Characterization of cotJ, a sE-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores

The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination. The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition. Here we report the cloning and characterization of an operon, cotJ, located at about 62 & on the B. subtilis genetic map, whose inactivation results in the production of spores with an altered pattern of coat polypeptides. The cotJ operon was identified by screening a random library of lacZ transcriptional fusions for a conditional (inducer-dependent) Lac1 phenotype in cells of a strain in which the structural gene (spoIIGB) for the early-acting, mother-cell-specific transcriptional factor sE was placed under the control of the IPTG (isopropyl-b-D-thiogalactopyranoside)-inducible Pspac promoter. Sequence analysis of cloned DNA from the cotJ region complemented by genetic experiments revealed a tricistronic operon preceded by a strong sE-like promoter. Expression of an SPb-borne cotJ-lacZ fusion commences at around h 2 of sporulation, as does expression of other sE-dependent genes, and shows an absolute requirement for sE. Studies with double-reporter strains bearing a cotJ-gusA fusion and lacZ fusions to other cot genes confirmed that expression of cotJ is initiated during sporulation prior to activation of genes known to encode coat structural proteins (with the sole exception of cotE). An in vitro-constructe