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.     

Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional homologues of hypA and hypB are not active in hydrogenase maturation

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of DeltahypA1 and DeltahypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.     

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.     

Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori

The nickel-containing enzymes hydrogenase and urease require accessory proteins in order to incorporate properly the nickel atom(s) into the active sites. The Helicobacter pylori genome contains the full complement of both urease and hydrogenase accessory proteins. Two of these, the hydrogenase accessory proteins HypA (encoded by hypA) and HypB (encoded by hypB), are required for the full activity of both the hydrogenase and the urease enzymes in H. pylori. Under normal growth conditions, hydrogenase activity is abolished in strains in which either hypA (HypA:kan) or hypB (HypB:kan) have been interrupted by a kanamycin resistance cassette. Urease activity in these strains is 40 (HypA:kan)- and 200 (HypB:kan)-fold lower than for the wild-type (wt) strain 43504. Nickel supplementation in the growth media restored urease activity to almost wt levels. Hydrogenase activity was restored to a lesser extent, as has been observed for hyp mutants in other (H(2)-oxidizing) bacteria. Expression levels of UreB (the urease large subunit) were not affected by inactivation of either hypA or hypB, as determined by immunoblotting. Urease activity was not affected by lesions in the genes for either the hydrogenase accessory proteins HypD or HypF or the hydrogenase large subunit structural gene, indicating that the urease deficiency was not caused by lack of hydrogenase activity. When crude extracts of wt, HypA:kan and HypB:kan were separated by anion exchange chromatography, the urease-containing fractions of the mutant strains contained about four (HypA:kan)- and five (HypB:kan)-fold less nickel than did the urease from wt, indicating that the lack of urease activity in these strains results from a nickel deficiency in the urease enzyme.     

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.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H(2)-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H(2)-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of (63)Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.     

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.     

The membrane-bound hydrogenase of Alcaligenes eutrophus: II. Localization and immunological comparison with other hydrogenase systems

Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing. Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane.The membrane-bound hydrogenases of A. eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas, Alcaligenes and Aquaspirillum. Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotrophicus GZ 29 was found only in a few hydrogen-oxidizing bacteria. Material cross-reacting to the soluble hydrogenase of A. eutrophus was detected in strains of A. eutrophus and A. ruhlandii only.Comparison of the membrane-bound hydrogenase of A. eutrophus, P. pseudoflava and X. autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only. The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.     

Electron microscopic localization of hydrogenase genes on the megaplasmid pHG 1 in Alcaligenes eutrophus

Summary Plasmids carrying hydrogenase genes in Alcaligenes eutrophus wild type H 16 and in two transposon Tn5 —induced mutants have been investigated by electron microscopy. Besides the pHG1 megaplasmid (458±27 kb) carrying genes coding for structural and regulatory properties of hydrogenases, small plasmids of unknown significance have been detected. The sizes of EcoRI fragments obtained from pHG1 were measured from electron micrographs. They were significantly different from sizes determined previously by agarose gel electrophoresis.Plasmid pHG1 isolated from the wild type H 16 was shown to contain two inverted repeats (IR 16-1 and IR 16-2) with sizes similar to known transposons.From electron microscopic hybridization studies, it was deduced that the sites of insertion of Tn5 into a regulation gene on pHG1 for both soluble and membrane-bound hydrogenase, and of Tn5-Mob into the gene coding for structural properties of the soluble hydrogenase, are about 67.2 kb apart. One of the inverted repeats (IR 16-1) was localized in between these sites.     

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism

Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO₂ fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO₂. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.     

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     

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.     

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.     

Plasmids required for utilization of molecular hydrogen by Alcaligenes eutrophus

Alcaligenes eutrophus and three other hydrogen bacteria exposed to plasmid-curing agents generated autotrophic-minus mutants at high frequency. These mutants were blocked in the metabolism of H₂ as an energy source and had normal levels of enzymes involved in CO₂ fixation. The loss of hydrogenase activity in A. eutrophus was accompanied by the loss or alteration of a plasmid that had molecular weight of approximately 200×10⁶. Mobilization of this plasmid from wild-type A. eutrophus strains into cured hydrogenase-minus derivatives restored hydrogenase function. It is concluded that A. eutrophus contains a large plasmid required for hydrogen metabolism and thereby autotrophic growth.Abbreviations Aut autotrophic - Hup hydrogen uptake - NTG N-methyl-N-nitro-N-nitrosoguanidine - RuBP ribulose bisphosphate - RuMP ribulose monophosphate - Kan kanamycin - Nal nalidixic acid - Rif rifampicin - Tet tetracycline     

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (βδ) and an NADH:acceptor oxidoreductase (αγ)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed. Received: 14 May 1996 / Accepted: 7 November 1996     

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF -hupS-hupL-hupM-hupD -hupF -hupG -hupH -huoJ -hupK -hypA-hypB-hupR1-hypC -hypD -hypE -ORF19 -ORF20 , all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup^? mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant