Singh S N, Srivastava R, Sinha V B, Srivastava B S
Division of Microbial Genetics, Central Drug Research Institute, Lucknow, India.
Microb Pathog. 1994 Aug;17(2):69-78. doi: 10.1006/mpat.1994.1053.
Mutants of Vibrio cholerae 01 strain 0395 (classical) mutated in genes encoding secretory or cell surface proteins were induced by TnphoA mutagenesis and were selected as blue colonies on L-agar plates containing 5-bromo-4-chloro-3-indolyl phosphate. Southern analysis of the total DNA from blue colonies showed that all mutants had TnphoA insertion in genomic DNA. These mutants were analysed for adherence, colonization and protein profile. Adherence to freshly isolated rabbit intestinal discs was affected in some mutants. The less adhesive mutants were examined for colonization of the intestine of infant mice. One mutant, designated T-87, was extremely poor at colonization and less diarrhaegenic than the parent strain. Analysis of T-87 by SDS-PAGE revealed that two proteins of 53 and 38 kDa were lacking. The 38 kDa protein was identified as OmpU. The 53 kDa protein was extracellular and cells treated with anti-53-kDa antibodies could not colonize the gut of infant mice. The expression of the 53 and 38 kDa proteins in T-87 was dependent of the growth medium. The data suggest that T-87 is mutated in a regulatory gene which regulates the expression of proteins involved in intestinal colonization.
通过TnphoA诱变诱导霍乱弧菌01菌株0395（古典生物型）中编码分泌蛋白或细胞表面蛋白的基因突变体，并在含有5-溴-4-氯-3-吲哚磷酸的L-琼脂平板上选择为蓝色菌落。对蓝色菌落的总DNA进行Southern分析表明，所有突变体在基因组DNA中都有TnphoA插入。对这些突变体进行了黏附、定殖和蛋白质谱分析。一些突变体对新鲜分离的兔肠盘的黏附受到影响。对黏附性较差的突变体进行了幼鼠肠道定殖检测。一个命名为T-87的突变体在定殖方面极差，并且比亲本菌株的致泻性更低。通过SDS-PAGE对T-87进行分析发现，缺少两种分别为53 kDa和38 kDa的蛋白质。38 kDa的蛋白质被鉴定为OmpU。53 kDa的蛋白质是细胞外蛋白，用抗53 kDa抗体处理的细胞不能在幼鼠肠道定殖。T-87中53 kDa和38 kDa蛋白质的表达取决于生长培养基。数据表明，T-87在一个调控基因中发生突变，该调控基因调节参与肠道定殖的蛋白质的表达。
