GENES ENCODING SPORE COAT POLYPEPTIDES FROM BACILLUS-SUBTILIS
GENES ENCODING SPORE COAT POLYPEPTIDES FROM BACILLUS-SUBTILIS
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DOI：
10.1016/0022-2836(87)90506-7
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发表时间：
1987-07-05
影响因子：
5.6
通讯作者：
LOSICK, R
中科院分区：
文献类型：
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作者：
DONOVAN, W;ZHENG, L;LOSICK, R
革兰氏阳性细菌枯草芽孢杆菌的内孢子被包裹在坚硬的蛋白质外壳中，称为外壳，由十几种或更多不同的多肽组成。我们克隆了编码Mr为65,000,59,000,12,000和11,000的孢子外壳蛋白的CotA， CotB， cotC和cotD结构基因。这些基因是用杂交探针克隆的，合成的寡核苷酸是在纯化的外壳蛋白部分nh2末端序列确定的基础上设计的。为了确定cot基因在染色体上的位置并研究它们的遗传功能，我们通过在每个基因的编码序列中插入一个氯霉素抗性决定因子（cat）来标记每个基因。然后，我们用相应的插入失活基因替换染色体上的每个野生型cot基因。遗传定位实验表明，cotA、cotB、cotC和cotD分别位于52°、290°。168.度。和200.度。，分别位于枯草芽孢杆菌染色体上。所有cot::cat插入突变体都不是Spo-，但发现cotD突变体的孢子萌发速度比野生型孢子慢一些，并且发现cotA突变体在野生型孢子细胞菌落的棕色色素特征的外观上被阻断。物理和基因实验证实，cotA与先前发现的一种名为pig的基因相同，这种基因已知负责孢子相关色素的产生。所有四个插入突变体的孢子都表现出野生型的外壳多肽模式，除了插入失活的cot基因的相应产物缺失。
Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell, known as the coat, that consists of a dozen or more different polypeptides. We have cloned structural genes designated CotA, CotB, cotC and cotD that encode spore coat proteins of Mr 65,000, 59,000, 12,000 and 11,000, respectively. These genes were cloned by using as hybridization probes synthetic oligonucleotides that were designed on the basis of partial NH2-terminal sequence determinations of the purified coat proteins. To determine the location of the cot genes on the chromosome and to study their function genetically, we tagged each gene by insertion of a chloramphenicol-resistance determinant (cat) within its coding sequence. We then replaced each wild-type cot gene in the chromosome with the corresponding, insertionally inactivated gene. Genetic mapping experiments showed that cotA, cotB, cotC and cotD were located at 52.degree., 290.degree. , 168.degree. and 200.degree. , respectively, on the B. subtilis chromosome. None of the cot::cat insertion mutants were Spo-, but spores of the cotD mutant were found to germinate somewhat more slowly than did wild-type spores, and the cotA mutant was found to be blocked in the appearance of the brown pigment characteristics of colonies of wild -type sporulating cells. Physical and genetic experiments established that cotA was identical to a previously identified gene called pig, known to be responsible for sporulation-associated pigment production. Spores from all four insertion mutants exhibited the wild-type pattern of coat polypeptides, except for the absence in each instance of the corresponding product of the cot gene that had been insertionally inactivated.