Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.     

The plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus

Abstract Alcaligenes eutrophus strain H16 harbors a 450 kilobase pairs (kb) conjugative plasmid which codes for the ability of the organism to grow lithoautotrophically on hydrogen and carbon dioxide (reviewed in [1]). The genes for hydrogen oxidation, designated hox , are clustered on plasmid pHG1 in a DNA region of approximately 100-kb in size ([2], Fig. 1). The hox genes and their organization have been analyzed by isolation of Hox-deficient mutants, by complementation analysis, by cloning of hox genes, identification of hox -encoded polypeptides and, most recently, by DNA sequencing. The hox cluster is flunked by the two structural gene regions, hoxS and hoxP ; it contains a regulatory locus, hoxC , and additional genes like hoxN and hoxM whose products play a role in the formation of catalytically active hydrogenase proteins. Of four indigenous 1.3-kb insertion elements, two copies of IS491 map in the hox gene cluster. These elements may be involved in rearrangements and deletions which occur particularly frequently in this region of the megaplasmid (Schwartz, Kortlüke and Friedrich, unpublished).     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange.

Alcaligenes hydrogenophilus was found to contain a soluble and a particulate hydrogenase whose control and structure differed in part from that in Alcaligenes eutrophus. One of at least two plasmids indigenous to A. hydrogenophilus determines hydrogenase genes (Hox). The interspecific exchange of Hox-encoding plasmids generated transconjugants which expressed the structural and regulatory Hox phenotype of the donor.     

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.     

Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria

Polyclonal, monospecific antibodies were produced against the two subunits (Mr 62,000, and Mr 31,000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodiffusion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits (Mr 63,000, 56,000, 30,000 and 26,000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All these proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration on three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.     

Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus

Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts). At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism. The soluble hydrogenase activity was stable at 37 degrees C. The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr). Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments. Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive. No cross-reactivity was detected above temperatures of 34 degrees C. The genetic information of Hox resides on a self-transmissible plasmid in A. eutrophus. Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant. This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

The H(2) sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H(2), which is available in low amounts in aerobic environments. H(2) sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H(2) sensor of R. eutropha is a cytoplasmic protein. Although capable of H(2) oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H(2) sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231-235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H(2) sensor is nickel dependent. The results suggest that H(2) sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H(2) binding or subsequent H(2) oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H(2)-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H(2) sensors represents a novel subclass of [NiFe] hydrogenases denoted as the "regulatory hydrogenases."     

Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii.

The membrane-bound hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii were purified extensively and compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons. The migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except A. vinelandii hydrogenase, which migrated further than the other three. The amino acid composition of each hydrogenase was determined, revealing substantial similarity among these enzymes. This was confirmed by calculation of S delta Q values, which ranged from 8.0 to 26.7 S delta Q units. S delta Q is defined as sigma j(Xi,j-Xk,j)2, where i and k identify the proteins compared and Xj is the content (residues per 100) of a given amino acid of type j. The hydrogenases of this study were also compared with an enzyme-linked immunosorbent assay. Antibody raised against B. japonicum hydrogenase cross-reacted with all four hydrogenases, but to various degrees and in the order B. japonicum greater than A. latus greater than A. eutrophus greater than A. vinelandii. Antibody raised against A. eutrophus hydrogenase also cross-reacted with all four hydrogenases, following the pattern of cross-reaction A. eutrophus greater than A. latus = B. japonicum greater than A. vinelandii. Antibody raised against B. japonicum hydrogenase inhibited B. japonicum hydrogenase activity to a greater extent than the A. eutrophus and A. latus activities; no inhibition of A. vinelandii hydrogenase activity was detected. The results of these experiments indicated remarkable homology of the hydrogenases from these four microorganisms.     

The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization

The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.     

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     

Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).     

Activation and active sites of nickel-containing hydrogenases

Hydrogenases that contain nickel and iron-sulphur clusters also have a regulatory mechanism, by which exposure to oxidants such as oxygen prevents their reaction with hydrogen. Treatment with reducing agents then causes reactivation. In some hydrogenases from Desulfovibrio species, there is evidence that there are at least two different deactivated states, which differ in their rates of reductive reactivation. The membrane-bound hydrogenase of D. desulfuricans, Norway strain, the periplasmic hydrogenase of D. gigas and the membrane-bound hydrogenase of Alcaligenes eutrophus can be isolated in a state (termed "Unready") which requires up to several hours for full activation by hydrogen. By contrast the soluble hydrogenases of D. desulfuricans and A. eutrophus can be reactivated relatively rapidly. In all of these enzymes, with the exception of the latter one, the existence of the activated and deactivated states can be correlated with different ESR-detectable forms of nickel. The possible functions of nickel and [Fe-4S] clusters in catalysis are discussed.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16.

The formation of the catalytically active membrane-bound hydrogenase (MBH) of Alcaligenes eutrophus H16 requires the genes for the small and large subunits of the enzyme (hoxK and hoxG, respectively) and an accompanying set of accessory genes (C. Kortl ke, K. Horstmann, E. Schwartz, M. Rohde, R. Binsack, and B. Friedrich, J. Bacteriol. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membrane-associated heterodimer depends on specific maturation processes mediated by hox genes.