Wanda S Y, Camilli A, Murchison H M, Curtiss R
Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
J Bacteriol. 1994 Dec;176(23):7206-12. doi: 10.1128/jb.176.23.7206-7212.1994.
An inhibitor of Streptococcus sobrinus endodextranase was detected in the extracellular fractions of UAB66 mutants identified following ethyl methanesulfonate mutagenesis as either devoid of dextranase activity (Dex-) or overproducing water-soluble glucan. The two groups of mutants had the same phenotype and displayed no dextranase activity in assays of extracellular fractions (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 34:1044-1055, 1981) and had been shown to be defective in adherence (Adh-) and capable of inhibiting adherence of wild-type strains during cocultivation in vitro (H. Murchison, S. Larrimore, and R. Curtiss III, Infect. Immun. 50:826-832, 1985) and in vivo in gnotobiotic rats (K. Takada, T. Shiota, R. Curtiss III, and S. M. Michalek, Infect. Immun. 50:833-843, 1985). By analysis of proteins in Western blots (immunoblots) and following blue dextran-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE), it was demonstrated that these Dex- mutants did synthesize enzymatically active dextranase. From the results of mixing experiments, it was determined that these Dex- Adh- mutants produced enhanced amounts of a cell surface-localized or a cell-associated dextranase inhibitor (Dei). Dei was heat stable but trypsin sensitive. By adding excess dextranase following BD-SDS-PAGE, Dei was detected as blue bands with apparent molecular masses of 43, 40, 37, 27, and 23 kDa. Dei competitively inhibits dextranase activity and is synthesized by wild-type S. sobrinus strains, with the amount varying depending upon growth medium and stage in the growth cycle. R. M. Hamelik and M. M. McCabe (Biochem. Biophys. Res. Commun. 106:875-880, 1982) previously described a Dei in a wild-type S. sobrinus strain.
在经甲磺酸乙酯诱变后鉴定出的UAB66突变体的细胞外组分中，检测到一种嗜酸性链球菌内切葡聚糖酶抑制剂，这些突变体要么缺乏葡聚糖酶活性（Dex-），要么过量产生水溶性葡聚糖。这两组突变体具有相同的表型，在细胞外组分检测中均未显示葡聚糖酶活性（H. 默奇森、S. 拉里莫尔和R. 柯蒂斯三世，《感染与免疫》34:1044 - 1055，1981年），并且已被证明在黏附方面存在缺陷（Adh-），在体外共培养期间能够抑制野生型菌株的黏附（H. 默奇森、S. 拉里莫尔和R. 柯蒂斯三世，《感染与免疫》50:826 - 832，1985年）以及在无菌大鼠体内（K. 高田、T. 椎田、R. 柯蒂斯三世和S. M. 米哈莱克，《感染与免疫》50:833 - 843，1985年）。通过蛋白质印迹（免疫印迹）分析以及蓝色葡聚糖 - 十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（BD - SDS - PAGE），证明这些Dex-突变体确实合成了具有酶活性的葡聚糖酶。从混合实验结果可知，这些Dex- Adh-突变体产生了大量细胞表面定位或细胞相关的葡聚糖酶抑制剂（Dei）。Dei热稳定但对胰蛋白酶敏感。在BD - SDS - PAGE后加入过量葡聚糖酶，Dei被检测为表观分子量分别为43、40、37、27和23 kDa的蓝色条带。Dei竞争性抑制葡聚糖酶活性，野生型嗜酸性链球菌菌株也能合成Dei，其产量因生长培养基和生长周期阶段而异。R. M. 哈梅利克和M. M. 麦凯布（《生物化学与生物物理研究通讯》106:875 - 880，1982年）之前在一株野生型嗜酸性链球菌菌株中描述过一种Dei。
