Department of Biochemistry, Tokyo Dental College, Chiba City, Japan.
J Oral Microbiol. 2011 Apr 14;3. doi: 10.3402/jom.v3i0.5495.
Streptococcus mutans surface-protein antigen (SpaP, PAc, or antigen I/II) has been well known to play an important role in initial attachment to tooth surfaces. However, strains with weak SpaP-expression were recently reported to be found in natural populations of S. mutans. The S. mutans gbpC-negative strain Z1, which we previously isolated from saliva and plaque samples, apparently expresses relatively low levels of SpaP protein compared to S. mutans strains MT8148 or UA159.
To elucidate the mechanism for weak SpaP-expression in this strain, the spaP gene region in strain Z1 was amplified by polymerase chain reaction (PCR) and analyzed.
Allelic exchange mutants between strains Z1 and UA159 involving the spaP gene region were constructed. The SpaP protein expressed in the mutants was detected with Coomasie Brilliant Blue (CBB)-staining and Western blot analysis following SDS-PAGE.
The 4689 bp spaP gene coding sequence for Z1 appeared to be intact. In contrast, a 20 bp nucleotide sequence appeared to be deleted from the region immediately upstream from the Z1 spaP gene when compared to the same region in UA159. The 216 bp and 237 bp intergenic fragments upstream from the spaP gene, respectively, from Z1 and UA159 were isolated, modified, and transformed into the other strain by allelic replacement. The resultant UA159-promoter region-mutant exhibited extremely weak SpaP-expression similar to that of strain Z1 and the Z1 complemented mutant expressed Spa protein levels like that of strain UA159.
These results suggest that weak SpaP-expression in strain Z1 resulted from a 20 bp-deletion in the spaP gene promoter region.
变异链球菌表面蛋白抗原（SpaP、PAc 或抗原 I/II）已被证实对初始黏附于牙面起重要作用。然而，最近有报道称，天然变异链球菌群体中存在 SpaP 表达较弱的菌株。我们之前从唾液和牙菌斑样本中分离出的变异链球菌 gbpC 阴性株 Z1，与 MT8148 或 UA159 等菌株相比，显然表达相对较低水平的 SpaP 蛋白。
阐明该菌株 SpaP 表达较弱的机制，通过聚合酶链反应（PCR）扩增 Z1 株的 spaP 基因区，并进行分析。
构建 Z1 株与 UA159 株之间 spaP 基因区的等位基因交换突变株。通过 Coomasie Brilliant Blue（CBB）染色和 SDS-PAGE 后的 Western blot 分析，检测突变株中表达的 SpaP 蛋白。
Z1 的 4689 bp spaP 基因编码序列似乎完整。相比之下，与 UA159 相同区域相比，Z1 spaP 基因上游的 20 个核苷酸序列似乎缺失。分别从 Z1 和 UA159 的 spaP 基因上游的 216 bp 和 237 bp 基因间片段被分离、修饰，并通过等位基因替换转化到另一个菌株中。结果，UA159 启动子区域突变株的 SpaP 表达非常弱，类似于 Z1 株，而 Z1 互补突变株的 Spa 蛋白表达水平与 UA159 株相似。
这些结果表明，Z1 株的 SpaP 表达较弱是由于 spaP 基因启动子区域的 20 个碱基缺失所致。
