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CotG is an abundant protein initially identified as an outer component of the Bacillus subtilis spore coat. It has an unusual structure characterized by several repeats of positively charged amino acids that are probably the outcome of multiple rounds of gene elongation events in an ancestral minigene. CotG is not highly conserved, and its orthologues are present in only two Bacillus and two Geobacillus species. In B. subtilis , CotG is the target of extensive phosphorylation by a still unidentified enzyme and has a role in the assembly of some outer coat proteins. We report now that most spore-forming bacilli contain a protein not homologous to CotG of B. subtilis but sharing a central “modular” region defined by a pronounced positive charge and randomly coiled tandem repeats. Conservation of the structural features in most spore-forming bacilli suggests a relevant role for the CotG-like protein family in the structure and function of the bacterial endospore. To expand our knowledge on the role of CotG, we dissected the B. subtilis protein by constructing deletion mutants that express specific regions of the protein and observed that they have different roles in the assembly of other coat proteins and in spore germination. IMPORTANCE CotG of B. subtilis is not highly conserved in the Bacillus genus; however, a CotG-like protein with a modular structure and chemical features similar to those of CotG is common in spore-forming bacilli, at least when CotH is also present. The conservation of CotG-like features when CotH is present suggests that the two proteins act together and may have a relevant role in the structure and function of the bacterial endospore. Dissection of the modular composition of CotG of B. subtilis by constructing mutants that express only some of the modules has allowed a first characterization of CotG modules and will be the basis for a more detailed functional analysis.

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

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 σE, SpoIIID, σK, and GerE (7, 8, 15, 24, 35, 40). σE 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 in references 7, 8, and 15). For instance, spores produced by a cotE mutant fail to assemble the electron-dense outer coat and the remaining coat structure appears to lack, in addition to CotE, several other abundant components (47). The results of a recent study indicate that specific regions in CotE are required for the assembly of different proteins and suggest that CotE might control the assembly of several outer-coat components by direct protein-protein interactions (2, 28). Most of the coat structural components are synthesized (under the control of σK and GerE) at a later stage in coat assembly, and it is only after σK is activated that assembly of the coat is unequivocally recognized by electron microscopy of sporulating cells (7, 8, 15). Activation of σK results in the expression of several genes coding for spore coat proteins and also results in transcription of the gerE gene (4), which encodes an ambivalent transcriptional regulator of coat gene expression. GerE acts together with σK to activate a late class of cot genes, but it also represses transcription of other cot genes (18, 19, 45, 46). These regulatory circuits suggest that the time and level of expression of the genes coding for coat structural components are important for the correct assembly of the coat structure (7, 8, 15). Proper assembly of the coat further relies on mechanisms such as translational control (34) and posttranslational modifications, including proteolytical processing of larger precursors, protein secretion, and protein cross-linking (reviewed in references 7, 8, and 15). These modifications may provide an additional level of control over the timing of assembly of specific components. For example, SafA is a morphogenetic protein of about 45 kDa produced under σE control from hour 2 of sporulation onwards but the main form of SafA detected in the coats is a smaller (approximately 30 kDa) species corresponding to the C-terminal region of the protein (32, 33). This smaller species is produced by internal translation initiation (34). In addition, the full-length and 30-kDa forms of SafA are processed by the YabG protease, which is produced under the control of σK (42, 43). The exact contribution of these mechanisms to the ordered assembly of the various coat components is poorly understood, and determination of their nature and contribution will ultimately rely on the functional and structural characterization of selected components (11, 29). To learn more about the mechanisms involved in the morphogenesis of the coat structure, we analyzed the assembly of the outer-coat protein CotB. The cotB gene was initially found (by reverse genetics) to encode an abundant spore coat component (6), later shown to be in the outer coat (47), which appears to be surface exposed (20). CotB has been utilized as a vehicle for the presentation of heterologous antigens at the spore surface, suggesting its potential use in vaccine development (9, 20). Thus, the study of the assembly of CotB may allow a more precise manipulation of CotB as a fusion partner for heterologous antigen presentation. Also, it will expand our knowledge of the protein-protein interactions underlying assembly of a complex multiprotein structure and may provide us with tools for nanoengineering applications involving the B. subtilis spore. The cotB gene forms a cluster with two cot genes, cotH and cotG (6, 30, 36). Expression of cotH is under the control of σK, whereas both cotG and cotB are expressed later under the dual control of σK and GerE (18, 30, 36, 46, 45). Assembly of CotB-66 was shown to require expression of both cotG and cotH (30, 36). We now show that cotB encodes a 46-kDa polypeptide (CotB-46) which is posttranslationally converted into a form of about 66 kDa (herein called CotB-66). This form of CotB (CotB-66) is equivalent to the 59-kDa protein previously reported by Donovan et al. (6). We show that formation of CotB-66 requires both cotG and cotH and that CotG does not accumulate in a cotH mutant. This suggests that the requirement for CotH or a CotH-dependent protein for CotB-66 formation results in part from its stabilization of CotG. We also found that CotB is present in complexes with CotG at the time when formation of CotB-66 is detected. Moreover, CotB was found to interact with itself and with CotG in a Saccharomyces cerevisiae two-hybrid assay. We suggest that formation of CotB-66 requires a direct interaction with CotG.

We describe the identification and characterization of a gene, herein designated cotG, encoding an abundant coat protein from the spores of Bacillus subtilis. The cotG open reading frame is 195 codons in length and is capable of encoding a polypeptide of 24 kDa that contains nine tandem copies of the 13-amino-acid long, approximately repeated sequence H/Y-K-K-S-Y-R/C-S/T-H/Y-K-K-S-R-S. cotG is located at 300 degrees on the genetic map close to another coat protein gene, cotB. The cotG and cotB genes are in divergent orientation and are separated by 1.3 kb. Like the promoter for cotB, the cotG promoter is induced at a late stage of sporulation under the control of the RNA polymerase sigma factor sigma K and the DNA-binding protein GerE. The -10 and -35 nucleotide sequences of the cotG promoter resemble those of other promoters recognized by sigma K-containing RNA polymerase, and centered 70 bp upstream of the apparent start site is a sequence that matches the consensus binding site for GerE. Spore coat proteins from a newly constructed cotG null mutant lack not only CotG but also CotB, a finding that suggests that CotG may be a morphogenetic protein that is required for the incorporation of CotB into the coat.

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by σ K , a sporulation-specific σ factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH . A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis , B. amyloliquefaciens , and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH . Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene.

Sporulating cells of the gram-positive bacterium Bacillus subtilis are partitioned into two cellular compartments called the mother cell and the forespore. Gene expression in the mother cell and the forespore is regulated differentially by the compartment-specific transcription factors sigma K and sigma G, respectively. Gene expression between the two compartments is also coordinated by a signal transduction pathway that couples the activation of sigma K (by processing of its inactive precursor pro-sigma K) in the mother cell to sigma G-directed gene expression in the forespore. To dissect the signal transduction pathway genetically, we previously isolated bypass of forespore mutations at loci called bofA and bofB that relieve the dependence of pro-sigma K processing on the action of sigma G. bofB mutations were previously shown to be allelic to the two-cistron sporulation operon spoIVF, which encodes the pro-sigma K-processing enzyme or its regulator. We now report that bofA mutations are located in a small open reading frame of 87 codons that encodes a putative integral membrane protein with three potential membrane-spanning domains. The possibility is discussed that BofA and the SpoIVF proteins form a heteromeric complex in the mother cell membrane that surrounds the forespore and that this complex mediates the intercompartmental coupling of pro-sigma K processing to events in the forespore.

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis . CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of σ K and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis . Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE . In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH . These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.