Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300؇ on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.Endospores of the gram-positive bacterium Bacillus subtilis are encased in a thick protein shell known as the coat (2). The coat is composed of 15 or more polypeptides arranged in an electron-dense outer layer and a lamellar inner layer. These layers protect the spore from bactericidal enzymes and chemicals, such as lysozyme and chloroform. So far, the genes for 13 of these coat proteins have been identified. These are located at diverse positions on the chromosome and code for polypeptides of 65 (CotA), 59 (CotB), 10 (CotC), 9 (CotD), 24 (CotE), 19 (CotF), 24 (CotG and CotJ), 41 (CotS), 10 (CotT), 19 (CotX), 26 (CotY), and 18 (CotZ) kDa (1,3,6,7,10,18,22,24). Certain proteins, such as CotD, are located in the inner coat, and others, such as CotA, CotB, CotC, and CotG, are located in the outer coat (18, 24). The coat is produced at a relatively late stage in the process of sporulation, when the developing spore (or forespore) is present as a free protoplast within the mother cell compartment of the sporangium (14). Coat proteins are organized on the outer surface of the membrane surrounding the forespore by the sporulation protein SpoIVA (8,17,21). SpoIVA controls the assembly of a ring of CotE proteins around the forespore (8). The CotE ring is thought to regulate the assembly of the proteins of the outer coat and is separated from the outer surface of the forespore membrane by a small gap, which is believed to be the site at which the inner coat will be assembled (8). The production of coat proteins is governed by a regulatory cascade of four transcription factors acting in the mother cell compartment of the sporangium in the sequence E , SpoIIID, K , and GerE (26). E and K are RNA polymerase sigma factors, whereas SpoIIID and GerE are DNA-binding proteins that act in conjunction with E -and K -containing forms of RNA polymerase, respectively (4,9,13,23,25).We report the identification of a gene, herein called cotH, located at 300Њ on the B. subtilis chromosomal map, where it is clustered with two previously described cot genes, cotB and cotG (7,18). cotH codes for a 42.8-kDa protein, apparently located in the inner laye...

Spores formed by wild-type Bacillus subtilis are encased in a multilayered protein structure (called the coat) formed by the ordered assembly of over 30 polypeptides. One polypeptide (CotB) is a surface-exposed coat component that has been used as a vehicle for the display of heterologous antigens at the spore surface. The cotB gene was initially identified by reverse genetics as encoding an abundant coat component. cotB is predicted to code for a 43-kDa polypeptide, but the form that prevails in the spore coat has a molecular mass of about 66 kDa (herein designated CotB-66). Here we show that in good agreement with its predicted size, expression of cotB in Escherichia coli results in the accumulation of a 46-kDa protein (CotB-46). Expression of cotB in sporulating cells of B. subtilis also results in a 46-kDa polypeptide which appears to be rapidly converted into CotB-66. These results suggest that soon after synthesis, CotB undergoes a posttranslational modification. Assembly of CotB-66 has been shown to depend on expression of both the cotH and cotG loci. We found that CotB-46 is the predominant form found in extracts prepared from sporulating cells or in spore coat preparations of cotH or cotG mutants. Therefore, both cotH and cotG are required for the efficient conversion of CotB-46 into CotB-66 but are dispensable for the association of CotB-46 with the spore coat. We also show that CotG does not accumulate in sporulating cells of a cotH mutant, suggesting that CotH (or a CotH-controlled factor) stabilizes the otherwise unstable CotG. Thus, the need for CotH for formation of CotB-66 results in part from its role in the stabilization of CotG. We also found that CotB-46 is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, using a yeast two-hybrid system, we found evidence that CotB directly interacts with CotG and that both CotB and CotG self-interact. We suggest that an interaction between CotG and CotB is required for the formation of CotB-66, which may represent a multimeric form of CotB.During the process of sporulation in the gram-positive soil bacterium Bacillus subtilis the developing spore is encased in a complex protein structure called the coat, which confers resistance to several physicochemical agents and contributes to the response of spores to the presence of germinants (7,8,15). The coat is formed by over 30 polypeptides, ranging in size from about 6 to about 70 kDa, which are assembled into a lamellar inner coat and a thick electron-dense outer coat (7,8,15). With only one possible exception (38), synthesis of the coat structural components is restricted to the mother cell chamber of the sporulating cell and is temporally governed by a cascade of transcription factors in the order E , SpoIIID, K , and GerE (7,8,15,24,35,40).E and SpoIIID drive synthesis of a class of morphogenetic proteins that (irrespective of their association with the final coat structure) appear to guide the assembly of several structural components into the spore coat (reviewed i...