Conjugative junctions in RP4-mediated mating of Escherichia coli

The physical association of bacteria during conjugation mediated by the Inc Pc alpha plasmid RP4 was investigated. Escherichia coli mating aggregates p repared on semisolid medium were ultrarapidly frozen using copper block fre ezing, followed by freeze substitution, thin sectioning, and transmission e lectron microscopy. In matings where the donor bacteria contained conjugati ve plasmids, distinctive junctions were observed between the outer membrane s of the aggregates of mating cells, An electron-dense layer linked the sti ffly parallel outer membranes in the junction zone, but there were no cytop lasmic bridges nor apparent breaks in the cell walls or membranes. In contr ol experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). I n matings where donor strains carried Tra2 only or Tra2 plus the pilin-proc essing protease TraF, junctions were found but they were shorter and more i nterrupted than the wild type. If the donor strain had the pilin gene knock ed out (trbC), junctions were still found. Thus, it appears that the electr on-dense layer between the outer membranes of the conjugating cells is not composed of pilin.