Molecular cloning and characterization of a cluster of genes involved in the spore coat morphogenesis of Bacillus subtilis

Jianke Zhang, Purdue University

Abstract

In response to nutrient limitation Bacillus subtilis initiates a developmental program to form a highly resistant and dormant endospore. In the last stages of sporulation, a thick coat is formed outside the developing spore. The Bacillus subtilis spore coat is arranged in three morphological layers and composed of twelve to fifteen polypeptides plus an insoluble protein fraction. 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 HPLC. This peptide was sequenced and its gene designated cotZ was cloned by reverse genetics. The cotZ gene was clustered with four other genes designated cotV, cotW, cotX, and cotY. These genes were mapped to 107$\sp\circ$ between thiB and metA on the B. subtilis chromosome. Deletion of the cotZ gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotZ, cotXY, cotZXY genes were heat- and lysozyme-resistant but readily clumped indicating surface hydrophobicity changes. There was a less densely staining outer coat in spores produced by the cotZ mutant and the spores of the cotZXY triple mutant had an incomplete outer coat. These results indicate localization of these proteins close to the spore surface. In addition, these spores responded to B. subtilis germinants more rapidly than did those produced by the parental strain. The deduced sequence of the CotX and CotY proteins were very similar and both were rich in cysteine. CotX is cross-linked by disulfide bonds on spore coat. The cotVWZXY gene cluster is organized in two multicistronic operons as well as two overlapping monocistronic transcription units. Transcription by $\sigma\sp{\rm K}$-RNA polymerase of at least one of these genes involves mother cell-specific DNA binding protein GerE. This complex coregulation implies that these polypeptides all function as part of the coat insoluble fraction and that each is synthesized according to requirements for correct assembly of the coat.

Degree

Ph.D.

Advisors

Aronson, Purdue University.

Subject Area

Molecular biology|Microbiology|Biochemistry

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