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

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.     

Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH.

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.     

Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH.

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit

A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3′-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase. The truncated gene was transferred to the in situ position on the chromosome. Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity. The activity was not restored by high nickel concentrations in the medium. The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of ⁶³Ni²⁺. These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal. Received: 31 July 1995 / Accepted: 3 November 1995     

Stimulation by nickel of soil microbial urease activity and urease and hydrogenase activities in soybeans grown in a low-nickel soil

Summary The concentration of nickel in some soils may be insufficient to meet the requirements of enzymes such as urease in soybeans and hydrogenase in Rhizobium. In an initial evaluation of nickel availability, several soils were examined for nickel content and microbial urease activity. Total and extractable nickel were determined by atomic emission spectrometry. Purified glucose and urea were added to soils to stimulate microbial growth and urease activity, the latter of which was monitored by the rate of decomposition of¹⁴C urea. Nickel also was added to some samples to determine if the indigenous supply was limiting. In one low-nickel soil (total Ni 13 ppm) urease activity increased 150% in response to additional nickel, while other soils (total Ni 22–3491 ppm) failed to respond to nickel. However, additional nickel did stimulate urease activity (up to 109%) in 3 out of 10 soils to which purified CaCO₃ was added. Presumably the rise in pH associated with this treatment decreased nickel availability. Additions of Co, Mn, Fe, or Cu had no consistent effect on urease activity, thus indicating that the response to Ni was specific. Nickel fertilization increased leaf urease and nodule hydrogenase activity of soybeans grown in low-nickel soil, however, yield was not improved. These results may have practical implications in the nutrition of plants and micro-organisms that metabolize H₂ and urea.     

Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A.

The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM).     

Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH-and MBH-specific antigen. Growing the cells in the presence of ⁶³Ni²⁺ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.     

Protein interactions and localization of the Escherichia coli accessory protein HypA during nickel insertion to [NiFe] hydrogenase

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event.     

The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein.

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.     

Involvement of the GroE chaperonins in the nickel-dependent anaerobic biosynthesis of NiFe-hydrogenases of Escherichia coli.

We analyzed the involvement of chaperonins GroES and GroEL in the biosynthesis of the three hydrogenase isoenzymes, HYD1, HYD2, and HYD3, of Escherichia coli. These hydrogenases are NiFe-containing, membrane-bound enzymes composed of small and large subunits, each of which is proteolytically processed during biosynthesis. Total hydrogenase activity was found to be reduced by up to 60% in groES and groEL thermosensitive mutant strains. This effect was specific because it was not seen for another oligomeric, membrane-bound metalloenzyme, i.e., nitrate reductase. Analyses of the single hydrogenase isoenzymes revealed that a temperature shift during the growth of groE mutants led to an absence of HYD1 activity and to an accumulation of the precursor of the large subunit of HYD3, whereas only marginal effects on the processing of HYD2 and its activity were observed under these conditions. A decrease in total hydrogenase activity, together with accumulation of the precursors of the large subunits of HYD2 and HYD3, was also found to occur in a nickel uptake mutant (nik). The phenotype of this nik mutant was suppressed by supplementation of the growth medium with nickel ions. On the contrary, Ni2+ no longer restored hydrogenase activity and processing of the large subunit of HYD3 when the nik and groE mutations were combined in one strain. This finding suggests the involvement of these chaperonins in the biosynthesis of a functional HYD3 isoenzyme via the incorporation of nickel. In agreement with these in vivo results, we demonstrated a specific binding of GroEL to the precursor of the large subunit of HYD3 in vitro. Collectively, our results are consistent with chaperonin-dependent incorporation of nickel into the precursor of the large subunit of HYD3 as a prerequisite of its proteolytic processing and the acquisition of enzymatic activity.     

Adsorption and uptake of nickel in Methanothrix concilii

During growth of the aceticlastic methanogen Methanothrix concilii, ⁶³Ni was incorporated primarily into two soluble proteins, methylcoenzyme M reductase and carbon monoxide dehydrogenase. During short-term studies, an uptake system for nickel was present which saturated, with kinetic constants of apparent K _m=91 M and V _max=23 nmol/min·mg^-1 dry wt. Nickel specificity was demonstrated in competition studies, in which magnesium, calcium, or manganese had little influence on ⁶³Ni uptake; cobalt, however, did compete. Although the uptake of nickel was blocked by extreme temperatures (6°C or 100°C) and by air, the complete abolition of methanogenesis by various ionophores, N,N-dicyclohexylcarbodiimide, bromoethanesulfonate, or pre-treatment of the cells at 80°C, had little effect on uptake. The kinetic characteristics of short-term nickel uptake in cells could be reproduced in purified sheath preparations, indicating that the predominant mechanism for nickel uptake in short-term studies was an energy-independent, semi-specific cell-surface absorption.National Research Council publication No. 29033