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J. Bacteriol. doi:10.1128/JB.01776-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mutations in rsmG, Encoding a 16S rRNA Methyltransferase, Result in Low-level Streptomycin Resistance and Antibiotic Overproduction in Streptomyces coelicolor A3(2)

National Food Research Institute, Tsukuba, Ibaraki 305-8642, and Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

^* To whom correspondence should be addressed. Email: kochi{at}affrc.go.jp.

	Abstract

Certain str mutations that confer high- or low-level streptomycin resistance result in overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type S. coelicolor resulted in acquisition of streptomycin resistance and overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the rsmG strain exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, HPLC analysis showed that the rsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of E. coli). Like certain rpsL mutants, the rsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the rsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in rsmG and rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1001,000-fold higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.

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