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

Abstract

SummaryThe 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 (JH⌬Eg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JH⌬23H lacking only the N-terminal histidinerich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JH⌬23H 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 M 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 M NiCl 2 . 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 O 2 , 10% partial pressure H 2 ) HypB was detected, and its expression preceded hydrogenase synthesis by 3-6 h. 63 Ni 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 expressionspecific 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 JH⌬23H 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.

The Identification, Purification, and Characterization of CooJ

“…HypB is required for in vivo (24) and in vitro (25) nickel insertion because deletions of HypB yield inactive hydrogenase (24, 25). The histidine-rich region of HypB has been shown to store nickel in B. japonicum (26).In this work, we report the identification, purification, and characterization of the nickel-binding protein, CooJ, from R. rubrum. The purification of wild type CooJ was complicated by the co-purification of CODH and several other proteins until detergent and chaotropic agents separated CooJ from these proteins.…”

“…HypB is required for in vivo (24) and in vitro (25) nickel insertion because deletions of HypB yield inactive hydrogenase (24,25). The histidine-rich region of HypB has been shown to store nickel in B. japonicum (26).…”

Molecular Biology of Microbial Hydrogenases

“…HupUV does indeed regulate the transcription of the hup genes (see above) and needs Ni to be active. It is rather HypB, a nickel-binding GTPase necessary for incorporation of Ni into the H 2 ase apoprotein that relays the signal to the regulatory proteins (Olson et al, 1997;Olson and Maier, 2000). On the other hand, the homologous HoxBC (RH) protein in R. eutropha, which contains Ni (Pierik et al, 1998), is nearly independent of HypB .…”

“…CP: carbamoyl phosphate. present at the N terminus of HypB enables the protein to store nickel (Rey et al, 1994;Fu et al, 1995;Olson et al, 1997, Olson andMaier, 2000). In other organisms, such as E. coli or R. eutropha, the absence of this nickelstorage function has been assumed to be compensated by a highly effi cient nickel transport system (Eitinger and Mandrand-Berthelot, 2000).…”

“…Whereas HypA functions in the maturation of H 2 ase-3 of E. coli, a homologue encoded by hybF in the hyb operon operates in H 2 ase-1 and -2 maturation . All HypB proteins have a conserved GTP-binding motif at the carboxyl terminus, and GTPase activity has been shown to be essential for Ni insertion Olson et al, 1997;Olson and Maier, 2000). In some organisms, such as Rhizobium leguminosarum, R. capsulatus, Azotobacter vinelandii or Bradyrhizobium japonicum, a histidine-rich domain Figure 5.…”