Becker Petra, Hakenbeck Regine, Henrich Bernhard
Fachbereich Biologie, Abteilung Mikrobiologie, Technische Universität Kaiserslautern, P.O. Box 3049, 67653 Kaiserslautern, Germany.
Antimicrob Agents Chemother. 2009 May;53(5):2034-41. doi: 10.1128/AAC.01485-08. Epub 2009 Mar 9.
Vancoresmycin is a novel tetramic acid antibiotic, probably interfering with functions of the cytoplasmic membrane. To investigate its mode of action, mutants of Streptococcus pneumoniae exhibiting reduced susceptibility to vancoresmycin were isolated at a low frequency. Four of them were further examined and showed similar pleiotropic phenotypes, including reduced growth rate, early autolysis, and chain formation. In one mutant, the level of transcripts from a single locus encoding the potential ABC transporter Spr0812-Spr0813 was increased sixfold. The corresponding DNA sequence revealed a nonsense mutation (C1744T) in spr0813, leading to the formation of a truncated permease lacking 2 of the 10 predicted transmembrane helices. As demonstrated by deletion and transformation analysis and reconstructing the spr0813(C1744T) mutation in the wild-type background, this nucleotide exchange was sufficient to cause reduced susceptibility to vancoresmycin and higher amounts of spr0812-spr0813 mRNA. Mapping and reporter assays of the cognate promoter P(abc) showed that spr0812 and spr0813 are cotranscribed with a preceding small gene and that the spr0813(C1744T) mutation does not affect the activity of P(abc). Due to the similarity of Spr0812-Spr0813 to bacitracin transporters of Streptococcus mutans and Bacillus spp., the bacitracin susceptibility of spr0813 mutants was examined. Both the spr0813(C1744T) nonsense mutation and the deletion of the transporter genes led to a clearly increased sensitivity to bacitracin. Thus, the intact transporter is required for intrinsic resistance to bacitracin, whereas reduced vancoresmycin susceptibility is mediated by the truncated permease.
万古霉素是一种新型四胺酸抗生素，可能会干扰细胞质膜的功能。为了研究其作用模式，以低频率分离出了对万古霉素敏感性降低的肺炎链球菌突变体。对其中4个突变体进行了进一步检测，结果显示它们具有相似的多效性表型，包括生长速率降低、早期自溶和链形成。在一个突变体中，编码潜在ABC转运蛋白Spr0812 - Spr0813的单个基因座的转录本水平增加了6倍。相应的DNA序列显示spr0813中存在无义突变（C1744T），导致形成一种截短的通透酶，该通透酶缺少10个预测跨膜螺旋中的2个。通过缺失和转化分析以及在野生型背景中重建spr0813（C1744T）突变证明，这种核苷酸交换足以导致对万古霉素的敏感性降低以及spr0812 - spr0813 mRNA水平升高。同源启动子P（abc）的定位和报告基因检测表明，spr0812和spr0813与前面的一个小基因共转录，并且spr0813（C1744T）突变不影响P（abc）的活性。由于Spr0812 - Spr0813与变形链球菌和芽孢杆菌属的杆菌肽转运蛋白相似，因此检测了spr0813突变体对杆菌肽的敏感性。spr0813（C1744T）无义突变和转运蛋白基因的缺失均导致对杆菌肽的敏感性明显增加。因此，完整的转运蛋白是对杆菌肽固有抗性所必需的，而对万古霉素敏感性降低是由截短的通透酶介导的。
