Nickel accumulation and storage in Bradyrhizobium japonicum

Hydrogenase-derepressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl2, and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown and derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunoprecipitation of 63Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms.     

Nickel in the catalytically active hydrogenase of Alcaligenes eutrophus.

Nickel is a constituent of soluble and particulate hydrogenase of Alcaligenes eutrophus. Incorporation of 63Ni2+ revealed that almost the total nickel taken up by the cells was bound to the protein. Chromatography of a crude extract on diethylaminoethyl cellulose demonstrated an association of 63Ni2+ with soluble and particulate hydrogenase, supported by further analysis like polyacrylamide gel electrophoresis. Unspecific binding of 63Ni2+ to the protein was excluded by comparison with a mutant extract free of hydrogenase protein. X-ray fluorescence analysis of the homogeneous soluble hydrogenase indicated the presence of 2 mol of nickel per mol of enzyme, whereas the amount of nickel determined by incorporation of 63Ni2+ was calculated to be approximately 1 mol/mol of enzyme. Cells grown under nickel limitation contained catalytically inactive, but serologically active, soluble and particulate hydrogenase. The immunochemical reactions were only partially identical with the enzyme from nickel-cultivated cells indicating a structural modification of the proteins in the absence of nickel. It is concluded that nickel is essential for the catalytic activity of hydrogenase and not involved as a regulatory component in the synthesis of this enzyme.     

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.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus.

The nickel-dependent chemolithoautotrophic growth of Alcaligenes eutrophus is apparently due to a requirement of nickel for active hydrogenase formation. Cells grown heterotrophically with fructose and glycerol revealed a specific activity of soluble and membrane-bound hydrogenase which was severalfold higher than the normal autotrophic level. The omission of nickel from the medium did not affect heterotrophic growth, but the soluble hydrogenase activity was reduced significantly. In the presence of ethylenediaminetetraacetic acid (EDTA), almost no hydrogenase activity was detected. The addition of nickel allowed active hydrogenase formation even when EDTA was present. When chloramphenicol was added simultaneously with nickel to an EDTA-containing medium, almost no hydrogenase activity was found. This indicates that nickel ions are involved in a process which requires protein synthesis and not the direct reactivation of a preformed inactive protein. The formation of the membrane-bound hydrogenase also appeared to be nickel dependent. Autotrophic CO2 assimilation did not specifically require nickel ions, since formate was utilized in the presence of EDTA and the activity of ribulosebisphosphate carboxylase was not affected under these conditions.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hupc mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme

A double mutant (JH103K10) was created from hydrogenase constitutive mutant (JH103) by replacement of a chromosomal 0.60 kb nickel metabolism related locus with a kanamycin resistance gene. The double mutant required 10 to 20 times more nickel (Ni) to achieve near parental strain levels of hydrogenase activity. In the absence of nickel, both JH103K10 and JH103 synthesized high levels of (inactive) hydrogenase apoprotein (large subunit, 65 kDa). With nickel, the double mutant JH103K10 synthesized the same level of hydrogenase apoenzyme (65-kDa subunit) as the JH103 parent strain; however, whole cell hydrogenase activity in JH103K10 was less than half of that in JH103, and the CPM (due to ⁶³Ni in hydrogenase) of membranes and the calculated ratio of nickel per unit of hydrogenase enzyme of the double mutant were 40% of that in JH103. Therefore, the difference in hydrogenase activities between the double mutant and the Hup^ck strain can be accounted for by different abilities of the strains to incorporate nickel into the hydrogenase apoenzyme. The addition of nickel ions to previously Ni-starved and then chloramphenicol-treated Bradyrhizobium japonicum whole cells (JH103 and JH103K10) resulted in (an in vivo) restoration of hydrogenase activity, suggesting that the apoprotein synthesized in the Ni-free cultures could be activated by addition of nickel even in the absence of protein synthesis. The extent of reconstitution of active hydrogenase by nickel was greater in the absence of chloramphenicol. Hydrogenase apoprotein could not be activated by nickel in vitro even with the addition of ATP. The successful in vivo but not in vitro results suggest that enzymatic but cell-disruption labile factors are required for Ni incorporation into hydrogenase.     

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).     

A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway

The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in DeltaslyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.     

Effect of chelating agents on hydrogenase in Azotobacter chroococcum. Evidence that nickel is required for hydrogenase synthesis.

The chelating agents EDTA, o-phenanthroline, nitrilotriacetic acid (NTA), ethylenediamine-bis(o-hydroxyphenylacetic acid) (EDDA) or dimethylglyoxime prevented the expression of hydrogenase activity in batch cultures of nitrogen-fixing Azotobacter chroococcum, but did not inhibit preformed enzyme. The inhibition was reversed either by adding a mixture of trace elements (Cu2+, Mn2+, Zn2+, Co2+) or Ni2+ or, to a lesser degree, Co2+ alone. Ni2+ or Ni2+ + Fe2+ also enhanced the rate of hydrogenase derepression in A. chroococcum in the absence of any added chelator, if the medium was first extracted with 8-hydroxyquinoline. A. chroococcum accumulated 63Ni2+ by an energy-independent mechanism. Both, Ni2+ uptake and hydrogenase synthesis were equally inhibited by either NTA, EDTA, EDDA or dimethylglyoxime. The evidence suggests a role for Ni2+ in hydrogenase synthesis.     

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.     

Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.     

Dissimilatory nitrate reduction by a strain ofClostridium butyricum isolated from estuarine sediments

Nitrate dissimilation in chemostat grown cultures ofClostridium butyricum SS6 has been investigated. Sucrose limited cultures grown on nitrate produced nitrite as the principal end-product of nitrate reduction whilst under nitrate-limiting conditions ammonia accumulated in the spent media. Nitrate reduction was accompanied by the synthesis of a soluble nitrate reductase (123 nmol·NADH oxidised · min^-1 · mg protein^-1) and in addition, under N-limiting conditions, a soluble nitrite reductase (56 nmol NADH oxidised min^-1 · mg protein^-1). Corresponding ammonia grown cultures synthesised neither enzyme. Concurrent with the dissimilation of nitrate to nitrite and ammonia cell population densities increased by 18% (C-limitation) and 32% (N-limitation). Spent media analyses of the fermentation products from ammonia and nitrate grown cells showed the accumulation of acetate in nitrate dissimilating cultures. Molar ratios of acetate/butyrate increased by a factor of 5 (C-limitation) to 12 (N-limitation) upon adding nitrate to the growth medium. In C-limited cultures, grown on nitrate, hydrogenase activity was 340 nmol · min^-1 · mg protein^-1 and under N-limitation this increased to 906 nmol · min^-1 · mg protein^-1. Since N-limited cultures are electron acceptor limited, the increase in hydrogenase activity enables excess electrons to be spilled by this route.     

Proline alleviates heavy metal stress inScenedesmus armatus

Growth and some metabolic activities ofScenedesmus armatus grown in the presence of different heavy metals (Cd, Mn and Ni) with and without exogenously added proline (Pro) were monitored. The growth ofS. armatus cells (cell concentration, pigment and dry mass) was inhibited by all these heavy metals. Addition of Pro to the culture medium minimized the toxic effect of the metals. The growth rate was somewhat higher in Pro-containing cultures and started to decline I d later than in cultures containing heavy metals alone.S. armatus cells accumulated the added Pro in response to heavy metals. The accumulation correlated with protein content. Cd was the strongest inducer of Pro accumulation, Mn being the weakest. Cells accumulated nickel more than cadmium and manganese. Heavy metal-treated cells had increased peroxidase and catalase activities.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Purification and properties of the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

A soluble hydrogenase has been isolated from Desulfovibrio desulfuricans (strain Norway 4) grown on Postgate's medium. The enzyme differs significantly from a membrane-bound hydrogenase previously purified from the same organism grown on Starkey's medium. The enzyme consisted of two subunits of 56 kDa and 29 kDa compared with masses of 60 kDa and 27 kDa for the membrane-bound enzyme. Analysis of preparations of the soluble enzyme by various methods gave values of 5-10 iron atoms, 6 labile sulphur atoms and 0.45-0.8 nickel atom per molecule. The enzyme was unusual in that it contained selenium, in quantities equivalent to nickel. The highly purified active enzyme produced no electron-spin-resonance (ESR) signals in the oxidized state. ESR signals due to a [3Fe-xS] cluster and nickel were observed only in some of the less active fractions of the enzyme, demonstrating that neither of these ESR-detectable components is a prerequisite for hydrogenase activity. Treatment of D. desulfuricans (Norway) cells with EDTA released a minor fraction with hydrogenase activity, which might indicate the presence of a periplasmic enzyme.