Evaluation of the recA-based containment system in Rhizobiummeliloti GR4

Abstract

Rhizobium meliloti recA strains have been reported as safe hosts for deliberate release experiments due to their reduced survival in soil microcosms. Using R. meliloti integration vectors, two GUS-positive RecA + and RecA − isogenic derivatives of R. meliloti strain GR4 were constructed. In contrast to previously reported RecA − strains, the RecA − 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 microcosms. In addition, the recA mutant showed delayed nodulation on axenically grown alfalfa plants, and its competitive nodulation ability was severely affected. This defect could be corrected by a genetic manipulation based on the use of a multicopy nifA-expressing plasmid. Another novel phenotype associated with the recA mutation in R. meliloti GR4 was an increased rate of plasmid loss. When inoculated in non-sterile soil microcosms, RecA + and RecA − strains established at high cell densities and no significant differences in their survival were found after 150 days incubation. Our results show that symbiotic performance of a R. meliloti GR4 recA strain may be significantly altered whereas its relative survival in a test soil where it was to be released was not significantly reduced with respect to RecA + strains.