Nishimura Kenji, Hosaka Takeshi, Tokuyama Shinji, Okamoto Susumu, Ochi Kozo
National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan.
J Bacteriol. 2007 May;189(10):3876-83. doi: 10.1128/JB.01776-06. Epub 2007 Mar 23.
Certain str mutations that confer high- or low-level streptomycin resistance result in the 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 Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the 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 strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG 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 the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.
某些赋予高水平或低水平链霉素抗性的str突变会导致链霉菌属过量产生抗生素。赋予高水平抗性的str突变发生在编码核糖体蛋白S12的rpsL内，而导致低水平抗性的突变则不太为人所知。我们利用比较基因组测序确定低水平抗性是由rsmG突变引起的，rsmG编码一种含有S-腺苷甲硫氨酸（SAM）结合基序的依赖SAM的16S rRNA甲基转移酶。从野生型天蓝色链霉菌中删除rsmG会导致获得链霉素抗性并过量产生抗生素放线紫红素。将野生型rsmG导入缺失突变体完全消除了rsmG缺失的影响，证实rsmG突变是观察到的表型的基础。与早期使用自发rsmG突变体的工作一致，携带DeltarsmG的菌株表现出增加的SAM合成酶活性，这介导了抗生素的过量产生。此外，高效液相色谱分析表明，DeltarsmG突变体在16S rRNA中缺乏7-甲基鸟苷修饰（可能在对应于大肠杆菌G527的G518位置）。与某些rpsL突变体一样，DeltarsmG突变体在生长后期表现出增强的蛋白质合成活性。然而，与rpsL突变体不同，DeltarsmG突变体既没有表现出70S核糖体复合物更高的稳定性，也没有表现出核糖体循环因子表达增加，这表明rsmG和rpsL突变体中蛋白质合成增加的机制不同。最后，自发rsmG突变出现的频率比rpsL突变高1000倍。这些发现为rRNA修饰在激活链霉菌次生代谢中的作用提供了新的见解。
