Comparison of proteins involved in pilus synthesis and mating pair stabilization from the related plasmids F and R100-1: insights into the mechanism of conjugation

Abstract

F and R100-1 are closely related, derepressed, conjugative plasmids from the IncFI and IncFII incompatibility groups, respectively. Heteroduplex mapping and genetic analyses have revealed that the transfer regions are extremely similar between the two plasmids. Plasmid specificity can occur at the level of relaxosome formation, regulation, and surface exclusion between the two transfer systems. There are also differences in pilus serology, pilus-specific phage sensitivity, and requirements for OmpA and lipopolysaccharide components in the recipient cell. These phenotypic differences were exploited in this study to yield new information about the mechanism of pilus synthesis, mating pair stabilization, and surface and/or entry exclusion, which are collectively involved in mating pair formation (Mpf). The sequence of the remainder of the transfer region of R100-1 (trbA to traS) has been completed, and the complete sequence is compared to that of F. The differences between the two transfer regions include insertions and deletions, gene duplications, and mosaicism within genes, although the genes essential for Mpf are conserved in both plasmids. F+ cells carrying defined mutations in each of the Mpf genes were complemented with the homologous genes from R100-1. Our results indicate that the specificity in recipient cell recognition and entry exclusion are mediated by TraN and TraG, respectively, and not by the pilus.

FIG. 1

Comparison of the F and R100-1 transfer operons with the chimeric plasmids used in this study depicted below. Detailed information on the clones is given in Table 2.

FIG. 2

The effect of pilin acetylation on structural characteristics of pili. Immunogold electron microscopy was carried out as described in Materials and Methods. The binding of MAb JEL92 to pili elaborated by MC4100 cells harboring either pOX38-Km (A) or pOX38-traX482 (B) is shown. Arrows indicate the terminal knobs or vesicular materials coated with gold particles. Magnification is ×14,500, and the size bar represents 1 μm.