MECHANISM OF RETROTRANSFER IN CONJUGATION - PRIOR TRANSFER OF THE CONJUGATIVE PLASMID IS REQUIRED

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient, Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrot ransfer, Two distinct models have been generally considered for the me chanism of retrotransfer, In the two-way conduction model, no transfer of the conjugative plasmid is required, The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions, In the one-way conduction model, t ransfer of the conjugative plasmid to the recipient is required to all ow the synthesis of a new conjugation bridge for the transfer of DNA f rom recipient to donor, We have tested these models by the constructio n of a mutant of the self-transmissible, IncP plasmid RK2lac that allo ws the establishment of the conjugation bridge but is incapable of sel f-transfer, Four nucleotides of the nic region of the origin of transf er (oriT) were changed directly in the 67-kb plasmid RK2lac by a simpl e adaptation of the vector-mediated excision (VEX) strategy for precis ion mutagenesis of large plasmids (E. K. Ayres, V. J. Thomson, G. Meri no, D. Balderes, and D. H. Figurski, J. Mel. Biol, 230:174-185, 1993), The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT (+) plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type, Whereas the wild-type RK2l ac oriT(+) plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT 1 mutant is severely defective in retrotransfer, Therefore, retrotrans fer requires prior transfer of the conjugative plasmid to the recipien t, The results prove that retrotransfer occurs by two sequential DNA t ransfer events.