Expression of hydrogenase in Hupc strains of Bradyrhizobium japonicum

Plasmid-borne hup-lacZ transcriptional fusion constructs were introduced into three separate mutant strains of Bradyrhizobium japonicum which express hydrogenase constitutively (Hup(c) strains SR470, SR473 and JH101) in both autotrophic and heterotrophic environments. The lacZ structural gene linked directly to the regulatory region upstream of the hydrogenase structural gene encompassing -149 bases expressed beta-gal at a constant, high level, in response to various concentrations of Ni (0 micromolar to 1 micromolar). beta-Gal activity was expressed at a constant level in response to variations in concentration of O2 (0%-10%) and H2 (0%-10%) as well. The cis-acting region required to express hydrogenase constitutively is located between -149 and -98 bases. This is also the site of nickel, oxygen and hydrogen-dependent regulatory action in the wild-type strain. It is postulated that a single mutation in Hup(c) strains affects the transacting factor which would normally by responsive to Ni, O2 and H2.