ComC is required for the processing and translocation of ComGC, a pilin‐like competence protein of Bacillus subtilis

Abstract

ComGC is a cell surface-localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N-terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH-solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross-linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

“…We have shown previously that ComGC is processed by a pathway that is dependent on ComC (Chung and Dubnau, 1994). This observation has now been extended to ComGD, GE and GG (Fig.…”

“…Processing of pre-ComGC is followed by the translocation of the mature protein from its position as an integral membrane protein to a new location outside the cell membrane, although roughly half of the processed molecules remain as NaOH-insoluble membrane-spanning proteins (Chung and Dubnau, 1994). In the present study, we have observed that a previously undetected fraction of the mature protein is released when the cell wall is hydrolysed by treatment with lysozyme or mutanolysin in an osmoprotective medium.…”

“…Indeed, we have shown that ComGC is processed by a pathway that requires the product of comC (Chung and Dubnau, 1994). As loss-of-function mutations in comC eliminate DNA binding, it appears that processing of at least one of the pilin-like proteins is required for transformation and, specifically, for the first step in this process.…”

“…Based on cross-linking experiments, we have reported previously that ComGC forms homodimers in vivo (Chung and Dubnau, 1994). Competent cells were treated with N-ethylmaleimide (NEM) before lysis in order to block the in vitro formation of disulphide bonds.…”

“…Unprocessed ComGC (in a comC background) is localized as an integral membrane protein, with its C-terminus outside the membrane (Breitling and Dubnau, 1990;Chung and Dubnau, 1994). Upon cleavage, a portion of the total ComGC pool is translocated to the outside of the membrane (Chung and Dubnau, 1994).…”