Localized mutagenesis in Rhizobium japonicum

Summary The slow-growing soybean symbiont, Rhizobium japonicum, has not readily been accessible so far to classical mutational analysis of genes responsible for symbiotic nitrogen fixation. We have overcome part of this problem by the successful application of a site-directed mutagenesis technique to this organism. The following steps are involved: (i) local Tn5 mutagenesis, in E. coli, of cloned R. japonicum DNA (e.g. the nifDK operon); (ii) conjugational transfer of the mutated DNA into R. japonicum using vectors which are unable to replicate there; (iii) selection of R. japonicum exconjugants which have exchanged their wild-type genomic DNA region for the Tn5-containing fragment by homologous recombination. While using this technique it appeared mandatory to distinguish double-crossover-events (true replacements) from single-crossover events (replicon fusions or cointegrations). Only the true replacement mutants were genetically stable; their phenotypes were determined with respect to nodulation (Nod) and nitrogen fixation (Fix) by plant infection tests. Tn5 mutations within nifD and nifK caused a Nod⁺ Fix^- phenotype, whereas mutants with insertions in the immediate vicinity on either side of nifDK were found to be Nod⁺ Fix⁺, suggesting that genes flanking nifDK may not be involved in the nitrogen fixing symbiosis. Nodule reisolates were found to carry Tn5 at their original locations.