COMPLETE SEQUENCE OF THE INCP-BETA PLASMID R751 - IMPLICATIONS FOR EVOLUTION AND ORGANIZATION OF THE INCP BACKBONE

The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial spec ies. To facilitate study of these plasmids we have compiled the comple te sequence of the IncP beta plasmid R751. Comparison with the sequenc e of the IncP alpha plasmids confirms the conservation of the IncP bac kbone of replication, conjugative transfer and stable inheritance func tions between the two branches of this family. As in the IncP alpha ge nome the DNA of this backbone appears to have been enriched for the GC CG/CGGC motifs characteristic of the genome of organisms with a high G +C content, such as P, aeruginosa, suggesting that IncP beta plasmids have been subjected during their evolution to similar mutational and s elective forces as IncP alpha plasmids and may have evolved in pseudom onad hosts. The IncP genome is consistently interrupted by insertion o f phenotypic markers and/ or transposable elements between oriV and tr fA and between the tra and tub operons. The R751 genome reveals a fami ly of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryp tic transposon Tn4321 revealed that it is not a member of the Tn21 fam ily as we had proposed previously from an inspection of its ends. Rath er it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distr ibuted throughout the prokaryotes. The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidentified p henotype. The most striking feature of the IncP alpha plasmids, the gl obal regulation of replication and transfer by the KorA and KorB prote ins encoded in the central control operon, is conserved between the tw o plasmids although there appear to be significant differences in the specificity of repressor-operator interactions. The importance of thes e global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts whe re the surrounding region, either a protein coding or intergenic seque nce, has diverged considerably. There appears to be no equivalent of t he parABCDE region which in the IncP alpha plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioni ng functions. However, we found that the continuous sector from co-ord inate 0 to 9100 bp, encoding the co-regulated klc and kle operons as w ell as the central control region, could confer a high degree of segre gational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was first revealed by study of the IncP alpha plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions. (C) 1998 Ac ademic Press.