Interaction of sugar metabolism with antibiotic susceptibility in Staphylococcus aureus

我们研究了葡萄球菌中糖代谢与万古霉素和甲氧西林敏感性的相互作用，并有以下几个发现：1）PTS突变体的构建和这些突变体的表征：我们发现了可能的17个PTS系统，例如葡萄糖和果糖的特异性PTS，因此我们构建了17个PTS突变体。2）ccpA基因突变体的构建及其特性研究：构建了ccpA基因突变体，该突变体参与葡萄糖代谢的整体调控。CcpA突变体降低了对甲氧西林的抗性水平。3）GlmS-ribozyme的分析：为了了解glmS-mRNA中核酶活性的关键区域，我们构建了不同长度的含有启动子区的DNA片段，并将这些片段克隆到一个载体中，用于XylE报告基因的分析。结果，负责核酶活性的区域是glmS-orf上游约300个区域。为了评价核酶的活性，我们建立了实时PCR方法，发现添加N-乙酰葡萄糖胺可以在特定位点切割glmS-mRNA，同时也消化glmS-mRNA本身。4）添加各种糖的表达谱的微阵列分析：我们分析了S.金黄色葡萄球菌通过添加各种糖，并发现特定的PTS为每种糖。此外，我们还评估了受每种糖影响的特定基因。

We investigated the interaction of sugar metabolism with the susceptibility to vancomycin and methicillin in Staphylococcus aureus, and had several findings described below.1) Construction of PTS mutants and characterization of these mutants : We found possible 17 PTS systems such as specific PTS for glucose and for fructose, so we constructed 17 PTS mutants. We measured the susceptibility to antibiotis, and found no significant difference between the mutant and the parent.2) Construction of ccpA-mutants and its characterization: We constructed the mutant of ccpA, which is involved in global regulation of glucose metabolism. CcpA-mutant reduced the resistance level to methicillin. From the microarray analysis, we found the genes regulated by CcpA.3) Analysis of GlmS-ribozyme: To know the critical regions for ribozyme activity in glmS-mRNA, we constructed the various length of DNA fragment containing promoter region, and cloned these fragments in one vector for XylE-reporter analysis. As a result, the region responsible for ribozyme activity is about 300 by region upstream of glmS-orf. To evaluate the ribozyme activity, we set up the method using real-Time PCR, and found that glmS-mRNA was cleaved in specific sites by additon of N-acetylglucosamine, and also digested glmS-mRNA itself.4) Microarray analysis of the expression profile by addition of various kinds of sugars: We analyzed the expression of all genes in S. aureus by addition of various kinds of sugars, and found specific PTS for each sugar. Also, we evaluated the specific genes affected by each sugar.