Conservation of the genetic switch between replication and transfer genes of IncP plasmids but divergence of the replication functions which are major host-range determinants.

The trfA operon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. The trb operon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncP beta plasmid R751 presented here with the equivalent IncP alpha sequence identifies conserved features of the organization and regulation of the trfA operon and the region controlling expression of the trb operon. As in IncP alpha plasmids, these operons are transcribed from a bidirectional promoter region consisting of trfAp for the trfA operon and trbAp and trbBp for the trb operon. The KorA-dependent switch between the trfA and trbA promoters is conserved as is the trbA gene encoding the third IncP global regulator. The intergenic region between trbA and trbB shows very little sequence identity between the two plasmids but the spacing, the KorB operator, the trbB promoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters the trbB ribosome binding site are all conserved. The trfA operon encodes two ORFs. The first ORF is highly conserved and encodes a putative single-stranded DNA binding protein (Ssb). The second, trfA, contains two translational starts as in the IncP alpha plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids. TrfA2 is very similar to the IncP alpha product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncP alpha TrfA1. This region has been implicated in the ability of IncP alpha plasmids to replicate efficiently in Pseudomonas aeruginosa. A TcR derivative of R751 was constructed and shown not to establish itself efficiently in P. aeruginosa at 37 degrees C, although it did establish itself inefficiently at lower temperatures, underlining the importance of this region in the adaptation of the plasmid to the host.