Elsevier

Plasmid

Volume 48, Issue 2, September 2002, Pages 104-116
Plasmid

Rhizobium etli CFN42 contains at least three plasmids of the repABC family: a structural and evolutionary analysis

Communicated by D. Chattoraj
https://doi.org/10.1016/S0147-619X(02)00119-1Get rights and content

Abstract

In this paper, we report the identification of replication/partition regions of plasmid p42a and p42b of Rhizobium etli CFN42. Sequence analysis reveals that both replication/partition regions belong to the repABC family. Phylogenetic analysis of all the complete repABC replication/partition regions reported to date, shows that repABC plasmids coexisting in the same strain arose most likely by lateral transfer instead of by duplication followed by divergence. A model explaining how new incompatibility groups originate, is proposed.

Introduction

Rhizobium etli is a soil bacterium with the ability to recognize and induce nitrogen-fixing nodules on bean plant roots. Most of the genes involved in this symbiotic interaction reside on a large plasmid of low copy number called the symbiotic plasmid or pSym. This plasmid may coexist with up to 10 large “cryptic” plasmids (Garcı́a-de los Santos et al., 1996). Beside the pSym (p42d), R. etli CFN42 contains five other plasmids, of which p42b is also indispensable for nodulation (Garcı́a-de los Santos and Brom, 1997). Some of the other plasmids have a role in the symbiotic process: cured derivatives of plasmid p42c or p42f and large deletions of p42e are drastically affected in competitiveness for nodulation (Brom et al., 1992, Brom et al., 2000). The six plasmids of R. etli CFN42 are able to replicate in Agrobacterium but one of them (p42a) exhibits incompatibility towards pTiC58, an Agrobacterium plasmid harboring pathogenicity determinants (Garcı́a-de los Santos and Brom, 1997). This is not an exceptional case since incompatibility has been observed previously between Rhizobium and Agrobacterium plasmids (Hooykaas et al., 1985; O'Connell et al., 1987).

Recently, the complete sequence of several basic replicons of the Rhizobiaceae family have been reported, most of them belonging to the emerging repABC family (see Table 1). The repABC plasmids are characterized by the presence of three genes, repA, repB, and repC encoded in the same DNA strand, and by the presence of a conserved large intergenic sequence between the repB and repC genes (Fig. 1). A genetic analysis of the replication/partition region of the R. etli Sym plasmid showed that the repA, B, and C genes are organized as an operon (Ramı́rez-Romero et al., 2000). The repA and repB products, which are homologous to the A and B proteins of the sop/par partition system of F and P1 plasmids, are involved in the partitioning of the pSym plasmid and in the regulation of plasmid copy number. RepC is essential for replication and is considered to be the initiator protein (Bartosik et al., 1998; Ramı́rez-Romero et al., 2000; Tabata et al., 1989).

The repABC family is not limited to the Rhizobiaceae, since a new repABC plasmid (pTAV320) was recently isolated from Paracoccus versutus which is a member of the Rhodobacter group (Bartosik et al., 1998).

Two cis-acting incompatibility sites in the same relative positions have been described for the Sym plasmid of R. etli and for pTAV320 of P. versutus. One site is located in the intergenic sequence between repB and repC genes (incα or inc1), and the other within the first 500 bp downstream of repC (incβ or inc2). Additionally, RepA has been identified as a trans-acting incompatibility factor in both plasmids (Bartosik et al., 2001; Ramı́rez-Romero et al., 2000).

Two lines of evidence demonstrate that repABC plasmids embrace more than one incompatibility group. First, hybridization studies indicate that some Rhizobia strains contain more than one repABC plasmid (Rigottier-Gois et al., 1998). Second, the analysis of genome sequences of three members of the Rhizobiaceae family shows that they encompass more than one replicon of the repABC type (Galibert et al., 2001; Goodner et al., 2001; Kaneko et al., 2000). In accordance here, we show that plasmids p42a and p42b of R. etli CFN42 belong to the repABC family.

To explain how new incompatibility groups arise within the same plasmid family, two alternative models can be suggested. In brief, The first model propose that plasmids of the same family but of different incompatibility groups arise within the same strain through the formation of a bireplicon intermediary containing two replication regions of the same incompatibility group. In this form, one of the replication systems of the bireplicon can change, until a functional new incompatibility group emerges, without risking other plasmid-encoded functions. Later, the bireplicon would be resolved into two plasmids of the same plasmid family but belonging to different incompatibility groups (Sykora, 1992). The second model proposes that in the first stage, two strains containing the same plasmid evolve independently. Changes in the plasmid sequences involved in replication and/or partition generate different incompatibility groups by divergent evolution. Next, a plasmid of one of these strains is mobilized to another strain already containing its own member of the involved plasmid family. The resident plasmid and the incoming plasmid can then coexist without interference.

The phylogenetic analysis of the collection of 17 sequenced members of the repABC family gives the opportunity to discriminate between the two models. This collection includes different incompatibility groups and, most important, it includes the replication/ partition sequences of four strains with more than one repABC replicon. These plasmids and strains are: plasmids p42a, p42b, and p42d of R. etli; plasmids pTiC58, pAtC58, and the linear chromosome of A. tumefaciens C58; plasmids pSyma and pSymb of S. meliloti 1021; and plasmids pMLa and pMLb of Mesorhizobium loti MAFF303099 (see Table 1).

To determine which of the models outlined above describes more realistically the evolution of the repABC plasmids, phylogenetic trees of each of the repABC products were constructed and their topologies compared. In summary, if the proteins of plasmids belonging to one strain (and for this reason, of different incompatibility group) form a tight cluster it indicates that the replication regions of those plasmids originated in the same strain or in closely related strains. This tree topology would support the first model described here (the bireplicon intermediary model). In contrast, if the proteins of plasmids of the same strains do not share the same cluster, and at the same time plasmids belonging to the same incompatibility group are clustered together, this would indicate that the acquisition of the replication/partition regions of those plasmids was through horizontal transfer (separately diverging model).

In this paper, we show that the repABC plasmids contained in one strain are not closely related, indicating that the plasmids were acquired by lateral transfer from an unrelated strain or species.

Section snippets

Bacterial strains and growth conditions

E. coli DH5α (Hanahan, 1983) and S17-1(Simon et al., 1983) strains were grown at 37 °C in Luria–Bertani medium. Two recA derivatives strains of R. etli CFN42 were utilized: CFNX101 a strain harboring p42d, and CFNX107 lacking plasmid p42d (Martı́nez-Salazar et al., 1991). These strains and their transconjugant derivatives were grown at 30 °C in PY medium (Noel et al., 1984). Antibiotics were added at the following concentrations (in μgml−1): Nalidixic acid 20, kanamycin 30, chloramphenicol 25,

Isolation and nucleotide sequence of the replication/partition region of plasmid p42a

A cosmid library of plasmid p42a, constructed in a mobilizable vector able to replicate in Rhizobium, was mated into R. etli CFN42. A plasmid profile analysis of the transconjugant strains showed that two overlapping cosmids exhibited incompatibility towards endogenous plasmid p42a, indicating that they contained either trans-acting incompatibility factors or cis-acting targets sites for incompatibility determinants encoded in plasmid p42a.

Among other fragments both cosmids share three EcoRI

Acknowledgements

We thank David Romero, Michael Dunn, and Brenda Valderrama for their critical comments, and Angeles Pérez-Oseguera, Nora Soberón, Patricia Bustos, and Rosa E. Gómez for their skilful technical support. We also thank Paul Gaytán and Eugenio López for the synthesis of oligonucleotides, and Dr. Atsuhiro Oka for plasmid pAO244. This work was supported by DGAPA-PAPIIT grant IN214898.

References (43)

  • D Bartosik et al.

    Identification of the partition site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1

    J. Bacteriol.

    (2001)
  • N.J Brewin et al.

    Plasmid-mediated transferer of host-range specificity between two strains of Rhizobium leguminosarum

    J. Gen. Microbiol.

    (1980)
  • S Brom et al.

    Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance

    J. Bacteriol.

    (1992)
  • D.H Demezas et al.

    Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum

    Mol. Ecol.

    (1995)
  • C Freiberg et al.

    Molecular basis of symbiosis between Rhizobium and legumes

    Nature

    (1997)
  • F Galibert et al.

    The composite genome of the legume symbiont Sinorhizobium meliloti

    Science

    (2001)
  • A Garcı́a-de los Santos et al.

    Rhizobium plasmids in bacteria–legume interactions

    World J. Microbiol. Biotechnol.

    (1996)
  • A Garcı́a-de los Santos et al.

    Characterization of two plasmid-borne lps beta loci of Rhizobium etli required for lipopolysaccharide synthesis and for optimal interaction with plants

    Mol. Plant Microbe Interact.

    (1997)
  • E Geniaux et al.

    Comparison of geographically distant population of Rhizobium isolated from root nodules of Phaseolus vulgaris

    Mol. Ecol.

    (1993)
  • B Goodner et al.

    Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58

    Science

    (2001)
  • D Hanahan

    Studies of transformation of E. coli with plasmids

    J. Mol. Biol.

    (1983)
  • Cited by (33)

    • Mutations in an antisense RNA, involved in the replication control of a repABC plasmid, that disrupt plasmid incompatibility and mediate plasmid speciation

      2015, Plasmid
      Citation Excerpt :

      Over time, this will lead to the generation of different incompatibility groups. If a plasmid of one of these strains is mobilized to another strain already containing a plasmid with the same common ancestor, both plasmids can then coexist without interference due to the divergent evolution (Cevallos et al., 2002). In this work, we show that a third hypothesis is possible: the acquisition of mutations allows the generation of new plasmid species in the same strain without disruption of the replication/segregation properties of the participating plasmids.

    • RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon

      2013, Plasmid
      Citation Excerpt :

      This modified operon sequence was introduced into a vector (pDOABTi) that is incapable of replicating in Rhizobium spp but contains the complete segregation system of Agrobacterium plasmid pTiC58. It is important to mention that this segregation system is compatible with plasmid p42d; in other words, the pTi segregation elements introduced into R. etli do not influence the performance of the p42d replication/segregation system (García-de los Santos and Brom, 1997; Cevallos et al., 2002). The resultant construct, designated pDOABTi-ΔA1N, was introduced into an R. etli strain lacking p42d (CFNX107), generating CFNX107 (pDOABTi-ΔA1N [CmR]).

    • RepABC-based replication systems of Rhizobium leguminosarum bv. trifolii TA1 plasmids: Incompatibility and evolutionary analyses

      2011, Plasmid
      Citation Excerpt :

      The existence of such multipartite genomes raises several questions regarding the multiple replicon maintenance and segregation during the cell cycle. It was proposed that repABC plasmids coexisting in the same strain arose most probably by separate events of lateral gene transfer, further forcing the appearance of different new incompatibility groups and allowing co-residence of plasmids equipped with the same replication/partition machinery in one bacterial cell (Cevallos et al., 2002). The varying degree of adaptation to the host genome observed both on the level of the various incompatibility groups, as well as the differences in repABC operons coexisting in the same species, support this hypothesis.

    View all citing articles on Scopus
    View full text