Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis

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Abstract
Symbiotic and auxotrophic mutants (20 each) of R. meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), were isolated by transposon Tn5 mutagenesis. [The mutants were characterized physically and genetically.] A suicide plasmid mutagenesis procedure was used to generate Tn5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random Tn5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). A combination of physical and genetic criteria were used to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 element. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was .apprx. 10-8 and the frequency of precise excision was < 10-9. In .times. 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These latter strains exhibited anomalous conjugation properties, and therefore the symbiotic phenotype could not be correlated with a Tn5 insertion. Both physical and genetic tests were required to fully characterize transposon-induced mutations.