Rhizobium japonicum USDA191 is a member of a new group of Rhizobium japonicum strains found in China. This strain is one of several strains shown to be salt-tolerant and fast-growing; it is unique in being the only strain of this group that effectively nodulates American soybean cultivars. For these reasons strain USDA 191 was chosen for further study and comparison to the common American Rhizobium japonicum isolate USDA 1 10. Strain USDA 19 1 has a doubling time of 3.2 h in complex medium and grows in concentrations of up to 0.4 M-NaCl, while strain USDAl 10, which has a doubling time of 12 h, is severely inhibited in media containing 0.1 MNaCl. Under salt stress conditions, intracellular levels of K+ and glutamate were shown to increase. A comparison based on carbohydrate metabolism, DNA homology and protein patterns on polyacrylamide gels reveals that strain USDA191 is more closely related to the fastgrowing rhizobia than to Rhizobium japonicum. However, the strain retains capacity to nodulate American soybean and cowpea cultivars effectively.

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A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobiumjaponicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% 02). The tritium exchange assay was used to screen a variety of different strains of R. jponicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H2. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H2 uptake efficiency of intact nodules.The enzyme, hydrogenase, has been reported to play an important role in the energy metabolism of many microorganisms (2,5,8,13). Schubert and Evans (4,11,12) suggested that strains of Rhizobium spp. which possess a hydrogenase are more efficient in utilization of photosynthate during symbiotic N2 fixation by recycling the H2 produced by nitrogenase. The procedure used by these authors as well as by others for determining hydrogenase activity involves measuring the H2 uptake from whole nodules inoculated with the respective strains or bacteroids isolated from these nodules. This procedure is both tedious and time-consuming and requires the utilization of the reducing power produced from H2 by hydrogenase. O'Gara and Shanmugam (10) recently described a procedure for determining the H2 uptake capacity of Rhizobium trifolii mutant strains using free-living cultures. In this communication, a rapid procedure for determining hydrogenase activity, involving 'H2 exchange, using free-living cultures of Rhizobium japonicum is presented. MATERIALS AND METHODSBacterial Strains and Growth Conditions. R. japonicum strains 31, 76, 110, 117, 123, 138, 142 was also used for preparing inoculum for the induction experiments described below. It has the following composition: (g.1-1) mannitol, 1.0; yeast extract, 1.0; KH2PO4, 0.3; Na2HPO4, 0.3; MgSO4 -7H20, 0.1; CaCl2, 0.5; and trace metals (mgl-') H3BO3, 10.0; ZnS04 2H20, 1.0; FeCl3, 1.0; CuSO4 5H20, 0.5; MnCl2; 0.5; Na2MoO4 2H20, 0.1; biotin, 0.2. The pH was adjusted to 6.8. Liquid cultures were grown at 30 C starting from a 2% (v/v) inoculum. Growth was followed by measuring A using a KlettSummerson colorimeter fitted with a green filter (no. 54) or by measuring A at 420 nm in a Gilford model N spectrophotometer (1-cm light path) and also by determining the increase of cell protein (3). Protein was routinely assayed by the procedure of Lowry et al. (7) using BSA as a standard.Hydrogenase Induction. Cultures for use as inoculum were grown aerobically in MY medium at 25 C for 48 hr in a rotary shaker and washed in N2-free medium. A 5% inoculum was used to inoculate the following medium (2.0 ml in 7 1-ml serum ...