Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA).

To select for bacterial strains with enhanced phenotypes, random fragments of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector. The resulting plasmids are integrated by recombination into the homologous DNA region of the original strain. Integration gives rise to a nontandem direct duplication of the corresponding DNA region separated by the vector moiety of the plasmid. Recombination between the direct repeats leads to tandem duplication and further amplification of the entire integrated DNA, including the vector. Bacteria harboring the amplified DNA are selected by increasing the dosage of an antibiotic corresponding to a resistance marker of the integrated vector. Pooled strains carrying amplifications are then challenged with a selective pressure for the desired phenotype. After repeated selection cycles, the most fit strains are isolated. We used this process, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation. Derivatives containing randomly amplified DNA regions of the symbiotic plasmid of Rhizobium tropici CFN299 strain were generated. Pools of amplified strains were inoculated onto various tropical legumes. After several cycles of selection through plants, amplified derivatives showing an increased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained.