GENES ESSENTIAL FOR NOD FACTOR PRODUCTION AND NODULATION ARE LOCATED ON A SYMBIOTIC AMPLICON (AMPRTRCFN299PC60) IN RHIZOBIUM-TROPICI

Amplifiable DNA regions (amplicons) have been identified in the genome of Rhizobium etli, Here we report the isolation and molecular charact erization of a symbiotic amplicon of Rhizobium tropici, To search for symbiotic amplicons, a cartridge containing a kanamycin resistance mar ker that responds to gene dosage and conditional origins of replicatio n and transfer was inserted in the nodulation region of the symbiotic plasmid (pSym) of R. tropici CFN299, Derivatives harboring amplificati ons were selected by increasing the concentration of kanamycin in the cell culture. The amplified DNA region was mobilized into Escherichia coli and then into Agrobacterium tumefaciens. The 60-kb symbiotic ampl icon, which we termed AMPRtrCFN299pc60, contains several nodulation an d nitrogen fixation genes and is flanked by a novel insertion sequence ISRtr1, Amplification of AMPRtrCFN299pc60 through homologous recombin ation between ISRtr1 repeats increased the amount of Nod factors. Stri kingly, the conjugal transfer of the amplicon into a plasmidless A. tu mefaciens strain confers on the transconjugant the ability to produce R. tropici Nod factors and to nodulate Phaseolus vulgaris, indicating that R. tropici genes essential for the nodulation process are confine d to an ampliable DNA region of the pSym.