Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli.

Zuber, Mohammed; Harker, Alan R.; Sultana, M A; Evans, Harold J.

doi:10.1073/pnas.83.20.7668

Cited by 19 publications

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“…But, as described in other organisms, this does not result in the formation of catalytically active C. GRZESZIK a n d OTHERS enzyme (Zuber et al, 1986;Voordouw et al, 1985;Karube et al, 1983). In cells of E. coli LHYl grown under standard conditions, all four SH subunits were detectable but were present in the form of inclusion bodies, and under conditions which avoided the formation of inclusion bodies the small subunits were exposed to proteolytic degradation.…”

Section: Ae L S V R a L G D A A V L S F S A E S I K P L I T E H P Ementioning

confidence: 92%

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

et al. 1997

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The dissociation of the soluble NAD-reducing hydrogenase of Rhodococcus opacus M R l l into two dimeric proteins with different catalytic activities and cofactor composition is unique among the NAD-reducing hydrogenases studied so far. The genes of the soluble hydrogenase were localized on a 7-4 kbp Asnl fragment of the linear plasmid pHG2Ol via heterologous hybridization. Analysis of the nucleotide sequence of this fragment revealed the seven open reading frames ORF1, hoxF, -U, -Y, -H, -Wand ORF7. The six latter ORFs belong to the gene cluster of the soluble hydrogenase. Their gene products are highly homologous to those of the NAD-reducing enzyme of Alcaligenes eutrophus H16. The genes hoxF, -U, -Y and -H encode the subunits a, 7, 6 and B, respectively. The gene hoxW encodes a putative protease, which may be essential for C-terminal processing of the / 3 subunit. Finally, ORF7 encodes a protein which has similarities to CAMP-and cGMP-binding protein kinases, but its function is not known. ORFI, which lies upstream of the hydrogenase gene cluster, encodes a putative transposase found in IS elements of other bacteria. Northern hybridizations and primer extensions using total RNA of autotrophically and heterotrophically grown cells of R. opacus M R l l indicated that the hydrogenase genes are under control of a like promoter located at the right end of ORFI and are even transcribed under heterotrophic conditions a t a low level. Furthermore, this promoter was shown to be active in the recombinant Escherichia coli strain LHYl harbouring the 7.4 kbp Asnl fragment, resulting in overexpression of the hydrogenase genes. Although all four subunits of the soluble hydrogenase were shown via Western immunoblots to be synthesized in E. coli, no active enzyme was detectable.Keywords : Rhodococcus opacus MR11, NAD-reducing hydrogenase, box genes INTRODUCTIONRhodococcus opacus strain M R l l (formerly Nocardia opaca 1 b) is a Gram-positive, facultative chemolithoautotrophic bacterium which can grow on carbon dioxide and gaseous H, as the sole carbon and energy sources. Physiologically, it belongs to the knallgas bacteria, a group which is composed of phylogenetically diverse organisms. For the activation of H,, all knallgas bacteria contain hydrogenases, which are of two basic types : the membrane-bound hydrogenases (MBHs) , which are coupled to the electron transfer chain and are The GenBank accession number for the nucleotide sequence reported in this paper is U70364.not capable of reducing NAD, and the soluble hydrogenases (SHs), which are localized in the cytoplasm and catalyse the transfer of electrons directly to NAD (Aragno & Schlegel, 1992;Schneider et al., 1984a;Zaborosch et al., 1989; Schink & Schlegel, 1979). The MBHs belong to the more common group of NiFehydrogenases consisting of one large and one small subunit, whereas the SHs belong to a family of less abundant multimeric NiFe-hydrogenases (Friedrich & Schwartz, 1993 ;Wu & Mandrand, 1993). The majority of the knallgas bacteria contain only the MBH, but a few, ...

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Section: Ae L S V R a L G D A A V L S F S A E S I K P L I T E H P Ementioning

confidence: 92%

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

et al. 1997

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“…The plasmid pMZ610, containing a 5.9-kbp HindIII fragment encoding both subunits of the membrane-bound hydrogenase of B. japonicum, was made available (48). The strong antigenic cross-reactivity between the B. japonicum enzyme and that from R. capsulatus (37) (47,48). Lanes: 1, pMZ610 cut with HindIII; 2, pRHP8, Pstl; 3, pRHP20, EcoRI; 4, pRHP8, EcoRI; 5, pRHP4, EcoRI; 6, total B100 chromosomal DNA, HindIII; 7, total B100 chromosomal DNA, EcoRI.…”

Section: Methodsmentioning

confidence: 99%

Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus

Love

Borghese

et al. 1989

J Bacteriol

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Mutants of Rhodobacter capsulatus unable to grow photoautotrophically with H2 and CO2 were isolated.Those lacking uptake hydrogenase activity as measured by H2-dependent methylene blue reduction were analyzed genetically and used in complementation studies for the isolation of the wild-type genes. Results of further subcloning and transposon TnS mutagenesis suggest the involvement of a minimum of five genes. Hybridization to the 2.2-kilobase-pair SstI fragment that lies within the coding region for the large and small subunits of Bradyrhizobium japonicum uptake hydrogenase showed one region of strong homology among the R. capsulatus fragments isolated, which we interpret to mean that one or both structural genes were among the genes isolated.The nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus produces and consumes molecular hydrogen. In this diazotroph, H2 production occurs primarily as a function of the nitrogenase enzyme complex (26, 43) and its consumption via an uptake hydrogenase (6, 40). Although hydrogenase catalyzes the reversible oxidation of H2 to protons and electrons, the enzyme found in R. capsulatus appears to be physiologically important only in an H2 uptake capacity (5, 40). The directionality of the enzyme underscores its essential role in photoautotrophic (19) and chemoautotrophic (31) growth and in recycling H2 generated during N2 reduction (40).Because H2 metabolism influences the efficiency of nitrogen fixation (14, 15), emphasis has been placed on gaining a clearer understanding of the regulation and function of hydrogenases in the diazotrophs (7,14,21). In addition, the potential application of the phototrophs for H2 production from biomass at the expense of light energy (44) provides a second impetus for analysis of this enzyme system. The enzyme has been purified from R. capsulatus and was initially reported to be a monomer (6). Later purification yielded a preparation with higher specific activity and showed the hydrogenase to be a dimer with subunits of 67,000 and 31,000 daltons (Da) (37). As yet, the number of genes essential for uptake hydrogenase biosynthesis and function has not been determined, although at least two have been isolated (7). Recently, the hydrogenase control system in Alcaligenes eutrophus has been implicated in the regulation of five polypeptides in addition to the hydrogenase peptides (28). Thus, it is possible that a regulon may be necessary for the formation, maturation, and metal metabolism of uptake hydrogenase and electron flux from this enzyme.To obtain a more complete picture of the hydrogenase genetic system in R. capsulatus, we isolated mutants unable to grow photoautotrophically and unable to reduce methylene blue with H2. After transductional analysis, complementing fragments were obtained from a pLAFR1 cosmid library. Subcloning and complementation revealed the presence of a minimum of five genes essential for restoration of * Corresponding author. t Missouri Agricultural Experiment Station journal series no. 10644.the mutant phenotype...

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“…2A) were used as hybridization probes to screen 1,500 clones of the gene bank by the colony hybridization method. The 5.9-kb HindIII fragment contains the 60-kilodalton subunit gene for the B. japonicum hydrogenase (43), and the 5.0-kb EcoRI fragment also contains essential genes for hydrogen uptake in B. japonicum (17). Two clones were identified, and the corresponding recombinant cosmids (pAL618 and pAL704) were isolated.…”

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“…Both cosmids pHU1 * Corresponding author. and pHU52, however, contained the genes for the two polypeptide subunits of the hydrogenase (43).…”

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“…After gel electrophoresis and blotting, EcoRI digests from these cosmids and from total DNA of strain UPM791 were hybridized to probe DNAs from pHU1 to confirm that the cosmids did contain sequences homologous to B. japonicum hup DNA and that the hybridizing fragments were present in the R. leguminosarum genome. In addition to the DNA probes mentioned above, the 2.9-kb EcoRI fragment from pHU1, containing the gene coding for the 30-kilodalton subunit of the B. japonicum hydrogenase (43), was also used as a hybridization probe (Fig. 1).…”

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Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum

et al. 1987

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A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHUl containing hup genes of Bradyrhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHUl (5.9-kb HindIll, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. keguminosarum hup-specific region closely parallels that of B. japonwum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed TnS mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the TnS insertion strains in symbiosis with peas. Transposon TnS insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.Certain strains of Bradyrhizobium japonicum and Rhizobium leguminosarum (Hup+ strains) induce, in symbiosis with soybeans and peas, respectively, the synthesis of an H2 uptake system which catalyzes the recycling of H2 generated by the nitrogenase complex as an obligate by-product of the nitrogen fixation process (11,13). This H2 uptake system has been studied in detail in free-living and symbiotic cells of Hup+ strains of B. japonicum (13,22,26). The first component of the system is a membrane-bound hydrogenase which contains nickel and two polypeptide subunits. Hydrogen is activated by the hydrogenase and oxidized with 02 to water through an electron transport chain, some of whose components may be specifically involved in the H2 oxidation. The oxidation of H2 in legume nodules has been shown to increase N2 fixation and legume productivity (13,14).Hydrogen uptake (hup) genes are not expressed in normal cultures of rhizobia. However, derepression of hup genes in free-living B. japonicum has been shown under certain culture conditions including low-carbon medium and an atmosphere with H2, C02, and low 02 (27)

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Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli.

Cited by 19 publications

References 14 publications

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum

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