Genetic transfer and expression of plasmid RP4::TOL in Sinorhizabium meliloti, Bradyrhizobium japonicum and B-elkanii

Sinorhizobium species generally obviate the need for nitrogen fertilizer since they supply their legume hosts with biological fixed nitrogen, Our working hypothesis is that Sinorhizobium species can perhaps be genetically modified to perform rhizosphere-stimulated bioremediation, Plasmid RP4::TOL has gene clusters encoding the metabolic pathways for benzene, toluene and xylene degradation. Originally from Pseudomonas, this plasmid, in Escherichia coli C600, was successfully transferred to symbiotically elite strains of alfalfa-nodulating Sinorhizobium meliloti USDA 1936. Reciprocal crosses with E, coli RR28, followed by analysis of DNA by restriction enzymes verified S, meliloti USDA 1936 (RP4::TOL) exconjugant, Reciprocal crosses involving B, japonicum USDA I-110 (RP4::TOL) exconjugant and B. elkanii USDA 61 (RP4,,TOL) exconjugant as donors with E. coli RR28 as a recipient produced exconjugant bearing derivative plasmids having a major part of the TOL region deleted, Therefore, soybean-nodulating, B, japonicum USDA I-110 and B, elkanii USDA 61, were not suitable hosts for stable maintenance of this plasmid, Colony size of S, meliloti (RP4::TOL) exconjugant and B, elkanii (RP4,,TOL) exconjugant were enhanced by the addition of 1.0 mM meta-toluic acid in minimal salts media. Although in untreated sterile soil, S, meliloti USDA 1936 (RP4,:TOL) exconjugant formed more nodules per plant than S, meliloti USDA 1936, it was determined to be symbiotically a competent in nitrogen fixation, based on plant dry weight and acetylene reduction. In liquid culture 77% reduction in toluate was recorded in 10 d by this exconjugant compared with only 15% reduction by the parent culture, Alfalfa plant tin situ) studies revealed almost 100% reduction in the concentration of 1.0 mM m-toluate by S, meliloti USDA 1936 (RP4:,TOL) exconjugant when tested after 8 weeks compared with 92% by the parent culture. Thus, a symbiotic combination of genetically-modified S. meliloti and the alfalfa plants were shown to have potential usefulness in the bioremediation of toxic hydrocarbons in soil. (C) 1999 Elsevier Science Ltd. All rights reserved.