Bacillus subtilis spore coat assembly requires cotH gene expression

Abstract

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...

Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis

“…Class 1 groups 28 genes, including cotH and the gerP operon (Behravan et al, 2000;Naclerio et al, 1996), whose expression is induced by 210 min and peaks at 270 min. Expression of the s K -dependent gerP operon is repressed by GerE (Behravan et al, 2000), and expression of cotH does not require GerE, although it is not presently known whether the expression of cotH is repressed by GerE or not (Naclerio et al, 1996). Class 2 groups 72 genes whose expression is also induced by 210 min, but continues to rise during the duration of the experiment, at least until 330 min (Fig.…”

GerE-independent expression of cotH leads to CotC accumulation in the mother cell compartment during Bacillus subtilis sporulation

“…Additionally, CotH, in conjunction with CotE, is required for efficient spore germination (Naclerio et al, 1996). Recent studies have shown that the previously observed role of CotH in the assembly of the outer coat components CotC, CotG and CotB (Naclerio et al, 1996) is to stabilize CotC (Isticato et al, 2004) and CotG, which in turn is needed for dimerization and assembly of CotB (Zilhao et al, 2004).…”

“…In contrast, the outer coat proteins are assembled on the outside of the CotE structure (Henriques & Moran, 2000;Driks, 2002). Additional proteins with morphogenetic functions are needed for coat formation: SpoVID, produced under s E control, has the dual role of directing SafA to the forming spore and maintaining the CotE ring around the forespore (Henriques & Moran, 2000;Ozin et al, 2001;Driks, 2002); CotH, produced under s K control, plays a role in the assembly of various outer coat components and in the development of the lysozyme resistance typical of the mature spore (Naclerio et al, 1996;Zilhao et al, 1999). Additionally, CotH, in conjunction with CotE, is required for efficient spore germination (Naclerio et al, 1996).…”

“…Additional proteins with morphogenetic functions are needed for coat formation: SpoVID, produced under s E control, has the dual role of directing SafA to the forming spore and maintaining the CotE ring around the forespore (Henriques & Moran, 2000;Ozin et al, 2001;Driks, 2002); CotH, produced under s K control, plays a role in the assembly of various outer coat components and in the development of the lysozyme resistance typical of the mature spore (Naclerio et al, 1996;Zilhao et al, 1999). Additionally, CotH, in conjunction with CotE, is required for efficient spore germination (Naclerio et al, 1996). Recent studies have shown that the previously observed role of CotH in the assembly of the outer coat components CotC, CotG and CotB (Naclerio et al, 1996) is to stabilize CotC (Isticato et al, 2004) and CotG, which in turn is needed for dimerization and assembly of CotB (Zilhao et al, 2004).…”

cse15, cse60, and csk22 are new members of mother-cell-specific sporulation regulons in Bacillus subtilis

“…Conversion of pro-E to its active form, E , which occurs exclusively in the mother cell, inaugurates the program of gene expression in this compartment (19,35,44,68). The E form of RNA polymerase (E E ) directs the transcription of a heterogeneous group of genes controlling functions as diverse as engulfment (23,56,58,62), germination (49,70), cortex synthesis (2,9,14,43,52,54,60,64), and coat morphogenesis (3,18,28,49,54,60,70), as well as genes that may assist in the control of the metabolic state of the mother cell (7,8,26,37). E E also drives expression of at least two regulatory genes, spoIIID (40,62,68) and sigK (39,68).…”