Nickel Affects Activity More Than Expression of Hydrogenase Protein in <Emphasis Type="Italic">Frankia</Emphasis>

The effects of nickel on hydrogen uptake and the post-translational processing of the large subunit of the hydrogenase protein in three Frankia strains (one isolated from an Alnus-Frankia symbiosis and two from Casuarina-Frankia associations) were investigated. All three strains responded to the addition of nickel with an increase in hydrogen uptake. Additional nickel did not affect nitrogenase activity, however evolved hydrogen was detected in Frankia KB5 in the absence of additional nickel, indicating that hydrogenase was not active. No increase in the processing rate of the hydrogenase large subunit was found with increasing nickel concentrations for any of the strains, indicating that the strategy for regulating hydrogenase in Frankia is different from that in other microorganisms. Received: 23 April 2001 / Accepted: 29 May 2001     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Biodiversity of Hydrogenases in Frankia

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

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.     

Use of a Reverse Micelle System for Study of Oligomeric Structure of NAD<Superscript>+</Superscript>-Reducing Hydrogenase from <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> H16

Inclusion of an oligomeric enzyme, NAD⁺-dependent hydrogenase from the hydrogen-oxidizing bacterium Ralstonia eutropha, into a system of reverse micelles of different sizes resulted in its dissociation into catalytically active heterodimers and subunits, which were characterized in reactions with various substrates. It was found that: 1) the native tetrameric form of this enzyme catalyzes all types of studied reactions; 2) hydrogenase dimer, HoxHY, is a minimal structural unit catalyzing hydrogenase reaction with an artificial electron donor, reduced methyl viologen; 3) all structural fragments containing FMN and NAD⁺/NADH-binding sites exhibit catalytic activity in diaphorase reactions with one- and two-electron acceptors; 4) small subunits, HoxY and HoxU also exhibit activity in diaphorase reactions with artificial acceptors. These results can be considered as indirect evidence that the second FMN molecule may be associated with one of the small subunits (HoxY or HoxU) of the hydrogenase from R. eutropha.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 782–789.Original Russian Text Copyright © 2005 by Tikhonova, Kurkin, Klyachko, Popov.     

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 biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Localization of hydrogenase in the purple sulphur bacterium Thiocapsa roseopersicina

The localization of hydrogenase in the phototrophic bacterium Thiocapsa roseopersicina was investigated by subcellular fractionations, and transmission electron microscopic immunocytochemistry. By using sonicated cells and measuring in vitro hydrogenase activities in soluble and membrane fractions, respectively, a weak hydrophobic interaction between the hydrogenase enzyme and the T. roseopersicina membranes was observed. Polyclonal antisera directed against the purified hydrogenase were raised in rabbits and exhibited one band in native-PAGE/Western immunoblot analysis. Native-PAGE/activity stain confirmed the identity of this band as being hydrogenase. Immunocytolocalization experiments using ultrathin sections showed an internal localization of the hydrogenase enzyme. A higher specific labeling was associated with chromatophores, indicating a possible coupling of hydrogenase with the photosynthetic membranes in the T. roseopersicina cells.     

A method to study zhizosphere processes in thin soil layers of different proximity to roots

Frankia is the diverse bacterial genus that fixes nitrogen within root nodules of actinorhizal trees and shrubs. Systematic and ecological studies of Frankia have been hindered by the lack of morphological, biochemical, or other markers to readily distinguish strains. Recently, nucleotide sequence of 16 S RNA from the small ribosomal subunit has been used to classify and identify a variety of microorganisms. We report nucleotide sequences from portions of the 16 S ribosomal RNA from Frankia strains AcnI1 isolated from Alnus viridis ssp. crispa (Ait.) Turrill and PtI1 isolated from Purshia tridentata (Pursh) DC. The number of nucleotide base substitutions and gaps we find more than doubles the previously reported sequence diversity for the same variable regions within other strains of Frankia.     

CYTOPLASMIC BIOSYNTHESIS OF LIGHT-HARVESTING POLYPEPTIDES IN THE GREEN FLAGELLATE MANTONIELLA SQUAMATA (MICROMONADOPHYCEAE)1

The biosynthesis of the light-harvesting complex (LHC) polypeptides of the green flagellate Mantoniella squamata (Manton et Parke) Desikachary (Micromonadophyceae, Chlorophyta) was examined by in vivo polypeptide labeling and immunoprecipitation of in vitro translation products. Using protein synthesis inhibitors, the LHC polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes and not in the chloroplasts of cells. Poly (A)+ RNA was isolated and proteins were synthesized by a rabbit reticulocyte lysate system, with antisera raised against M. squamata LHC used for immunoprecipitation from the translation products. One polypeptide 3-5 kDa larger than mature LHC polypeptides was immunoprecipitated. These studies indicate that although the LHC of M. squamata is quite different from the LHC of most green plants, the LHC polypeptides are synthesized as precursors in the cytoplasm of the cell and suggest that the genes encoding these polypeptides are located in the nucleus.     

Biosynthesis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O₂-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O₂-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.     

Localization of the F420-reducing hydrogenase in Methanococcus voltae cells by immuno-gold technique

The F₄₂₀-reducing hydrogenase of Methanococcus voltae, which takes part in the terminal reduction step of methanogenesis, was localized in situ in ultrathin sections. This result was obtained by the immuno-gold technique using a high titer antiserum raised against the purified enzyme. Its specifity for the hydrogenase was shown by Western blot analysis. The hydrogenase of M. voltae was found to be membrane-associated.Abbreviations ELISA Enzyme linked immuno sorbent assay - F₄₂₀ 8-hydroxy-5-deazaflavin     

A phloem-specific,lectin-like protein is located in pine sieve-element plastids by immunocytochemistry

Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.Abbreviations DAB diamino benzidine tetrachloride - FITC fluorescein isothiocyanate - kDa kilodalton - PBS phosphate-buffered saline - PP phloem polypeptide(s) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Characterization of a native subunit of the NAD-linked hydrogenase isolated from a mutant of Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of Alcaligenes eutrophus H16 consists of four non-identical subunits. From the mutant strain HF14, defective in this enzyme, a protein was isolated that reacted with specific antibodies raised against the wild-type hydrogenase; the reaction type was of partial identity. The same protein was also tested with specific antibodies raised against each of the four denatured subunits of the wild-type hydrogenase and was found to be completely identical with the second largest subunit; it reacted weakly with the antibody against the largest subunit and not at all with the antibody against the small subunits. In SDS-polyacrylamide gel electrophoresis the protein of the mutant migrated as a single polypeptide and corresponded to the second largest subunit of soluble hydrogenase with Mr = 56,000. The mutant enzyme strongly differed from the wild-type hydrogenase in its binding behaviour to chromatographic gels. It had pronounced hydrophobic properties and bound strongly to phenyl-Sepharose; it had high affinity to triazin dye gels. Enzymatically the polypeptide was totally inactive with NAD as electron acceptor, but showed weak residual activities with methylene blue, ferricyanide and cytochrome c. The protein also contained nickel; however, because of the instability of this polypeptide the amount varied between 0.2-1.4 nickel per enzyme molecule. As shown by ESR studies, the iron is organized in a [4Fe-4S] cluster but is partially present also in the 3Fe-form. No nickel signal could be detected. The role of the polypeptide subunit for hydrogen activation in the intact hydrogenase is discussed.     

Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16

The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.