Complete sequence of the IncPβ plasmid R751: implications for evolution and organisation of the IncP backbone¹

Abstract

The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species. To facilitate study of these plasmids we have compiled the complete sequence of the IncPβ plasmid R751. Comparison with the sequence of the IncPα plasmids confirms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family. As in the IncPα genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G+C content, such as P. aeruginosa, suggesting that IncPβ plasmids have been subjected during their evolution to similar mutational and selective forces as IncPα plasmids and may have evolved in pseudomonad hosts. The IncP genome is consistently interrupted by insertion of phenotypic markers and/or transposable elements between oriV and trfA and between the tra and trb operons. The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends. Rather it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed 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 phenotype. The most striking feature of the IncPα plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be significant differences in the specificity of repressor-operator interactions. The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably. There appears to be no equivalent of the parABCDE region which in the IncPα plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioning functions. However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was first revealed by study of the IncPα plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions.

Introduction

Plasmids form a vital part of the bacterial genome and can constitute up to 10% or more of the DNA in a bacterial cell. Plasmids can transfer by a variety of means including conjugation, transformation and transduction. Most bacteria are capable of becoming hosts to a large array of different plasmids commonly found in other strains, other species or even other genera. Plasmids thus represent a large genetic resource for diversity and adaptation of bacteria. Although plasmids can facilitate the exchange and recombination of chromosomal DNA their particular strength is that they allow the spread of genes independently of the need for homologous recombination in the recipient bacterium. Plasmids which can transfer between and maintain themselves in diverse species thus may have a particularly important role in plasmid-mediated gene spread. The IncP plasmids are the most intensively studied self-transmissible promiscuous plasmids Thomas and Smith 1987, Pansegrau et al 1994. In recent years many new members of the IncP group have been discovered in bacteria from aquatic and soil environments Burlage et al 1990, Gotz et al 1996, Junker and Cook 1997, Speakman et al 1997, Top et al 1995, Wyndham et al 1988.

The complete nucleotide sequence of the archetype of the IncPα subgroup was compiled recently (Pansegrau et al., 1994). This has underpinned the study of plasmid replication, transfer, stable inheritance and coordinate regulation so that now the IncP plasmids form a paradigm for many aspects of plasmid biology. The complete sequence has also allowed study of the plasmid genome as a whole. It has shown that the backbone of the plasmid has a particular nucleotide signature, prevalence of GCCG and CGGC sequences, which is a characteristic of Pseudomonas sensu strictu and is deficient in many palindromic hexanucleotides, possibly due to selection against restriction targets during ancestral transfers between authentic pseudomonads (Wilkins et al., 1996). The compilation also emphasised the existence of a number of genes and operons for which no replication, maintenance or transfer function has been demonstrated. To pursue this analysis we have exploited the fact that the IncP plasmids consist of two well separated subgroups Chikami et al 1985, Sakanyan et al 1983, Villarroel et al 1983, Yakobson and Guiney 1983 which nevertheless demonstrate not only cross-complementation of some key functions between the two subgroups, but also similar organisation of all the replication, transfer and control functions (Smith & Thomas, 1987).

To understand the evolution of this group a detailed knowledge of the main archetypes of this family is needed. Many of the newly isolated IncP plasmids appear to be more closely related to the IncPβ plasmids. To underpin the analysis of these plasmids we have completed the sequence of R751 (Jobanputra & Datta, 1974), the best studied IncPβ plasmid. R751 is known to have a similar backbone of replication and transfer functions to the well studied IncPα plasmids RK2 and RP4 Smith and Thomas 1987, Smith and Thomas 1989, Pansegrau and Lanka 1987, Pansegrau et al 1994, but the complete sequence reported here reveals novel features of the genome as a whole as well as new information about specific segments of the plasmid and its functional organisation.