Evidence for a Third Uptake Hydrogenase Phenotype among the Soybean Bradyrhizobia

The existence of a hydrogen uptake host-regulated (Hup-hr) phenotype was established among the soybean bradyrhizobia. The Hup-hr phenotype is characterized by the expression of uptake hydrogenase activity in symbiosis with cowpea but not soybean. Uptake hydrogenase induction is not possible under free-living cultural conditions by using techniques developed for uptake hydrogenase-positive (Hup) Bradyrhizobium japonicum. Hydrogen oxidation by Hup-hr phenotype USDA 61 in cowpea symbioses was significant because hydrogen evolution from nitrogen-fixing nodules was not detected. An examination for uptake hydrogenase activity in soybean and cowpea with 123 strains diverse in origin and serology identified 16 Hup and 28 Hup-hr phenotype strains; the remainder appeared to be Hup. The Hup-hr phenotype was associated with serogroups 31, 76, and 94, while strains belonging to serogroups 6, 31, 110, 122, 123, and 38/115 were Hup. Hup strains of the 123 serogroup typed positive with USDA 129-specific antiserum. The presence of the uptake hydrogenase protein in cowpea bacteroids of Hup strains was demonstrated with immunoblot analyses by using antibodies against the 65-kDa subunit of uptake hydrogenase purified from strain SR470. However, the hydrogenase protein of Hup-hr strains was not detected. Results of Southern hybridization analyses with pHU1 showed the region of DNA with hydrogenase genes among Hup strains to be similar. Hybridization was also obtained with Hup-hr strains by using a variety of cloned DNA as probes including hydrogenase structural genes. Both hydrogenase structural genes also hybridized with the DNA of four Hup strains.