• β–glucuronidase;
  • integration vector;
  • luciferase;
  • recA;
  • Rhizobium meliloti;
  • Rhizobium leguminosarum biovar. viciae


A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.