Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H2

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO₂ and H₂ (Aut⁻) were isolated. Five Aut⁻ mutants lacked hydrogen uptake activity (Hup⁻). The other seven Aut⁻ mutants possessed wild-type levels of hydrogen uptake activity (Hup⁺), both in free-living culture and symbiotically. Three of the Hup⁻ mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup⁻ only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O₂. All five Hup⁻ mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut⁻ Hup⁺ mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut⁻ Hup⁺ mutant, had CO₂ fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut⁻ mutant. The Aut⁻ Hup⁻ mutants that were Hup⁻ both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup⁻ mutants.