Molecular Analysis of Multiple Forms of the Streptococcus mutans Dextranase

用分子遗传学的方法对变形链球菌葡聚糖酶的多种形态进行了分子分析。为了找出致龋菌葡聚糖酶(Dex)的共同特征，测定了唐氏链球菌葡聚糖酶基因的全核苷酸序列，并与变形链球菌和远缘链霉菌进行了比较。同源性分析表明，Dex基因由一个保守区和两个可变区组成。此外，在地塞米松活性染色中还检测到三种地塞米松具有多种活性形式。因此，我们推测，保守区域对Dex活性是必不可少的，N-末端和C-末端的可变区是Dex的多种形式的原因。在此基础上，我们通过遗传操作在变形链球菌Dex的两个可变区构建了突变体，并检测了对Dex的多种形式和活性的影响。C末端可变区内的突变体表现出具有Dex活性的多种形式。同时缺失C末端可变区和部分保守区的突变体，不仅失去了多种形式，而且失去了活性。另一方面，在具有Dex活性的C端突变体的N端可变区内的突变体并没有表现出多种形式和活性。这些结果表明，DEX的多种形式是导致C末端可变区降解的原因，并表明保守区包含活性和底物结合位点等催化部位。

Molecular analysis of multiple forms of the Streptococcus mutans dextranase was studied by the approach of molecular genetics. In order to find out common characteristics of dextrnase (Dex) of cariogenic bacteria, complete nucleotide sequence of the S. downei dextranase gene was determined and compared with those of S. mutans and S. sobrinus. Homology analysis showed that the Dex was constituted by a conserved region and two variable regions. In addition, Dex of three species was detected as active multiple forms in Dex active staining. We, therefore, speculated that the conserved region was essential for Dex activity and the variable regions of N- and C-terminals were responsible for the multiple forms of the Dex.. On the basis of this speculation, we constructed mutants at two variable regions on the S. mutans Dex by genetic manipulation and examined influence to the multiple forms and activity of the Dex. Mutants within a C-terminal variable region showed the multiple forms with the Dex activity. Mutants defective both the C-terminal variable region and a part of the conserved regions disappeared not only the multiple forms but also the activity. On the other hand, mutants within a N-terminal variable region of the C-terminal mutant with Dex activity did not show both the multiple forms and the activity. These results indicated that the multiple forms of the Dex was responsible for degradation of the variable region of C-terminus and suggested that the conserved region would contain the catalytic sites such as active and substrate binding sites.