Analysis of pilin polymerization in a uropathogenic Escherichia coli strain containing two pyelonephritis-associated (pap)gene clusters.

E. coli strain C1212 expresses two distinct pili on its cell surface that is encoded by separate pyelonephritis-associated pili (pap) gene clusters. These gene clusters have been demonstrated to produce expression of pili composed of either 17 kilodalton (kd) or 21 pilin monomers. The potential for copolymerization of the two pilin monomers, resulting in production of hybrid pili, was examined here. Pilin structural genes (A-B) and assembly genes (C-G) from each cluster were isolated and ligated to pBR325 and pACYC184 plasmid vectors, respectively. These vectors, harboring specific pap genes, were utilized to produce organisms carrying a set of pilin structural genes from one pap gene cluster and a set of assembly genes from the other (heterologous) pap gene cluster. Pilin monomers expressed by these organisms were examined by immunoelectron microscopy with 6nm and 18nm-diameter gold particles conjugated to a pap monoclonal antibody. Individual pilin monomers were also harvested and purified from overnight cultures of uropathogenic E. coli expressing either pilin-17 or pilin-21. Copolymerization of the two monomers was examined (in vitro) through specific iodination of pilin monomers, incubation and polymerization of monomer mixtures with 0.1M MgCl2, column chromatography of mixtures on Sepharose CL-6B and Sephadex G-100, and Immunoblots of pilin-17 and pilin-21 immunoprecipitates. Data gathered from complementation analysis of heterologous gene clusters and immunoprecipitates suggest that in vitro copolymerization of pilin monomers and hybrid pilin production can occur.