Expression of hydrogenase activity in free-living Rhizobium japonicum

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Abstract
A medium is described on which selected R. japonicum strains express hydrogenase (H2 uptake) activity under free-living conditions. Low concentrations of C substrates, decreased O2 tension and the quantity of combined N in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H2 under conditions such that the cells did not exhibit nitrogenase activity. H2 uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H2 oxidized this gas when grown in free-living cultures. In comparison 6 randomly chosen strains forming nodules that lost H2 in air either showed no or low capacity to take up H2 under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H2. This method enabled the isolation of a spontaneous R. japonicum mutant strain that lost the ability to utilize H2. This mutant strain forms nodules that evolve H2 but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the N2-fixing process of legumes.