TrfA-dependent inner membrane-associated plasmid RK2 DNA synthesis and association of TrfA with membranes of different gram-negative hosts

TrfA, the replication initiator protein of broad-host-range plasmid RK2, wa s tested for its ability to bind to the membrane of four different gram-neg ative hosts in addition to Escherichia coli: Pseudomonas aeruginosa, Pseudo monas putida, Salmonella enterica serovar Typhimurium, and Rhodobacter spha eroides. Cells harboring TrfA-encoding plasmids were fractionated into solu ble, inner membrane, and outer membrane fractions. The fractions were subje cted to Western blotting, and the blots were probed with antibody to the Tr fA proteins. TrfA was found to fractionate with the cell membranes of all s pecies tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without ad ded template or enzymes), only the inner membrane fraction was capable of e xtensive synthesis that was inhibited by anti-TrfA antibody in a manner sim ilar to that of the original host species, E. coli. In addition, although D NA synthesis did occur in the outer membrane fraction, it was much less ext ensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membr ane fraction of one representative species, P. aeruginosa, was characterist ic of supercoil and intermediate forms of the plasmid, Extensive DNA synthe sis was observed in the soluble fraction of another representative species, R. sphaeroides, but it was completely unaffected by anti-TrfA antibody, su ggesting that such synthesis was due to repair and/or nonspecific chain ext ension of plasmid DNA fragments.