Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Abstract

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.

Phosphorylation of spore coat proteins by a family of atypical protein kinases

“…Therefore, loss of CotH phosphorylation would result in CotG degradation. This mechanism may explain why CotG does not accumulate in spores generated from cells depleted of CotH (26,36). In any event, it is clear that phosphorylation is an important, but overlooked, mechanism in spore biology.…”

“…The cotB encodes a predicted 43-kDa polypeptide that migrates in SDS/PAGE with an apparent molecular mass of 66 kDa in sporulating bacteria (36). Interestingly, CotB migrates as a 43-kDa species in cotH-null spores, suggesting that CotH promotes the formation of the more slowly migrating 66-kDa form of CotB (26,36). CotB has a C-terminal domain that is enriched in Ser, Lys, and Arg residues (Fig.…”

“…CotH appears to maintain the integrity of the spore coat because loss of CotH in B. subtilis is detrimental to the germination efficiency of the spores (24)(25)(26). However, the molecular mechanisms through which this occurs are unclear.…”

“…6B). We hypothesized that the CotB modification observed in vivo was due to phosphorylation of CotB by CotH, thus accounting for the apparent 23-kDa decrease in molecular mass of CotB in cotH-null spores (26,36). We purified full-length recombinant CotB from E. coli and performed in vitro kinase assays.…”

“…The protein is rich in Ser residues (39 Ser) and is highly basic, because nearly half of its residues are Arg, Lys, or His. A recent proteomics experiment has shown that B. subtilis CotG is phosphorylated on multiple residues within the tandem repeats (26). In fact, the CotG ortholog in B. anthracis, ExsB, has been described as the most extensively phosphorylated bacterial protein characterized to date (37).…”