Analysis of Conjugal Transfer Region of Plasmid R64.

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Bacterial conjugation is a complex process whereby plasmid DNA is transferred from one cell to another by direct cell-to-cell contact. This process is usually mediated by conjugative plasmids. R64 is a 122-kb conjugative plasmid belonging to the incompatibility group I1.By constructing a series of deletion-derivatives of plasmid R64, a DNA region responsible for the conjugal transfer was located within a 54-kb region to the right of replication region. To clarify the gene organization of R64 transfer region, we determined its entire nucleotide sequence. R64 transfer region was shown to consist of 54, 089 bp although sequences of only one strand were determined in some regions. At least 39 open reading frames (ORFs) are found over the entire sequence of the transfer region except for 2.2-kb gap region without ORF. All genes except nikAB genes are situated in the same deirection. traABCD genes are present at one end of the transfer region and traBC genes appear to function as positive regulators of transfer gene expression. pilI-V genes consist of an operon responsible for thin pilus formation. pilV gene, the last one of this operon, is under the control of shufflon. R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any two inverted repeats results in a complex DNA rearrangement. The shufflon is thought to determine the recipient specificity in R64 liquid mating, by selecting one of the seven C-termini of the pilV gene. DNA inversions are mediated by rci gene lacated adjacent to shufflon region. Three transfer genes, traEFG, seem to constitute an operon with rci. nuc gene encoding an EDTA-resistant nuclease and sog gene encoding DNA primase make an operon together with traH-P. This operon is under control of traBC gene. At the other end of R64 transfer region, oriT operon is present, which constitutes an origin of transfer, oriT, and nikAB genes.