The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel‐dependent transcriptional regulation of hydrogenase

Abstract

The HypB protein from Bradyrhizobium japonicum is a metal‐binding GTPase required for hydrogenase expression. In‐frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JHΔEg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JHΔ23H lacking only the N‐terminal histidine‐rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JHΔ23H was low in comparison to the wild type in 10–50 nM nickel levels, but could be cured to nearly wild‐type levels by including 50 μM nickel during the derepression incubation. Studies on strains harbouring the hup promoter–lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild‐type promoter activity in 50 μM NiCl₂. Further evidence that HypB is required for hup promoter‐binding activity was obtained from gel‐shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O₂, 10% partial pressure H₂) HypB was detected, and its expression preceded hydrogenase synthesis by 3–6 h. ⁶³Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild‐type strain at all time points tested during the derepression incubation. Wild‐type cultures that received nickel during the HypB expression‐specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JHΔ23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal‐binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.