Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation

DNA sequences responsible for the development and maintenance of symbiotic nitrogen fixation have been identified and isolated from Rhizobium trifolii. Symbiotically-defective strains were generated by random mutagenesis with the transposon Tn5. The defective genes which give rise to the mutant phenotype have been cloned into bacterial plasmids and used as hybridization probes to isolate the corresponding wild-type genes from a clone bank of R. trifolii DNA. Symbiotic genes cloned in this manner are able to correct the lesion caused by the insertion of the transposon in their respective mutants and so restore the nitrogen fixation phenotype. The correction of the mutation is shown to occur by two distinguishable mechanisms--either by complementation or by homologous recombination. This approach provides a reliable method for isolation and mapping of bacterial DNA sequences involved in symbiotic nitrogen fixation.