CLONING AND CHARACTERIZATION OF A CLUSTER OF GENES ENCODING POLYPEPTIDES PRESENT IN THE INSOLUBLE FRACTION OF THE SPORE COAT OF BACILLUS-SUBTILIS

The Bacillus subtilis spore coat is composed of at least 15 polypeptid es plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat prot ein and is resistant to solubilization by a variety of reagents, imply ing extensive cross-linking. A dodecapeptide was purified from this fr action by formic acid hydrolysis and reverse-phase high-performance li quid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodec apeptide at its amino end was clustered with four other genes designat ed cotV, cotW, cotY, and cotZ. These genes were mapped to 107-degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both prot eins are cysteine rich, and CotY antigen was present in spore coat ext racts as disulfide cross-linked multimers. There was little CotX antig en in the spore coat soluble fraction, and deletion of this gene resul ted in a 30% reduction in the spore coat insoluble fraction. Spores pr oduced by strains with deletions of the cotX, cotYZ, or cotXYZ genes w ere 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 p roduced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These prot eins, as part of the coat insoluble fraction, appear to be localized t o the outer coat and influence spore hydrophobicity as well as the acc essibility of germinants.