GREENHOUSE AND FIELD EVALUATIONS OF AN AUTOSELECTIVE SYSTEM BASED ON AN ESSENTIAL THYMIDYLATE SYNTHASE GENE FOR IMPROVED MAINTENANCE OF PLASMID VECTORS IN MODIFIED RHIZOBIUM-MELILOTI

The stability of the thy autoselective system, based on an essential t hymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-g rown alfalfa, The thy autoselective system consists of a free-replicat ing, broad-host-range plasmid vector containing a copy of the thyA gen e from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(-)), Under greenhouse conditions, Thy(-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of th e host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(-) rhizobia contained only T hy(+) revertants. In soil, Thy(-) rhizobia were compromised and failed to nodulate alfalfa, Thy(-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. T he thy autoselective system was tested in the field with Thy(-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistanc e genes and marked with constitutively expressed lacZY, At 80 days aft er sowing, most rhizobia isolated from the nodules of field-grown alfa lfa inoculated with Rm42T(pPR602) contained pPR602, The thy autoselect ive system proved useful to ensure maintenance of the plasmid vector u nder greenhouse and field conditions in R. meliloti.