Two distinct functions of ComW in stabilization and activation of the alternative sigma factor ComX in Streptococcus pneumoniae

Abstract

Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (sigma(X)), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the sigma(X) protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of sigma(X). As sigma(X) is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of sigma(X) depends on ComW. The clpP comW double mutant accumulated an amount of sigma(X) higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for sigma(X) synthesis, it acts both in stabilization of sigma(X) and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of sigma(X), but only the N terminus is important for stimulating its activity.

FIG. 1.

Effect of comW mutation on the transcription of comX. Beta-galactosidase activities in CPM3 (comX lacZ; WT), CPM4 (comX lacZ; ΔcomX), and CP1724 (comX lacZ; ΔcomW) were assayed at the indicated times after CSP treatment. Competence of CPM3 is also shown (▵). Transformation of CPM4 and CP1724 yielded fewer than 100 Nov^r/ml. Error bars, standard deviation among three replicate experiments. M.U., Miller units.

FIG. 2.

Accumulation of the σ^X protein requires ComW. The WT (CP1250) and a comW mutant (CP1376), a C-His-tagged ComW mutant (CP1802; 0- to 25-min samples in panel C), and an N-His-tagged ComW mutant (CP1805; 0- to 25-min samples in panel D) were treated with CSP (at time zero), and samples were collected as described in Materials and Methods at the indicated times (in minutes). An extract representing 1 ml of culture was loaded into each well. Samples of CP1376 from two independent preparations were loaded onto different gels (A and B). The gels were assayed by Western blotting using an antibody against σ^X.

FIG. 3.

Quantification of σ^X in various genetic backgrounds. (A) σ^X standard curve prepared from a 15-min extract of CP1250 (WT). A sample (100%) was prepared from CP1250 at 15 min post-CSP treatment, and then different amounts (dilutions of 1:5, 1:7.5, 1:10, 1:12.5, 1:15, or 1:20) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. The relationship between the integrated density values (IDV) of enhanced chemiluminescence assay bands and relative amounts of protein is shown. (B) σ^X standard curve prepared from an 80-min extract from CP1815 (clpP comW double mutant). The extract was serially diluted in a blank extract of CPM8 (ΔcomX) (dilutions of 1:2.5, 1:3.75, 1:5, 1:7.5, or 1:10) before assay by SDS-PAGE and Western blotting. (C) Immunoblotting image of σ^X in a wild-type strain and various mutant strains. The relative amount of σ^X in each strain is shown, with a range. The intensities of CP1376, CP1802, and CP1805 were compared to the CP1250 standard curve (A), while those of CP1250, CP1815, and CP1359 were compared to the CP1815 standard curve (B).

FIG. 4.

Deletion of both comW and clpP leads to the accumulation of σ^X after competence induction. The wild-type (CP1250; 0- to 60-min samples in panel A), a comX mutant (CPM8), a clpP single mutant (CP1359; 0- to -80-min samples in panel B), and a comW clpP double mutant (CP1815; 0- to 80-min samples in panel C) were induced by CSP (at time zero), and samples were withdrawn at the indicated times (in min) after induction. The indicated amount of each extract (0.1 ml of original culture per μl extract) were assayed by Western blotting analysis and probed with anti-ComX antibody.

FIG. 5.

Effects of terminal extensions of the ComW protein on competence and σ^X accumulation. The sequences of WT ComW (middle), amino-terminally His-tagged ComW (CP1805; top), and carboxyl-terminally His-tagged ComW (CP1802; bottom). His tags introduced into the wild-type ComW are underlined; boldface residues in N-His-ComW indicate a factor Xa cleavage site; boldface residues in C-His-ComW indicate a V5 epitope. Omitted 53 residues: IEEEYGPTFGDNFDWEHVHFKFLIYYLVRYGIGCRKDFIVYHYRVAYRLYLEK. The corresponding competence phenotype (mean relative number of transformants ± standard deviation) and relative amount of σ^X (mean and range) observed 15 min after CSP induction are also shown. aa, amino acids.