The establishment of a nitrogen-fixing symbiotic relationship between soil-dwelling bacteria (rhizobia) and legumes is a valuable source of bioavailable nitrogen in the biosphere. The genes required for the establishment and maintenance of this symbiosis may be encoded on plasmids or within the chromosome of the rhizobial microsymbiont as a Symbiosis Island (SI). Previous studies have found that Mesorhizobium ciceri bv. biserrulae WSM1271, which forms a nitrogen-fixing symbiosis with the pasture legume Biserrula pelecinus, transferred its tripartite SI to at least two presumably non-symbiotic Mesorhizobium spp., converting them into B. pelecinus-nodulating rhizobia. These newly evolved symbionts were not as effective in nitrogen fixation with B. pelecinus as WSM1271, even though they had acquired the entire SI from WSM1271. Importantly, WSM1271 harbours a plasmid (pMESCI01) that is absent from WSM2073 and WSM2075. This plasmid may carry additional determinants required for highly effective symbiotic interactions with B. pelecinus. Therefore, analysis of the potential symbiotic role and transmissibility of this plasmid may help explain the differences in nitrogen fixation of these three strains with B. pelecinus.
Bioinformatic analysis of pMESCI01 revealed >50% of genes to have hypothetical functions only, with a diverse range of function predictions for the remainder of genes. Crucially, 12 putative nitrogen fixation genes were identified on pMESCI01 that could have a role in the symbiosis between WSM1271 and B. pelecinus. Homologs of known plasmid conjugal transfer type IV secretion systems (T4SS) or relaxases were not detected, but a potential origin of transfer (oriT) site was located, leaving open the question of whether pMESCI01 is self-transmissible.
To determine whether pMESCI01 is self-transmissible, the plasmid was marked with an Ω-Sp/Sm cassette, encoding spectinomycin and streptomycin resistance. Possible transconjugants from a subsequent conjugation experiment between WSM1271-pMESCI01::Ω-Sp/Sm and WSM2073Nm could not be screened, owing to unexpected spectinomycin resistance of the WSM2073Nm recipient. However, WSM2073Nm was confirmed to be highly sensitive to streptomycin, so the conjugation experiment can be repeated in future to assess self-transmissibility of pMESCI01 with pMESCI01::Ω-Sp/Sm.
To assess whether pMESCI01 was essential to nitrogen fixation in WSM1271 with B. pelecinus, the curing vector pSRKrepABC was constructed and plasmid-cured derivatives of WSM1271 were produced via a plasmid incompatibility approach. Phenotypic assessment of wild-type WSM1271 compared to the plasmid-cured derivatives inoculated onto B. pelecinus in a glasshouse trial revealed no difference in mean shoot dry weights or nodulation of plants across the treatments. This indicated that the plasmid-located genes are likely to not be essential to nitrogen fixation in WSM1271 with this host.
Alignment of the sequence of pMESCI01 with accessory plasmids from other fully-sequenced M. ciceri strains revealed a high percentage of homology across the plasmids from these strains, which originate from geographically diverse locations. This suggests that these M. ciceri plasmids are widely dispersed and may be a common feature of this species. While the work in this thesis indicates that pMESCI01 is not essential in the symbiosis between WSM1271 and B. pelecinus, genes on the plasmid may still impart important regulatory or metabolic benefits to WSM1271. Future investigations into the possible function of the large number of genes (432 in total) on pMESCI01 may help to address the role of this plasmid in WSM1271 and, by extension, the role of accessory plasmids in other M. ciceri strains. Therefore, continued studies of Mesorhizobium spp. would be important in determining the evolution, transmissibility and role of these plasmids
