Isolation of genes involved in nodulation competitiveness from Rhizobium leguminosarum bv. trifolii T24.

Rhizobium leguminosarum bv. trifolii T24 produces a potent anti-rhizobial compound, trifolitoxin, and exclusively nodulates clover roots when in mixed inoculum with trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii [Schwinghamer, E. A. & Belkengren R. P. (1968) Arch. Mikrobiol. 64, 130-145]. In the present study, the isolation of trifolitoxin production and resistance genes is described. A cosmid genomic library of T24 was prepared in pLAFR3. No trifolitoxin expression was observed in the resulting Escherichia coli cosmid clones. One cosmid clone was identified that restored trifolitoxin production and nodulation competitiveness in three nonproducing mutants of T24. The recombinant plasmid from this cosmid clone, pTFX1, also conferred trifolitoxin production and resistance when transferred to symbiotically effective strains of R. leguminosarum bvs. trifolii, phaseoli, and viceae. Cosmid pTFX1 also conferred expression of trifolitoxin production when present in strains of Rhizobium meliloti and Agrobacterium tumefaciens. No trifolitoxin expression was observed in strains of Bradyrhizobium japonicum or Rhizobium sp. (cowpea) with pTFX1. Southern blot analysis with a biotinylated pTFX1 probe did not suggest that these genes were plasmid-borne. Transfer of pTFX1 to T24 or its derivatives resulted in 6- to 10-fold higher level of trifolitoxin production than wild-type T24.