Nickel uptake in Bradyrhizobium japonicum

Free-living Bradyrhizobium japonicum growth heterotrophically with 1 μM Ni accumulated label. Strain SR470, a Hup(c) mutant, accumulated almost 10-fold more Ni on a per-cell basis that did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hup(c) mutant strain SR470 again accumulating significantly more Ni than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent K(m)s for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 μM, respectively. The V(max) values, however, were significantly different, 0.29 nmol of Ni/min per 10 cells for SR and 1.40 nmol of Ni/min per 10 cells for SR470. The uptake process was relatively specific for nickel; only Cu and Zn (10 μM) were found to appreciably inhibit the uptake of 1 μM Ni, while a 10-fold excess of Mg, Co, Fe, or Mn did not affect Ni uptake. The lack of inhibition by Mg indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4°C) and slightly by the ionophores nigerican and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased repspiratory activity. Nickel uptake showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the Ni taken up in 1 min by strain SR470 was not exchangeable with cold nickel.