GENERATION OF RHIZOBIUM STRAINS WITH IMPROVED SYMBIOTIC PROPERTIES BYRANDOM DNA AMPLIFICATION (RDA)

To select for bacterial strains with enhanced phenotypes, random fragm ents of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector. The resulting plasmids are integrated by r ecombination into the homologous DNA region of the original strain. In tegration gives rise to a nontandem direct duplication of the correspo nding DNA region separated by the vector moiety of the plasmid. Recomb ination between the direct repeats leads to tandem duplication and fur ther amplification of the entire integrated DNA, including the vector. Bacteria harboring the amplified DNA are selected by increasing the d osage of an antibiotic corresponding to a resistance marker of the int egrated vector. Pooled strains carrying amplifications are then challe nged with a selective pressure for the desired phenotype. After repeat ed selection cycles, the most fit strains are isolated. We used this p rocess, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation. Derivativ es 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 c ycles of selection through plants, amplified derivatives showing an in creased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained.