EVALUATION OF THE RECA-BASED CONTAINMENT SYSTEM IN RHIZOBIUM-MELILOTIGR4

Rhizobium meliloti recA strains have been reported as safe hosts for d eliberate release experiments due to their reduced survival in soil mi crocosms. Using R. meliloti integration vectors, two GUS-positive RecA (+) and RecA(-) isogenic derivatives of R. meliloti strain GR4 were co nstructed. In contrast to previously reported RecA(-) strains, the Rec A(-) strain GR4KLR showed similar doubling times as its isogenic RecA( +) strain GR4KLC. Accordingly, GR4KLR showed normal growth competition capacity when coinoculated with the parental strain in sterile soil m icrocosms. In addition, the recA mutant showed delayed nodulation on a xenically grown alfalfa plants, and its competitive nodulation ability was severely affected. This defect could be corrected by a genetic ma nipulation based on the use of a multicopy nifA-expressing plasmid. An other novel phenotype associated with the recA mutation in R. meliloti GR4 was an increased rate of plasmid loss. When inoculated in non-ste rile soil microcosms, RecA(+) and RecA(-) strains established at high cell densities and no significant differences in their survival were f ound after 150 days incubation. Our results show that symbiotic perfor mance of a R. meliloti GR4 recA strain may be significantly altered wh ereas its relative survival in a test soil where it was to be released was not significantly reduced with respect to RecA(+) strains.