Interactions among CotB, CotG, and CotH during Assembly of the Bacillus subtilis Spore Coat

Abstract

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...

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

“…Spores, prepared as previously described (Isticato et al, 2004), were collected after a 30 h incubation at 37 uC, washed four times and purified by lysozyme treatment as previously described (Cutting & Vander Horn, 1990;Nicholson & Setlow, 1990). The number of purified spores obtained was measured by direct counting with a Bürker chamber under an optical microscope (Olympus BH-2 with 406 lenses).…”

“…The number of purified spores obtained was measured by direct counting with a Bürker chamber under an optical microscope (Olympus BH-2 with 406 lenses). Aliquots of 10 10 spores suspended in 0?3 ml distilled water were used to extract coat proteins by 0?1 M NaOH treatment at 4 uC as previously reported (Isticato et al, 2004). The concentration of the extracted coat proteins was determined by the Bio-Rad DC (Detergent Compatible) Protein Assay to avoid potential interference by the NaOH present (0?2-0?6 mM final concentration) in the extraction buffer and 15 mg of total proteins was fractionated on 18 % denaturing polyacrylamide gels.…”

“…Proteins were then electrotransferred to nitrocellulose filters (Bio-Rad) and used for Western blot analysis by standard procedures. The analysis of sporulating cells was performed as previously described (Isticato et al, 2001(Isticato et al, , 2004. Samples were harvested at various times during sporulation and disrupted by sonication in 25 mM Tris (pH 7?5), 0?1 M NaCl, 1 mM EDTA, 15 % (v/v) glycerol and 0?1 mg phenylmethylsulfonyl fluoride ml 21 .…”

“…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).…”

“…Cross-linking of structural proteins is also believed to occur and results in the insolubilization of specific components (Kobayashi et al, 1994). At least two coat components assemble in the mature coat in either homo-or hetero-multimeric forms (Isticato et al, 2004;Zilhao et al, 2004).…”