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

ThetrfAoperon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. Thetrboperon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncPβ plasmid R751 presented here with the equivalent IncPα sequence identifies conserved features of the organization and regulation of thetrfAoperon and the region controlling expression of thetrboperon. As in IncPα plasmids, these operons are transcribed from a bidirectional promoter region consisting oftrfApfor thetrfAoperon andtrbApandtrbBpfor thetrboperon. The KorA-dependent switch between thetrfAandtrbApromoters is conserved as is thetrbAgene encoding the third IncP global regulator. The intergenic region betweentrbAandtrbBshows very little sequence identity between the two plasmids but the spacing, the KorB operator, thetrbBpromoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters thetrbBribosome binding site are all conserved. ThetrfAoperon 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 IncPα plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids. TrfA2 is very similar to the IncPα product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncPα TrfA1. This region has been implicated in the ability of IncPα plasmids to replicate efficiently inPseudomonas aeruginosa.A TcRderivative of R751 was constructed and shown not to establish itself efficiently inP. aeruginosaat 37°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.