Identification and analysis of rhizobial plasmid origins of transfer

Twelve Rhizobium leguminosarum isolates from France, Germany and the UK, each carrying between four and seven plasmids, were screened by PCR using primers designed to amplify partial traA and traC genes and the intergenic spacer (igs) between them, which is expected to contain oriT (the nick site for conjugal transfer). Five strains, 1062, RES-2, RES-6, RES-7 and RES-9, generated oriT-containing PCR fragments. Sequencing identified three types that are related to but different from other rhizobial plasmid oriT sequences in the database. Sequence comparisons revealed conserved motifs in the igs, including a 14-bp putative nic site, a stem-loop and a tra box. The RES-2, RES-6 and RES-9 PCR products were used as probes in Southern hybridisation studies to screen the 12 strains for related sequences. Eleven strains contain at least one homologous sequence, but of the 64 plasmids present among the 12 strains only 17 hybridised to the oriT probes. Four sequence variants of the repC plasmid replication initiation gene have previously been described in these strains, but there is no correlation between repC and oriT sequence distributions, and there is evidence for recombination to generate different repC-oriT combinations. Three plasmids, pYK32, pYK36 and pYK39, containing the oriT amplified from RES-2, RES-6 and RES-9, respectively, were constructed for functional analysis of the oriT sequence variation. Each plasmid was transformed into R. leguminosarum strains 1062, RES-2 and RES-9 to generate nine donor-plasmid combinations, and their mobilisation frequencies into Escherichia coli and Agrobacterium tumefaciens measured following biparental matings. All three plasmid constructs were mobilised at a similar frequency (10(-7) to 10(-8) per recipient) by each donor strain, suggesting that there is no discrimination by the transfer proteins between the different oriT sequences.