Elucidation of a unique regulatory mechanism for scrA gene expression in S.mutans

我们研究了S.mutans蔗糖PTS编码酶ii的scrA基因的调控机制。scrA基因的表达不仅受到蔗糖的调控，还受到山梨醇的调控。为了阐明这一特征调控的分子机制，我们开展了两个项目：1)鉴定基于scrA和scrB基因的推定orf。2)山梨醇转运基因的克隆。scrA基因下游的orf分别被鉴定为编码果糖激酶和磷酸甘露糖异构酶的scrK和pmi基因。Northern分析显示，这些组成操纵子的基因是独立于scrA基因转录的。另一个位于scrB基因下游的ORF （ds2ORF3）不参与scrA或scrB调控，但有趣的是，它参与了应激诱导的葡聚糖依赖性聚集。虽然具体的聚集机制尚不清楚，但我们已经建立了一种克隆直接参与聚集的基因的方法。在克隆山梨醇转运基因的过程中，还克隆了另外两个基因。其中一个被鉴定为编码丙酮酸甲酸裂解酶的pfl基因，该酶是链球菌糖代谢的关键酶。另一个与假定的葡萄糖苷转移酶正调控基因相似。由于这些基因对s.a mutans的龋齿性很重要，因此它们必须在未来与ds2ORF3一起进行表征。我们可以鉴定出变形链球菌糖代谢的所有必需基因。虽然我们无法阐明这一时期独特的scrA表达机制，但研究已经扩展到意想不到的方向。

We have investigated regulatory mechanisms of the scrA gene encoding EnzymeII of sucrose PTS from S.mutans. Expression of the scrA gene was regulated not only by sucrose but also by sorbitol. In order to elucidate molecular mechanisms of this characteristic regulation, we launched two projects :1) Characterization of putative ORFs franking the scrA and scrB genes. 2) Cloning of sorbitol transport genes.ORFs downstream from the scrA gene were identified as the scrK and pmi genes encoding fructokinase and phosphomannose isomerase respectively. Northern analysis revealed that these genes consisting an operon were transcribed independently of the scrA gene. Another ORF (ds2ORF3) downstream from the scrB gene did not involve in scrA or scrB regulation but interestingly involved stress-induced glucan-dependent aggregation. Although the mechanism of the aggregation in detail remains to be resolved, we could have established a method to clone genes directly involved in the aggregation.During a process to clone sorbitol transport genes, two other genes have been cloned. One was identified as the pfl gene encoding pyruvate formate-lyase that is the key enzyme of sugar metabolism of streptococci. The other was similar to the putative positive regulator gene of S.gordonii glucosyltransferase. Since these genes should be important for cariogenisity of S.mutans, they have to be characterized in future together with ds2ORF3.We could identify all genes essential for sucrose metabolism of S.mutans. Although we could not elucidate mechanisms of the unique scrA expression within this period, study has expanded to unexpected derections.