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Endospores of the Gram-positive bacterium Bacillus subtilis are encased in a tough protein shell known as the coat. The coat is composed of a dozen or more different structural proteins. We report the identification of and studies on the regulation of promoters governing the expression of coat protein (cot) genes designated B to E encoding polypeptides of 59, 12, 11 and 24 kDa, respectively. We show that transcription of genes B, C and D is governed by single promoters and that transcription of gene E is governed by tandem promoters designated P1 and P2. In extension of recent work on the transcription of cot gene A and the mother-cell regulatory genes gerE, sigK and spoIIID, we show that genes involved in coat formation are turned on in a regulatory cascade of at least four co-ordinately controlled gene sets. The cascade consists of: cotE as transcribed from its P1 promoter and spoIIID, which are turned on during hours three to four of sporulation; cotE as transcribed from its P2 promoter and sigK, which are turned on during hour five by the appearance of the product (a small DNA-binding protein) of spoIIID; cotA, cotD and gerE, which are turned on during hours five to six by the appearance of the product (sigma factor sigma K) of sigK; and cotB and cotC, which are turned on during hour seven by the appearance of the product (an inferred DNA-binding protein) of gerE. The cascade is hierarchical in that the first three gene sets each contain the regulatory gene that turns on the expression of the next gene set in the pathway. We also show that the level of expression of a member (cotC) of the terminal class of gene expression is strongly influenced by medium and that this effect directly or indirectly depends on the product of sporulation gene spoIV A.

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 degrees, 290 degrees, 168 degrees and 200 degrees, 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 characteristic 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.

Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is a morphogenic protein required in the assembly of the outer coat. The nucleotide sequence of cotE reveals an open reading frame capable of encoding a 181-residue-long polypeptide of 21 kD. A cotE mutant was created by replacing the chromosomal gene, which was located at 145 degrees on the chromosome, with an in vitro constructed, deletion-mutated gene. The resulting cotE mutant formed normal-looking (optically refractile) spores that were heat resistant but were sensitive to lysozyme and somewhat impaired in germination. Ultrastructural analysis indicated that the mutant spores lacked the electron-dense outer layer of the coat but retained a normal-looking inner coat. The mutant spores were pleiotropically deficient in several coat proteins, including the product of cotE and the products of previously cloned cot genes A-C. Based on experiments in which expression of the cotA and cotC genes was found to be unimpaired in cotE mutant cells, we infer that the cotE gene product is involved in the assembly of the products of cotA-cotC, and certain other proteins into the electron-dense outer layer of the coat.