Conjugal Transfer of Megaplasmid 2 between Rhizobium meliloti Strains in Alfalfa Nodules

A DNA fragment containing the RP4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of Rhizobium meliloti 0540 (Inf⁻ EPS⁻), resulting in PG101 (Inf⁻ EPS⁻). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and R. meliloti 2526 (Nod⁻). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 × 10⁻⁷. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 × 10⁻⁵. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.