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° 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.

Crystal or inclusion Source Other features CP p Under location coat Bacillus cereus T B. cereus T. germinated B. cereus, SO3 treated B. cereus, extracted with DTE B. cereus, extracted with DTE-SDS B. cereus 10LD (lysozymedependent germination) B. cereus 13LS (lysozyme sensitive) B. cereus 10ts (temp-sensitive GSH reductase)a B. thuringiensis var. alesti B. cereus var. fowler B. thuringiensis var. finitimus B. popilliae

The Bacilus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 1070 between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30%o reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.

A polyester embedding technique was used to study the early stages of spore formation in members of the genus Bacillus in order to investigate further the origin and nature of the initial spore septum and the resulting forespore envelope. Whereas previously, with a methacrylate procedure, this layer had appeared to be continuous with the cell wall, this study reveals it as a double layer of cytoplasmic membrane. Perisporal, membranous organelles connected both to the developing forspore envelope and to the cytoplasmic membrane were encountered in the four species studied. Similar organelles were prominent during growth at the sites of transverse septa formation. These were connected to, or continuous with, the cytoplasmic membrane and often adherent to the chromatin bodies of the dividing bacilli.

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