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- Title
The construction of recA-deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk-assessment studies.
- Authors
Selbitschka, W.; Pühler, A.; Simon, R.
- Abstract
A vector system was developed employing the <em>rec</em>A genes of <em>Rhizobium meliloti</em> and <em>Rhizobium leguminosarum</em> biovar. <em>viciae</em> 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 <em>rec</em>A genes of <em>R. meliloti</em> and <em>R. leguminosarumbv. viciae</em>. Consequently, the recombinant strains become recombination deficient (RecA-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptII) promoter of transposon Tn5. The system was used to construct <em>rec</em>A mutant strains of <em>R. meliloti</em> and <em>R. leguminosarum bv. viciae</em>, carrying the <em>Escherichia coli gusA</em> gene encoding β-glucuronidase as well as the firefly (<em>Photinus pyralis</em>)<em>luc</em> 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 <em>luc</em> 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 <em>rec</em>A mutants as model organisms in risk assessment studies with genetically engineered <em>Rhizobium</em> strains.
- Subjects
RISK assessment in genetic engineering; RHIZOBIUM meliloti; RHIZOBIUM leguminosarum; RECOMBINANT DNA; GENETIC markers; GENE expression; GENOMICS
- Publication
Molecular Ecology, 1992, Vol 1, Issue 1, p9
- ISSN
0962-1083
- Publication type
Article
- DOI
10.1111/j.1365-294X.1992.tb00150.x