A macromolecular complex formed by a pilin-like protein in competent Bacillus subtilis

Abstract

In competent Bacillus subtilis, the ComG proteins are required to allow exogenous DNA to access to membrane-bound receptor ComEA during transformation. Here we describe a multimeric complex containing the pilin-like protein ComGC. Due to similarities to the type 4 pilus and the type 2 secretion system pseudopilus, we have tentatively named it the "competence pseudopilus." The ComGC multimer is released from cells upon digestion of the cell wall with lysozyme and has a heterogeneous size, estimated to range between 40 and 100 monomers, covalently linked by disulfide bonds. We determined that the prepilin peptidase ComC, the thiol-disulfide oxidoreductase pair BdbDC, and all seven ComG proteins are necessary to form the pseudopilus. Furthermore, these proteins are also sufficient to form a functional complex, i.e. able to facilitate binding of exogenous DNA to ComEA. The initial steps of pseudopilus biogenesis include the processing of ComGC in the cytoplasmic membrane and consist of two independent events, proteolytic cleavage by ComC and formation of an intramolecular disulfide bond by BdbDC. The other ComG proteins are required to assemble the mature ComGC monomers in the membrane into a multimeric complex proposed to span the cell envelope. We discuss the possible role of the competence pseudopilus in DNA binding and uptake during transformation.

FIGURE 1. The pilin-like protein ComGC forms a multimeric complex associated with the cell envelope.

Left panel, anti-ComGC immunoblots with protoplast supernatant fractions (equivalent to 200 μl of culture) prepared from wild-type strain BD2528 (wt) and comG mutant BD2780. Samples, with or without treatment with reducing agents β-mercaptoethanol (β-ME) or dithiothreitol (DTT), were separated by SDS-PAGE. Right panel, anti-ComGC immunoblots from protoplast fractions (equivalent to 100 μl of cell culture) from wild-type strain BD2528, with or without reduction with β-mercaptoethanol.

FIGURE 2. Determination of hydrodynamic parameters of the ComGC complex.

A, gel filtration. The calibration curve for a Sephacryl S-500HR column was established as described under “Experimental Procedures.” Protoplast supernatant preparations from wild-type strain BD2528 were loaded and fractionated, and 6-ml fractions were collected and probed with anti-ComGC antibody after SDS-PAGE. The line underneath the immunoblot indicates the fractions considered to contain most of the ComGC complex. In the various fractions, which were concentrated by trichloroacetic acid precipitation prior to loading, the abundance of other proteins sometimes resulted in distortions of the lanes in SDS-PAGE. B, sucrose gradient centrifugation. Samples were run through a 12-ml 15–45% sucrose gradient. The calibration curve was established as described under “Experimental Procedures.” Protoplast supernatant preparations were loaded on the top of the gradient and centrifuged; 1-ml fractions were collected from the top, precipitated with trichloroacetic acid, and analyzed for the presence of ComGC by immunoblotting. The line underneath the immunoblot indicates the fractions considered to contain most of the ComGC complex. The signal at the bottom of the gradient (fraction 12) was considered to be precipitated ComGC material.

FIGURE 3. Factors necessary for ComGC complex formation.

Protoplast supernatant (PSn) and membrane fractions (M) from mutants in different late competence genes were analyzed by immunoblotting. A, wild-type strain (BD2528) and mutants in comG (BD2780), bdbDC (BD3002), comC (BD2708), comE (BD2718), and comF (BD2996). B, wild-type strain (BD2528) and mutants in each of the comG genes: comGA (BD2786), comGB (BD2787), comGD (BD2789), comGE (BD2790), comGF (BD2791), and comGG (BD2709); mutant in the comGA Walker A ATP-binding site (comGA-ATP⁻, BD4140). 1 μg of membrane protein was loaded in the lower panel (M).

FIGURE 4. Processing of ComGC in the membrane.

A, the transformation deficiency of a bdbC mutant can be partially relieved by oxidizing agents. Treatment of cells with 5 mM L-cystine enhanced transformation frequencies of the bdbC strain (BD2999); a less pronounced effect was seen with the strain lacking bdbD (BD3355) or the double mutant bdbDC (BD3002). B, the cysteine residues of ComGC are important for protein stability but not for cleavage by ComC. Immunoblot analysis of membrane preparations from strains expressing different forms of ComGC from an ectopic locus; to increase the ComGC signal in these strains, the comG operon was inactivated. In the left panel,1 μg of protein from the strain expressing wild-type ComGC (BD3944), and 10 μg of protein from strains expressing ComGC-C41S (BD3942), ComGC-C81S (BD3943), and ComGC-C41/81S (BD3945) were loaded; an uncleaved ComGC sample from a comC mutant (BD3845, 5 μg) was loaded as a standard. In the right panel, 0.5 μg of protein from the strain expressing wild-type ComGC (BD3944), and 10 μg of protein from strains expressing ComGC-C41/81S (BD3945) and ComGC-C41/81S comC (BD3999) were loaded. C, oxidation of ComGC does not depend on cleavage. Immunoblot analysis of membrane preparations (10 μg of protein and longer exposure in the left panel, 5 μg of protein in the right panel) from wild-type strain (BD2528), bdbDC (BD3002), comC (BD3844), and bdbDC comC (BD3845). Strain BD3944 (see B) is included as a cleaved ComGC standard in the last lane on the right.

FIGURE 5. Factors that are sufficient for ComGC complex formation.

Expression of comC, bdbDC, and the comG operon from IPTG-inducible promoters in a comK background is sufficient to form ComGC complex. In A: left panel, immunoblot analysis of protoplast supernatant preparations from wild-type strain BD2528 and IPTG-inducible strain BD3502, with or without β-mercaptoethanol reduction. Right panel, immunoblot analysis of fractions from gel-filtration chromatography: protoplast supernatant preparations from wild-type strain (BD2528) and IPTG-inducible strain BD3502 were separated in a Sephacryl S-500HR column. The distortion of the bands in this panel is due to intentional overloading, to enable detection of minor amounts of ComGC in some of the fractions. B, expression of comEA, comC, and the comG operon from inducible promoters (icomEA/C/G, strain BD4164) is sufficient to promote binding of radiolabeled DNA (compared with BD2121, p < 0.0001, Student’s t test). Expression of comEA from a xylose-inducible promoter (icomEA, BD4148) or of comC/comG by themselves (icomC/G, BD4149) does not promote DNA binding to the cells. The graph presents data from one representative experiment out of three independent experiments, performed in triplicate, with results shown as mean ± S.D. All strains are comK.