolor was found to strongly restrict DNA methylated in vivo by the Dam, Dcm and Hsd modification systems of Escherichia coli. Hsd-modified DNA was restricted as strongly as Dam-modified DNA, even though there are significantly fewer sites on the plasmid; Dcm-modified plasmid was restricted more strongly then either Dam- or Hsd-modified DNA. Restriction of plasmid DNA modified in vitro by different methylases also showed a greater dependence on the methylated sequence than on the number of methylated sites. Streptomyces coelicolor mutants were constructed that lacked genes identified as the likely candidates for encoding methyl-specific restriction nucleases (the products of the SCO4213, SCO4631 and SCO2863 genes, as well as the SCO3261-SCO3262 operon) that are located in the laterally acquired genomic islands of the S. coelicolor chromosome; these mutants showed partial alleviation of methylated DNA restriction. Cloning of these genes in the close relative Streptomyces lividans increased the restriction of methylated DNA by this species, confirming their role as part of the methyl-specific restriction system of S. coelicolor.

No abstract

No abstract

A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.