CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis

Abstract

Bacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation-specific and heat-labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo-regulator CotH contributes to the switch between the coat of 25 C and that of 42 C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.

CotG Mediates Spore Surface Permeability in Bacillus subtilis

“…To verify whether GHyb was functional, Western blotting experiments were performed on proteins extracted from purified spores of a wild-type strain and strains expressing GHyb with and without the kinase CotH. Anti-CotG antibody, raised against the N-terminal 14 residues of B. subtilis CotG ( 18 ), and anti-protein kinase C (anti-PKC) antibody, recognizing phosphorylated serine residues ( 18 ), were reacted against the extracted proteins. As previously reported, CotG was extracted from wild-type spores in two forms (CotG 36 and CotG 32 ), and both proteins were not found in extracts of spores of a strain lacking CotH ( 12 , 20 ) ( Fig.…”

“…CotC and CotU, two highly homologous small coat proteins that assemble on the spore coat in multiple forms ( 22 , 23 ), fail to assemble in a strain lacking CotH ( 17 ). Such an effect, due to the accumulation of unphosphorylated CotG in the mother cell cytoplasm ( 18 ), is not observed when the central repeats of CotG (G Δcentral ) were deleted ( 15 ). Western blotting experiments showed that all forms of CotC and CotU were present in coat extracts of strains carrying GHyb as the only CotG form, independently from the presence of CotH ( Fig.…”

“…CotG has a high net charge and is listed in the top 20 positively supercharged proteins present in data banks ( 16 ). Such a high charge is partially balanced by CotG's extensive, CotH-dependent phosphorylation ( 12 , 17 ), essential for CotG assembly on the spore coat ( 18 ). Unphosphorylated, and presumably unfolded, CotG molecules do not assemble on the spore and form inclusion body-like aggregates in the mother cell cytoplasm ( 18 ).…”

“…Such a high charge is partially balanced by CotG's extensive, CotH-dependent phosphorylation ( 12 , 17 ), essential for CotG assembly on the spore coat ( 18 ). Unphosphorylated, and presumably unfolded, CotG molecules do not assemble on the spore and form inclusion body-like aggregates in the mother cell cytoplasm ( 18 ). In a mutant strain lacking the kinase CotH, all CotG molecules are unphosphorylated, form massive aggregates that sequester other coat proteins, and as a consequence, cause the production of defective spores ( 17 , 18 ).…”

“…Unphosphorylated, and presumably unfolded, CotG molecules do not assemble on the spore and form inclusion body-like aggregates in the mother cell cytoplasm ( 18 ). In a mutant strain lacking the kinase CotH, all CotG molecules are unphosphorylated, form massive aggregates that sequester other coat proteins, and as a consequence, cause the production of defective spores ( 17 , 18 ). Such altered spores are not observed in a mutant lacking the kinase CotH but also with the central repeats of CotG deleted, indicating that the formation of cytoplasmic aggregates is due to the central part of CotG ( 17 , 18 ).…”