Materials and Methods

Cells of B. japonicum 3Ilbl 10 (US Department of Agriculture, Beltsville, MD, USA) and mutant derivatives nirK GRK13, norC GRC131 and nosZ GRZ25 (Mesa et al. 2001) were used. Plasmid isolations, restriction enzyme digestions, agarose gel electrophoresis, ligations and E. coli transformations were performed according to standard protocols (Sambrook et al. 1989). Seeds of Glycine max L. Merr. cv. Williams were surface-sterilized, germinated, planted in sterile Leonard jars, and grown in controlled environment chambers as previously described (Delgado et al. 1998). For nitrate treatments, the mineral nutrient solution was supplied with 4 mM KNO3. Plants were harvested 40-45 days after planting. Plant dry weight and reduced nitrogen content (Kjeldhal analysis) were determined on plants that had been heated at 85°C for 48 h. Bacteroids were prepared by grinding 1 g of nodules with 50 mM Tris-HCl buffer (pH 7.4) containing 250 mM mannitol (Delgado et al. 1998).

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