Results and Discussion

The chromosomal dct operons of R7A and Mesorhizobium sp. strain CJ1 complemented Rhizobium leguminosarum bv. viciae dctA, dctB and dctD mutants, indicating they are functionally intact. This was confirmed by sequencing. However the operons did not complement non-symbiotic mesorhizobia. Sequence analysis of the dct operon from a CJlDct+ variant indicated that the adaptive mutation did not occur in the operon. A DNA library of CJlDct+ was introduced into a Dct" non-symbiont and a cosmid pJWl isolated that conferred the ability to utilize succinate. pJWl was used as a probe against genomic DNA from several mesorhizobia and only hybridized to CJ1 and N18, the strains that give rise to Dct+ variants. pJWl hybridized to a plasmid in these strains, suggesting that there is some form of communication between the plasmid-encoded activating mutation and the chromosomally-encoded dct operon. Sequence obtained from the plasmid suggested it encodes the ability to synthesize nicotinamide and to catabolize rhizopine, in addition to the ability to utilize succinate. Tn5 mutagenesis indicated three genes may be involved in the switch to succinate utilization, a lacl family repressor and two genes of unknown function. Further work is aimed at characterizing the role of these genes and identifying the actual adaptive mutation.

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