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

Abstract

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-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-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 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 later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.