A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ς ^K and GerE during Development and Is Located in the Inner Coat Layer of Spores

Abstract

The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerEmutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotEmutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores.

Assembly of the CotSA coat protein into spores requires CotS in Bacillus subtilis

“…We obtained B. subtilis 168 (trpC2), 1G12 (leu-2, gerE36), 1S101 (trpC2, cotA: :cat), 1S102 (trpC2, cotB: :cat), 1S103 (trpC2, cotC: :cat), 1S104 (trpC2, cotD: :cat), 1S105 (trpC2, cotE: :cat), 1S106 (trpC2, cotF: :cat), and 1S108 (trpC2, cotT: :cat) from the Bacillus genetic stock center (Ohio State University) and used them in this study. Construction of the B. subtilis cotS mutant, CB701, has been described previously [7]. The B. subtilis strains were all grown in DS medium [12] and when necessary, tetracycline was added to a ¢nal concentration of 20 mg ml 3I .…”

“…The B. subtilis strains were all grown in DS medium [12] and when necessary, tetracycline was added to a ¢nal concentration of 20 mg ml 3I . Spore coat gene defective mutants and the gerE mutant of B. subtilis have been described in the previous paper [7]. Escherichia coli JM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, V3, v(lac-proAB), [FP, traD36, proAB, lacI q ZvM15]) was grown in an LB medium.…”

“…Whole proteins from sporulating cells and coat proteins were prepared as described previously [7]. Protein samples were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) (12.0% acrylamide) as described previously [16].…”

“…For immunoblotting, proteins were transferred onto a polyvinyl di£uoridine membrane (Immobilon; 0.45 mm pore size; Millipore), and detected by using rabbit immunoglobulin G (IgG) against CotSA, CotS, SigA, or SigK as the ¢rst antibody and donkey anti-rabbit IgG-horseradish peroxidase conjugate as the second antibody (Amersham). Antibodies against CotSA or CotS were prepared using recombinant proteins as described previously [7]. Anti-SigA and anti-SigK antibodies were provided by M. Fujita and Y. Sadaie (National Institute of Genetics, Mishima, Japan).…”

“…We cloned the cotS operon [10], which consists of cotSA (named ytxN in the B. subtilis genome project [11]), cotS, and ytxO in order. The CotS protein, encoded by the cotS gene, is synthesized under the regulation of SigK and GerE and is located within the spore coat, in particular in the inner coat of dormant spores of B. subtilis, as shown by immunoelectron microscopic observation [7]. Assuming that CotS and CotSA regulate assembly of the counterpart into coat layer and vice versa, in this paper CotSA was puri¢ed and we examined the e¡ect of CotS and CotSA on their assembly into the spore coat of B. subtilis during cell development.…”