RrgB antiserum is specific to RrgB. (A) Two RrgB peptides were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed separately with mouse preimmune serum (lane 1) or mouse anti-RrgB serum (lane 2). The two RrgB peptides (filled arrow) and a breakdown product (open arrow) were detected by the antiserum.

RrgA and RrgB are polymerized into pili. (A) Cell wall fractions of RrgA-HA and RrgB-HA S. pneumoniae TIGR4 form high-molecular-weight complexes when immunoblotted with anti-HA antisera. Wild-type (wt) TIGR4 (lane 1), RrgA-HA (lane 2), and RrgB-HA (lane 3) are shown. (B) The high-molecular-weight RrgA-HA complexes are not present in a strain with rrgB deleted and rrgC but are present when only rrgC is deleted. Wild-type TIGR4 (lane 1), RrgA-HA (lane 2), RrgA-HA ΔrrgB ΔrrgC (lane 3), and RrgA-HA ΔrrgC (lane 4) strains are shown.

Immunogold transmission electron microscopy visualization of RrgB and RrgA-HA incorporated into pilus-like structures on the surface of S. pneumoniae TIGR4. (A and B) Representative micrographs of TIGR4 labeled with mouse anti-RrgB antiserum and visualized with anti-mouse IgG Colloidal Gold 18-nm particles. (C to E) Representative micrographs of TIGR4 labeled with rabbit polyclonal anti-HA antibody and visualized with anti-rabbit IgG Colloidal Gold 12-nm particles. (C) RrgA-HA; (D) RrgA-HA ΔrrgB ΔrrgC; (E) RrgA-HA ΔrrgC. (F and G) S. pneumoniae RrgA-HA labeled with both mouse anti-RrgB antiserum and anti-mouse IgG Colloidal Gold 18-nm particles as well as rabbit polyclonal anti-HA antibody and anti-rabbit IgG Colloidal Gold 12-nm particles. RrgA, labeled with 12-nm gold particles (arrows), is found periodically within the pilus. (H) TIGR4 labeled with mouse preimmune serum. Magnifications: (A) ×10,500 (scale bar, 1 μm); (B, G, and H) ×19,000 (scale bar, 1 μm); (C to F) ×25,000 (scale bar, 0.5 μm).

Incorporation of RrgA into pili requires SrtD. (A) Cell wall fractions of wild-type and sortase mutant strains were examined by Western blotting with polyclonal anti-RrgA antiserum. Fractions were from wild-type TIGR4 (lane 1) and strains carrying mutations in rrgA (lane 2), srtA (lane 3), srtB (lane 4), srtC (lane 5), and srtD (lane 6). (B) Schematic of the RrgA chimeras examined by Western blotting. RrgA (black arrow and rectangles) containing the C-terminal CWSS of each gene listed (arrowheads) is shown. The RrgA-RrgB^CWSS chimera was generated by a suicide plasmid insertion-duplication mutation as illustrated. The integrated plasmid is a derivative of pAC1000 (9) containing rrgA′ (bases 1 to 2586) joined to ′rrgB (bases 1902 to 1999). The RrgA-RrgC^CWSS and RrgA-SP0082^CWSS chimeras were generated by double-crossover allelic exchange using pAC1000 derivatives, resulting in the replacement of the 3′ end of rrgA (bases 2586 to 2683) with ′rrgC (bases 1084 to 1150) and ′SP0082 (bases 2461 to 2574), respectively. (C) Western blots of the cell wall fractions of chimeric strains of AC1441, AC1442, and AC1443 in an otherwise-wild-type background (lane 1 in each panel) or in the sortase mutant backgrounds indicated above each lane. The cell wall fractions were immunoblotted with anti-RrgA polyclonal antibody. The intense band at approximately 120 kDa consists of two poorly resolved bands.