Evolution and assembly of a plasmid DNA conjugation apparatus

Intercellular transfer of plasmid DNA molecules is achieved by a large array of proteins which assemble into both cytoplasmic and membrane-associated complexes. The incompatibility group H plasmids R478, R27, and pHCM1 are large conjugative genetic elements (≥180 kbp), are harboured within pathogenic bacteria, and encode resistance to multiple antibiotics and heavy metals. The evolution and function of the H plasmid transfer apparatus was investigated by comparative sequence analyses, fluorescent labeling of transfer protein complexes, and biochemical characterization of protein interactions.; The 274,762 bp sequence of R478 was determined and used to define the minimal set of core H plasmid determinants (including replication, partitioning, and conjugative transfer) by a comparative analysis with R27 and pHCM1 sequences. No resistance determinants are predicted to be present in the ancestor common to these plasmids, and a comparison with related transfer sequences suggested that the H-type transfer determinants encoding mating pair formation (Mpf) functions are of a different lineage than those determinants encoding DNA-processing (Dtr) and membrane-targeting (coupling) functions.; An assembly of R27-encoded proteins was visualized through an Mpf protein-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci. Thirteen of the nineteen R27 transfer proteins were required for focus formation, and an individual focus possibly represents a subassembly comprised of some or all of these transfer proteins. These data support the notion that the transfer apparatus is a multi-component structure. The fluorescence assay also demonstrated that the NTP-binding motifs of the putative ATPase TrhC were necessary for conjugative transfer, but not for assembly of the fluorescent subcomplex, and that the temperature-sensitive phenotype of H plasmid transfer results from temperature-dependent expression of R27 transfer genes.; The proposed link between the Mpf and Dtr protein complexes is the coupling protein. This model was confirmed by bacterial two-hybrid and immunoprecipitation assays which characterized a direct interaction between the R27-encoded coupling protein TraG and the Mpf protein TrhB. Evolution of the H-type transfer system required the adaptation of unrelated Mpf and Dtr determinants by the coupling protein to form an apparatus functional for conjugative plasmid transfer.