Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and cuter membranes of donor cells: Implications for related macromolecule transport systems

During bacterial conjugation, the single-stranded DNA molecule is transferr ed through the cell envelopes of the donor and the recipient cell. A membra ne-spanning transfer apparatus encoded by conjugative plasmids has been pro posed to facilitate protein and DNA transport. For the IncP alpha plasmid R P4, a thorough sequence analysis of the gene products of the transfer regio ns Tra1 and Tra2 revealed typical features of mainly inner membrane protein s. We localized essential RP4 transfer functions to Escherichia coil cell f ractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specif ied by the Tra2 core region and by traF of the Tra1 region, was found in th e outer membrane fraction with one exception, the TrbB protein, which behav ed like a soluble protein. The membrane preparation from Mpf-containing cel ls had an additional membrane fraction whose density was intermediate betwe en those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is kno wn to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the so luble fraction, but also in the inner membrane fraction. This indicates tha t the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coil cell envelope, The Tra1 protein TraG was pr edominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.