Autotrophic growth of strains ofRhizobium and properties of isolated hydrogenase

Four strains ofRhizobium (R. trifolii RCL10,R. japonicum S19 and SB16, andRhizobium sp. NEA4) were demonstrated to grow lithoautotrophically with molecular hydrogen as sole electron donor and with ammonium or with N₂ as N source. All of them showed ribulose-1,5-bisphosphate carboxylase activity and hydrogenase (H₂-uptake) activity with methylene blue and oxygen as electron acceptors. ForR. japonicum SB 16, a doubling time under autotrophic conditions of 30 h and a specific hydrogenase activity (methylene blue reduction) in crude extracts of 1.4 U/mg protein were calculated.Rhizobium hydrogenase is a membrane-bound enzyme. It is mainly detectable in particulate cell fractions, it cross-reacts with the antibodies of the membrane-bound hydrogenase ofAlcaligenes eutrophus, and is unable to reduce NAD. The isolated hydrogenase is a relatively oxygen-sensitive enzyme with a half-life of three days when stored at 4°C under air.     

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     

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.     

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.     

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.     

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     

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H₂ and CO₂. All of them showed H₂ uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H₂+O₂ uptake (32.5 l/min. mg protein) as well as H₂ uptake in the presence of methylene blue (41.4 l/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O₂ concentration the maximal rates of H₂+O₂ uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H₂-gased samples.     

Identification of new peptides synthesized under the hydrogenase control system of Alcaligenes eutrophus

Total protein of Alcaligenes eutrophus was analyzed by two-dimensional protein map. Cells grown at 30° C expressed hydrogen-oxidizing (Hox) ability mediated by a soluble (Hos) and a particulate hydrogenase (Hop). Hox ability was not expressed at 37° C (HoxTs). The six subunits of the two hydrogenases were identified. Besides these six subunits eight peptides were not or hardly detected at 37° C. The mutant HF117 which expressed Hox ability at 37° C (HoxTr), formed the hydrogenase peptides and five of the eight peptides. These peptides designated B, C, E, F, and H were characterized by their isoelectric point and molecular mass (M _r); their M _r were 18 800, 45 400, 41 900, 39 400, and 40 600, respectively. The five peptides were not formed in regulatory Hox^– mutants, and not formed in mutants cured of plasmid pHG1, carrying the genetic information for hydrogenase formation. Strain HF160, carrying a Tn5 insertion in a gene essential for Hos expression specifically did not form the B-peptide. All peptides were found in the soluble fraction of cell extracts, the F-peptide was also detected in the particulate fraction. The function of the new Hox-peptides is presently unknown.Abbreviations PAGE polyacrylamide gelelectrophoresis - SDS sodium dodecylsulfate - Hox hydrogen oxidizing ability     

Manipulation of the genes for poly-β-hydroxybutyric acid synthesis in Alcaligenes eutrophus

Summary In order to establish the molecular breeding system in Alcaligenes eutrophus producing poly--hydroxybutyric acid (PHB), phbCAB genes from A. eutrophus were recombined into the E. coli-A. eutrophus shuttle vector and directly transferred into A. eutrophus by the electroporation. In A. eutrophus transformants, recombinant plasmids were stably maintained and enzyme activities for PHB biosyntheses were elevated 1.4–2.7 fold by the cloned genes.     

Overproduction of a stable acetaldehyde dehydrogenase with high affinity to acetaldehyde by Escherichia coli expressing the ACOD gene of Alcaligenes eutrophus

Summary The acoD gene of Alcaligenes eutrophus, which encodes a very stable NAD dependent aldehyde dehydrogenase with high affinity toward acetaldehyde (K _m = 4M), was overexpressed in Escherichia coli. Plasmid pDel087, a deletion derivative of a plasmid constructed recently (Priefert et al., 1992), conferred acetaldehyde dehydrogenase activity of 2.5 U/mg of protein to E. coli, which was about 8-fold higher than the activity in ethanol-grown cells of A. eutrophus.     

Micromorphology of Gram-negative hydrogen bacteria

The fine structure of the cell envelope, of membrane systems and of cytoplasmic inclusions of Gram-negative aerobic hydrogen bacteria has been studied. The results have been tabulated, and three main groups could be recognized: Group 1: Alcaligenes eutrophus, A. paradoxus, A. ruhlandii, Pseudomonas facilis, P. flava, P. pseudoflava, P. palleronii, and Aquaspirillum autotrophicum; Group 2: Corynebacterium autotrophicum and strains MA 2 and SA 35; Group 3: Paracoccus denitrificans. Special structures related to the chemoautotrophic way of life of the hydrogen bacteria were not observed.Abbreviations CM cytoplasmic membrane - OM outer membrane     

Isolation of hydrogenase regulatory mutants of hydrogen-oxidizing bacteria by a colony-screening method

Mutants derepressible for hydrogenases (Hox d) have been isolated from the wild type of Alcaligenes hydrogenophilus which is inducible for hydrogenases (Hox i). The mutants are able to form the hydrogenases during growth on gluconate under air while the wild type requires molecular hydrogen for hydrogenase systhesis.Mutant selection involved alternating growth under autotrophic and heterotrophic conditions. Mutants derepressed for hydrogenases after growth on gluconate were recognized by a new colony-screening method allowing differentiation between colonies of hydrogenase-containing and hydrogenase-free cells of aerobic hydrogen-oxidizing bacteria. The method is based on the ability of the colonies to reduce triphenyltetrazolium chloride in the presence of monoiodoacetate and gaseous hydrogen to its water-insoluble purple formazan. Endogenous dye reduction (under nitrogen) and the function of the cytoplasmic NAD-reducing hydrogenase were completely inhibited by monoiodoacetate. The applicability of the method has been demonstrated for wild type strains and mutants of various hydrogen-oxidizing bacteria. When mutants of A. hydrogenophilus and A. eutrophus H16 lacking the Hox-encoding plasmids pHG21-a and pHG1, respectively, were used as recipients and Hox d mutant M 201 of A. hydrogenophilus as a donor transconjugants appeared which had received the Hox d character and the megaplasmid pHG21-a.Abbreviations MIAc monoiodoacetate - TTC 2,3,5-triphenyl-2-tetrazolium chloride - Hox ability to oxidize hydrogen Dedicated to Gerhard Drews on the occasion of his 60th birthday, remembering the education and inspiration we received from our teacher Johannes Buder at the Martin-Luther University of Halle     

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.     

Structural aspects of the soluble NAD-dependent hydrogenase isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b

The soluble NAD-dependent hydrogenase (hydrogen-NAD oxidoreductase, EC 1.12.1.2), consisting of four non-identical subunits, was isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b and analyzed by a HPLC gel permeation technique and electron microscopy. The tetrameric enzyme particles from both origins, as determined from negatively stained electron microscopic samples, were found to be elongated and very similar in shape and size. The A. eutrophus enzyme was measured in more detail. It exhibited dimensions of 12.7 nm by 5.5 nm (axial ratio 2.3:1). Dissociation into smaller particles and unspecific aggregation combined with partial inactivation were observed in the presence of the inhibitor NADH. Kept in buffer without added nickel, the enzyme was partially dissociated. Reassociation of tetramers without restored enzyme activity was achieved by addition of 0.5 mM NiCl₂. A working model for the structural organization of the tetrameric enzyme particle is presented.     

Distribution analysis of hydrogenases in surface waters of marine and freshwater environments

Background

Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H₂ evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H₂ metabolism in marine environments is nitrogen fixation.

Principal Findings

We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H₂ uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean.

Significance

This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H₂-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles.     

Rhizobium japonicum hydrogenase: purification to homogeneity from soybean nodules, and molecular characterization

Rhizobium japonicum hydrogenase was purified to homogeneity from soybean root nodules by four column chromatography steps after solubilization from membranes by treatment with a nonionic detergent. The specific activity was from 40 to 65 mumol H2 oxidized min-1 mg protein-1 and was increased 450-fold relative to that in bacteroids. The yield of activity was from 7 to 12%. The molecular weight of the native enzyme was 104,000 as determined by sucrose density gradient centrifugation. Electrophoresis in the presence of sodium dodecyl sulfate revealed two subunits with molecular weights of 64,000 and 35,000, indicating an alpha beta subunit structure. The amino acid content of the protein indicated 20 cysteine residues. Analysis of the metal content indicated 0.59 +/- 0.06 mol Ni/mol hydrogenase and 6.5 +/- 1.2 mol Fe/mol hydrogenase. Antisera prepared to the hydrogenase cross-reacted with the enzyme in bacteroid extracts at all stages of the purification but did not cross-react with extracts of Alcaligenes eutrophus grown under chemolithotrophic conditions. The similarity of rhizobial hydrogenase to the particulate hydrogenases of A. eutrophus and A. latus is discussed.     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Purification of hydrogenases by affinity chromatography on Procion Red-agarose.

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.