FIG. 5.

Inducible expression of SpaH pilins. (A) Plasmid pPlac-spaH was generated by cloning the coding sequence of spaH under the control of an IPTG-inducible promoter of a C. diphtheriae/E. coli shuttle vector, pEKEx2. Conserved lysine 202 of the SpaH pilin motif was substituted by an alanine in a control plasmid used in C. (B) Corynebacteria harboring pPlac-spaH were induced by the addition of 1 mM IPTG, and samples with an equal number of cells were taken at various time intervals for treatment with muramidase. Solubilized pilins were boiled in SDS sample buffer, separated on 4 to 12% SDS-PAGE gels, and detected by immunoblotting with the specific antiserum, anti-SpaH (α-SpaH). (C) A parallel experiment as described in B was performed with corynebacteria harboring a control plasmid that expressed mutant SpaH pilins. SrtD was used as a lysis and loading control.