The housekeeping sortase SrtF is not required for pilus polymerization. Uniformly treated protein samples were collected from the culture medium (M) and cell wall fragments (W) of the C. diphtheriae wild-type (WT) strain or its isogenic derivatives expressing individual sortase (SrtA–F) prior to extraction with hot SDS sample buffer. Proteins were separated on SDS-PAGE and detected by immunoblotting with α-SpaA (A), α-SpaB (B), α-SpaC (C) or α-SpaD (D). The monomer (arrow) and high-molecular-weight products (e.g. Spa_HMW) of pilus polymerization, the position of molecular weight markers and the stacking gel portion of SDS-PAGE (stack) are indicated.

SrtF is required for the efficient cell wall anchoring of SpaA pili. Samples were treated exactly as in Fig. 1. Proteins were separated on 4–12% Tris-glycine gradient gels and detected by immunoblotting with α-SpaA. Anti-pentahistidine (α-His) was used for loading controls. The monomer (arrow) and high-molecular-weight products (e.g. Spa_HMW) of pilus polymerization, and the position of molecular weight markers are indicated.

Efficient assembly of SpaA pili on bacterial surface mediated by SrtF. Cells of the wild type (A) or its isogenic derivatives (B–F) were immobilized on carbon grids, stained with specific antiserum against SpaA (α-SpaA) and goat anti-rabbit IgG conjugated to 18 nm gold particles. Samples were viewed by transmission electron microscopy. Scale bars indicate the length of 0.2 μm.

Immunofluorescent detection of SpaA pili displayed on the bacterial surface. Corynebacteria were stained with a specific antibody against SpaA (α-SpaA) and AlexaFluor 488 chicken anti-rabbit IgG. Shown are the fluorescent, the nomarski DIC and the merged images (A). The samples were observed on a Zeiss LSM 510 confocal microscope. The results are shown as representatives of at least three independent experiments.
B. Quantification of fluorescence intensity in (A) was carried out using the Metamorph software. Intensity values shown with arbitrary units were averaged over 25 cells in each strain and expressed relative to the average fluorescence intensity of the wild type (WT).

SrtF is required for the efficient cell wall anchoring of the SpaD-type pili. Corynebacteria were stained with a specific antibody against SpaD (α-SpaD) and AlexaFluor 488 chicken anti-rabbit IgG. Shown are the fluorescent, the nomarski DIC and the merged images. The samples were observed on a Zeiss LSM 510 confocal microscope. The results are shown as representatives of at least three independent experiments.

SrtF is required for the efficient cell wall anchoring of the SpaH-type pili. Wild-type C. diphtheriae (A) and its isogenic derivatives ΔsrtA-C, F (designated as srtD⁺srtE⁺) (B), or ΔsrtF (C) were immobilized on carbon grids, stained with specific antiserum against SpaH and goat anti-rabbit IgG conjugated to 18 nm gold particles. Samples were viewed by transmission electron microscopy. Scale bars indicate the length of 0.2 μm.

Inducible expression of SpaA pilins.
A. Plasmid pPlac-spaA was generated by cloning the coding sequence of spaA under the control of an IPTG-inducible promoter of the E. coli/C. diphtheriae shuttle vector pEKEx2.
B. Cells of the isogenic ΔsrtF or ΔsrtF-ΔspaA strain harbouring pPlac-spaA were induced by the addition of 1 mM IPTG, and samples with an equal number of cells were taken at timed intervals for isolating medium (M) and cell wall fragments (W) as described in Fig. 1. Samples of the wild-type and ΔsrtF bacteria were collected at the end of induction. Solubilized pilins were boiled in SDS sample buffer and were separated on 4–12% Tris-glycine gradient gels and detected by immunoblotting with the specific antiserum α-SpaA. Anti-his (α-His) and no IPTG (−) were used as controls.
C. The same treatment as described in (B) was employed for cells of the wild type (WT), its isogenic ΔspaA or ΔspaA strain harbouring pPlac-spaA.

SrtF is required for efficient corynebacterial adherence to pharyngeal epithelial cells. The wild type (WT) and its isogenic deletion mutants were subjected to adhesion assays using pharyngeal epithelial cells (D562). Data are presented as percentage of adhesion relative to that of the WT. The results are presented as averages (±SD) from three independent experiments performed in triplicates.

The two-stage mechanism of pilus biogenesis: function of the housekeeping sortase. This working model, built on the most significant findings reported here, depicts the two stages of pilus polymerization and cell wall anchoring catalysed by a pilus-specific sortase and the housekeeping sortase in corynebacteria. As detailed in the text, pilus assembly requires two sortase molecules properly juxtaposed to capture the pilin precursors in the exoplasm and catalyse cycles of covalent linkage between pilin monomers. This is carried out by a pair of the pilus-specific sortases (black), the SrtA sortase for the heterotrimeric SpaABC pilus, each of which must form an acyl enzyme intermediate with the pilin precursors via the catalytic cysteine residue (Cys) and the threonine (Thr) residue of the pilin LPXTG motif. Pilus polymerization is catalysed by cross-linking Thr with the lysine of the pilin motif. Pilus polymerization is terminated when the resulting polymer is joined to the cross-bridge amino group of the lipid II precursor, which may be catalysed by the pilus-specific sortase. The main feature of the current model is the involvement of the housekeeping sortase enzyme (yellow). The key data in this paper show that this enzyme acts predominantly to terminate pilus polymerization (see text). In essence, when the housekeeping sortase captures a pilin precursor, it is in turn poised to receive and secure a pilus polymer assembled on a pilus-specific sortase (centre). Now, the pilus residing on the housekeeping sortase (not drawn in the figure) can only be transferred to the lipid II precursor and incorporated into the cell wall via transglycosylation reaction. Individual pilins are shown with letters A (SpaA), B (SpaB) and C (SpaC). Arrows indicate the direction of transfer of pilin and pilus polymers. Note that the tip pilin SpaC is believed to act as the normal nucleator for polymerization of the pilus shaft made of SpaA, on which the SpaB minor pilin is cross-linked by the SrtA sortase. How this is achieved remains one of the significant puzzles of pilus biogenesis.