The hydrogenase gene cluster ofRhizobium leguminosarum bv.viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity

Plasmid pAL618 contains the genetic determinants for H₂ uptake (hup) fromRhizobium leguminosarum bv.viciae, including a cluster of 17 genes namedhupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream ofhypE has now been sequenced, thus completing the sequence of the 20 441-bp insert DNA in plasmid pAL618. An open reading frame (designatedhypX) encoding a protein with a calculated M_r of 62 300 that exhibits extensive sequence similarity with HoxX fromAlcaligenes eutrophus (52% identity) andBradyrhizobium japonicum (57% identity) was identified 10 bp downstream ofhypE. Nodule bacteroids produced byhypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 µM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by ahypX gene provided in trans. From expression analysis ofhypX-lacZ fusion genes, it appears thathypX gene is transcribed from the FnrN-dependenthyp promoter, thus placinghypX in thehyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N₁₀-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C₁), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase-family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C₁ donor N₁₀-formyl-tetrahydrofolate and transfer of the C₁ to an unknown substrate, and catalysis of a reaction involving polarization of the C=O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.     

Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX₂C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX₂CX₈₁CX₂C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.     

The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli

The hyp operon of Escherichia coli comprises several genes which are required for the synthesis of all three hydrogenase isoenzymes. Deletions were introduced into each of the hypA-E genes, transferred to the chromosome and the resulting mutants were analysed for hydrogenase 1, 2 and 3 activity. The products of three of the genes, hypB, hypD and hypE were found to be essential for the synthesis of all three hydrogenase isoenzymes. A defect in hypB, as previously observed, could be complemented by high nickel concentrations in the medium, whereas the effects of mutants in the other genes could not. Lesions in hypA prevented development of hydrogenase 3 activity, did not influence the level of hydrogenase 1 but led to a considerable increase in hydrogenase 2 activity although the amount of hydrogenase 2 protein was not drastically altered. Lesions in hypC, on the other hand, led to a reduction of hydrogenase 1 activity and abolished hydrogenase 3 activity. HYPA and HYPC, besides being required for hydrogenase 3 formation, therefore may have a function in modulating the activities of the three isoenzymes with respect to each other and adjusting their levels to the requirement imposed by the physiological situation. Mutations in all five hyp genes prevented the apparent processing of the large subunits of all three hydrogenase isoenzymes. It is concluded that the products of the hypA-E genes play a role in nickel incorporation into hydrogenase apoprotein and/or processing of the constituent subunits of this enzyme. The importance of their roles is also reflected in their phylogenetic conservation in distantly related organisms.     

hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX. Received: 15 June 1998 / Accepted: 5 August 1998     

Engineering the Rhizobium leguminosarum bv. viciae Hydrogenase System for Expression in Free-Living Microaerobic Cells and Increased Symbiotic Hydrogenase Activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.     

Purification and characterization of the hydrogenase from Thiobacillus ferrooxidans

Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 ×g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in an 85% pure preparation with a specific activity of 6.0 U (mg protein)^–1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49° C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50–100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase. Received: 17 November 1995 / Accepted: 2 February 1996     

ThehupB gene of theAzotobacter chroococcum hydrogenase gene cluster is involved in nickel metabolism

InAzotobacter chroococcum the hydrogenase gene (hup) cluster spans about 14 kb of DNA. The genes coding for the small and large subunits,hupSL, are located at the 5 end, and a cluster of genes,hupABYCDE, resembling theEscherichia coli hyp operon, is located at the 3 end. In this study, we determined the effect of adding nickel to the medium used for the growth ofhup mutants. Hydrogenase activity was restored tohupA andhupB mutants, but nothupY, hupD, orhupE mutants, by the addition of nickel to the growth medium, suggesting that the products ofhupA andhupB are somehow involved in nickel metabolism. The restoration of hydrogenase activity to thehupB mutant required protein synthesis.     

Heterotrophic metabolism and regulation of uptake hydrogenase activity in symbiotic cyanobacteria

The H₂ uptake activity of three cyanobionts isolated fromCycas revoluta, C. circinalis andazolla filiculoides was shown to be related primarily to the growth rate and independent of the main mode of carbon nutrition. Significant H₂ uptake was found in the coralloid roots ofCycas revoluta andZamia furfuracea (3 and 22 times higher than the respective C₂H₂ reduction activities). The results attained allow us to conclude that in cyanobacteria, in contrast to most nitrogen-fixing heterotrophs, uptake hydrogenase activity is not repressed by carbon substrates and that cyanobacteria in association seem to be endowed with sufficient H₂ uptake capacity to recover all of the H₂ released during the process of N₂-fixation.     

Gene products of the hupGHIJ operon are involved in maturation of the iron-sulfur subunit of the [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (deltahupG, deltahupH, deltahupI, and deltahupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The deltahupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the deltahupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from deltahupH, deltahupI, and deltahupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the deltahupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.     

Localization of an uptake hydrogenase in anabaena

Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H₂ uptake was detected (0.60-1.44 μmoles/[mg of chlorophyll a per hour]) in all three strains when grown with N₂ as the sole source of nitrogen. H₂ uptake (in vivo and in vitro) was severely suppressed in cultures grown on NH₄⁺ and lacking heterocysts. H₂ uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N₂/CO₂, vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase.     

Duplication of hyp genes involved in maturation of [NiFe] hydrogenases in Alcaligenes eutrophus H16

  Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1. Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A. eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies. Mutants with lesions in both copies showed clear alterations in hydrogenase activities. Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H₂. Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium. Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor. Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes. HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms. Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site. Received: 15 June 1998 / Accepted: 5 August 1998     

Highly active immobilized hydrogenase from Desulfovibrio gigas

The hydrogenase from the sulfate reducer $\text{Desulfovibrio gigas}$ has been immobilized by covalent coupling onto a porous silica support. Two methods have been used: glutaraldehyde activation of aliphatic amino Spherosil and diazotation of aromatic amino Spherosil. The effect of cytochrome C₃ and CC₃ addition during coupling has been investigated. The highest enzymatic activity (4440 U/g support) and immobilization yield (29 %) was obtained when coupling hydrogenase in the presence of cytochrome C₃ or CC₃ with diazotized aromatic amino silica. This immobilized hydrogenase preparation which shows a very good resistance to oxygen inactivation seems suitable for hydrogen photoproduction by coupling with illuminated chloroplasts.