hydγ, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase

Stokkermans, J.P.W.G.

doi:10.1016/0378-1097(89)90041-4

Cited by 18 publications

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“…However, no hydrogenase activity using reduced ferredoxin as a substrate could be detected. Similar results have already been found for two [Fe] hydrogenase genes expressed in E. coli (43,48).…”

Section: Resultssupporting

confidence: 88%

“…The hydrogenase of C. acetobutylicum ATCC 824 is about 70% identical and 80% similar to [Fe] hydrogenases of the other clostridial species tested, i.e., C. acetobutylicum P262 (41) and C. pasteurianum W5 (31). Comparison of C. acetobutylicum ATCC 824 hydrogenase with hydrogenases of the Desulfovibrio species, i.e., D. fructosovorans (27), D. vulgaris Hildenborough (43,47), and D. vulgaris Monticello (49), gives a lower level of identity (about 40%), although more than 50% similarity is reached. The secondary structure of the hydrogenase of C. acetobutylicum ATCC 824 was estimated by the method of Garnier (16).…”

Section: Resultsmentioning

confidence: 88%

“…2) are homologous to clostridial consensus regions that correspond to elements recognized by B. subtilis (43) and E. coli (70) RNA polymerases (53). Young and coworkers (53) proposed an extended recognition sequence between Ϫ10 and Ϫ35 regions: 5Ј-AAtATga-3Ј.…”

Section: Resultsmentioning

confidence: 99%

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Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824

1996

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Clostridium acetobutylicum, a strictly anaerobic spore-forming bacterium, usually shows a biphasic batch fermentation pattern when grown on glucose. After producing acetate and butyrate, the organism switches to the formation of acetone, butanol, and ethanol shortly before entering the stationary phase (23). The change in carbon flow from acids to solvents is associated with a modification of the electron flow. In the acidogenic phase, ferredoxin is reduced by the pyruvate-ferredoxin oxidoreductase and by the NADH-ferredoxin reductase to oxidize the NADH produced in excess during glycolysis (21). Oxidized ferredoxin is regenerated by the hydrogenase, using the protons as electron acceptors. In the solventogenic phase, ethanol-and butanol-producing pathways require more NAD(P)H than can be produced during glycolysis. The reoxidation of reduced ferredoxin to produce NAD(P)H then competes with the oxidation of ferredoxin by hydrogenase, and as a consequence the rate of hydrogen production is decreased (23). Two other roles have been assigned to the hydrogenase of C. acetobutylicum: (i) at neutral pH it is involved in the removal of protons from the cytoplasm, limiting the expenditure of ATP to generate the proton motive force (20), and (ii) a decrease of its in vivo activity is sufficient to modify product distribution from acid toward alcohol formation. The latter role has been demonstrated in several manners: (i) by increasing hydrogen partial pressure (14, 44, 52); (ii) by sparging the culture with carbon monoxide (11,25,28,29), a reversible hydrogenase inhibitor (45); (iii) by limiting iron in cultures to decrease active hydrogenase concentration (24, 35); (iv) by addition of an artificial electron carrier (19, 34, 37); and (v) by growth on mixtures of glucose and glycerol used as the substrate (46). Although the regulation of hydrogenase activity at the enzymatic level has been clearly established (46), its regulation at the genetic level in the ATCC 824 strain has never been studied. Cloning of the hydrogenase gene is required before the molecular mechanism(s) of regulation of expression can be elucidated and is also necessary for the development of strains with low levels of expression of this gene.In this report, the cloning, sequencing, molecular characterization, and expression of the putative hydrogenase gene of C. acetobutylicum ATCC 824 are described. MATERIALS AND METHODSBacterial strains and plasmids. C. acetobutylicum ATCC 824 was obtained from the American Type Culture Collection (Rockville, Md.). Escherichia coli ER2275 [trp-31 his-1 tonA2 rpsL104 supE44 xyl-7 mtl-2 metB1 e14 ϩ ⌬(lac)U169 endA1 recA1 R(zgb-210::Tn10) Tet s ⌬(mcr-hsd-mrr)114::IS10/FЈ proAB lacI q Z ⌬M15 22::mini-Tn10 (Km r )] was used as a host for all cloning steps and was obtained from New England Biolabs (Beverly, Mass.). The plasmid pUC18 (51) was used for the construction of the genomic libraries. The plasmid pCPH11 containing an EcoRI insert with a fragment of the Clostridium pasteurianum hydrogenase gene (hydI) was kindly p...

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 88%

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Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824

1996

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“…Escherichia coli possesses four hydrogenases (1) (6), and a gene encoding a potential fourth one has been isolated (21). Thus, it is not surprising for D. fructosovorans to possess a fourth hydrogenase activity.…”

Section: Fig 2 Southern Blot Hybridizations Of Genomic Dnas Digestedmentioning

confidence: 99%

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

et al. 2002

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A strain devoid of the three hydrogenases characterized for Desulfovibrio fructosovorans was constructed using marker exchange mutagenesis. As expected, the H 2 -dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H ؉ -D 2 exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in D. fructosovorans.Molecular hydrogen plays an important role in the energygenerating metabolism of sulfate reducers belonging to the genus Desulfovibrio. Desulfovibrio species can alternatively utilize hydrogen as the sole source of electron and energy (2, 3) or can produce hydrogen when growing fermentatively on a suitable carbon source in the absence of sulfate as an electron acceptor (17). Furthermore, hydrogen is successively produced and consumed during the degradation of organic compounds in the presence of sulfate (7,22,23 [NiFeSe] on the basis of their metal contents), and the cellular location of hydrogenases vary considerably from one Desulfovibrio species to another (6,25). This diversity makes the role of these various hydrogenases difficult to determine.With the aim to study the role of hydrogenases in Desulfovibrio, we chose D. fructosovorans DSM 3604 (16) as a model. In this species, three hydrogenases have been already characterized: a periplasmic [NiFe] hydrogenase which represents about 1% of the total proteins (8, 20), a cytoplasmic NADPreducing hydrogenase (13), and a periplasmic [Fe] hydrogenase (4). In order to elucidate the relative importance of these various hydrogenases in the energy-generating metabolism of D. fructosovorans, deletions were first made by marker exchange mutagenesis of the genes encoding the [NiFe] hydrogenase (19) and the NADP-reducing hydrogenase (12). All mutants (single or double) showed significant growth on organic substrates as well as on medium containing H 2 as the sole energy source.Construction and molecular characterization of a triple mutant depleted of all three hydrogenases. In order to perform the marker exchange experiment, a 5-kb fragment containing the two structural genes (hydAB) coding for the [Fe] hydrogenase of D. fructosovorans (obtained by PCR amplification performed on genomic DNA by using oligonucleotides 1 [5Ј-AAACGGCGACGCCGTGGTCGGCAAGGTCAA-3Ј] and 2 [5Ј-CGATGTCGGTGCCCGGATATTT-3Ј]) was cloned in pMosblue-T-vector (Amersham) to give the recombinant plasmid pMBE9 (Fig. 1). A 1.3-kb fragment containing the gentamicin resistance gene (acc1) was obtained by PCR amplification performed on pML122 (10), using two oligonucleotides, one introducing a BspEI restriction site (in boldface) (Gm 1, 5Ј-TTAAATCCGGATGAAGGCACGAACCCAGTT-3Ј) and one located downstream from the BstEII restriction site (Gm 2, 5Ј-GACGCTTAGCACCTCTGATAGTT-3Ј). The amplification product was digested with BstEII and BspEI and cloned into pMBE9 with a deletion of the BstEII-BspEI fragment containing hydAB, to give pE9Gm (Fig. 1). This recombinant s...

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“…2). A potentially bidirectional transcription terminator has been shown to be present in the region of the Hildenborough sequence separating the hydAB and hydC genes (25,28) *** ** * ** ********** * **** *1*1* * * * * * ** **** * * * MSRIEMEKIFYEDHAPDPKADPDKLFFIQIDESK C IG C DS C QQY C PTGAIFGDTGDAHKI 61 PHIEA C IN C GQ C LTH C PENAIYEAQSWVPEVEKKLKDGKVKCIAMPAPAVRYALGDAFGM ** * * ** * ** **** ** **** **** * *** * ** ***************** PHEEL C IN C GQ LTH PVGAIYESQSWVTEIEKKIKAKDVKVIAMPAPAVRYALGDAFGL 121 PVGSVTTGKMLAALQKLGFAHCWDTEFTADVTIWEEGSEFVERLTKKSDMPLPQFTS _ P *** ****** ** *** **** ************ *** ***** * ******* ** * PVGTVTTGKMFSALKELGFDHCWDNEFTADVTIWEEGTEFVQRLTKKLDKPLPQFTS CC P A 181 GWQKYAETYYPELLPHFSTCKSPIGMNGALAKTYGAERMKYDPKQVYTVSIMP C IAKKYE ** ** * **** ** * ******* * ******* ***** ******** 1* ***** GWHKYVESLYPELFPHMSSCKSPIGMLGTLAKTYGADRMKYDRAKVYTVSIMP C TAKKYE A -241 GLRPELKSSGMRDIDATLTTRELAYMIKKAGIDFAKLPDGKRDSLMGESTGGATIFGVTG * ** * ** ***** *********** *** ******** ********** ***** GMRPQLWDSGHKDIDATIDTRELAYMIKKAKIDFTKLPDGKRDTLMGESTGGATLFGVTG 301 GVMEAALRFAYEAVTGKKPDSWDFKAVRGLDGIKEATVNVGGTDVKVAVVHGAKRFKQVC ******** ** ******* * *** **** * ********* ********** ** ** GVMEAALRYAYQAVTGKKPESMDFKGVRGLQGVKEATVNVGGVDVKVAVVHGARRFHDVC A 361 DDVKAGKSPYHFIEYMACPGG C V C GGGQPVMPGVLEAMDRTTTRLYAGLKKRLAMASANKA ***** * **** ****** 1*1*1* ** ********* ** ** ** ************* * ELVKAGKAPWHFIEFMACPGG C ViC GGGQPVMPGVLEAADRRSTRMYAGLKKRLAMASASRA (AAAAGCCCCCCGACGCGCAGCGCAGGGGGGCG GAAAA). The homology of hydC to the hydAB genes was discussed above and is indicated in Fig.…”

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confidence: 99%

Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello

1989

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The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of a and 0i subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) a subunit precedes that of the 13.5-kDa 0i subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both a subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed ,I subunits (124 and 123 amino acid residues, respectively) share 71 % sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was (1,20), and gram-negative photosynthetic bacteria such as Rhodobacter capsulatus (8). The presence of the [NiFe] hydrogenase in these bacteria has been proven by purification and characterization of the enzymes (1,6,10,13,14,18,20,22) and, more recently, by Southern blotting, cloning, and sequencing of the genes for this hydrogenase (8,9,21

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hydγ, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase

Cited by 18 publications

References 0 publications

Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824

Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824

Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello

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