Identification of a locus within the hydrogenase gene cluster involved in intracellular nickel metabolism in Bradyrhizobium japonicum

Abstract

A 0.6-kb fragment of DNA involved in intracellular Ni metabolism was isolated and cloned from a cosmid containing 23.2 kb of hydrogenase-related genes of Bradyrhizobium japonicum. This locus is located 8.3 kb upstream of the hydrogenase structural genes. The hydrogenase activity of a mutant with a gene-directed mutation at this locus (strain JHK7) showed dependency on nickel provided during hydrogenase derepression. The hydrogenase activity was only 20% of that in the wild-type strain, JH, at a concentration of 0.5 μM NiCl2. The hydrogenase activity in JH reached its maximum at 3 μM NiCl2, whereas the mutant (JHK7) reached wild-type levels of hydrogenase activity when derepressed in 50 μM NiCl2. Studies with the hup-lacZ transcriptional fusion plasmid pSY7 in JHK7 showed that the mutant JHK7 expressed less promoter activity under low-nickel conditions than did strain JH. The mutant accumulated less nickel during a 45-h hydrogenase derepression period than did the wild type. However, both JHK7 and the JH wild-type strain had the same short-term Ni transport rates, and the K(m)s for Ni of both strains were about 62 μM. When incubated under non-hydrogenase-derepression conditions, the mutant accumulated Ni at the same rate as strain JH. However, this stored source of nickel was unable to restore hydrogenase expression ability of the mutant to wild-type levels during derepression without nickel. The results indicate that the locus identified in B. japonicum is not involved in nickel- specific transport; indeed, it was not at all homologous to the 'nickel transporter' hoxN gene of Alcaligenes eutrophus. Rather, its product is probably involved in intracellular Ni incorporation into hydrogenase and the trans-acting regulator that is responsive to nickel levels.