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Query: EC:1.12.7.2 (hydrogenase)
 3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new gene whose product is required for the production of formate hydrogenlyase (FHL) has been identified in Escherichia coli. This gene, termed fhlB, maps between the frdA (94.4 min) and argI (96.6 min) genes on the E. coli chromosome and is transcribed in a clockwise direction toward argI. Biochemical analysis of an FhlB- mutant, strain SE-2011 [phi(fhlB-lacZ+)], revealed that the mutant lacks formate dehydrogenase activity associated with FHL (FDH-H) and hydrogenase activity. As a result of these defects, fermentative hydrogen production and hydrogen uptake reactions were undetectable in strain SE-2011. Fumarate reductase activity of this mutant was also reduced to about 15% of the levels of the parent (strain MC4100), and strain SE-2011 did not produce succinate as a fermentation end product. Regulation of expression of the fhlB gene, studied as production of beta-galactosidase activity by strain SE-2011, revealed that the operon is expressed at low levels under aerobic conditions. Under anaerobic growth conditions, this activity increased by two- to threefold. Addition of formate enhanced the differential rate of synthesis of the fhlB gene product to as high as 130 U of beta-galactosidase specific activity per microgram of cell protein, but only under anaerobic conditions. Formate-dependent expression of phi(fhlB-lacZ+) required the sigma 54 subunit of RNA polymerase and the fhlA gene product. The concentration of formate required for maximum expression of the fhlB gene was about 15 mM; this value decreased to about 3 mM in the presence of plasmid pSE-133, which carries the fhlA gene in a multicopy plasmid. DNA sequence analysis of the fhlA gene showed that the FhlA protein is 686 amino acids long and has an anhydrous molecular weight of 78,086. On the basis of sequence homology with other transcriptional activators such as NtrC, HydG, and Klebsiella pneumoniae NifA proteins, the FhlA protein was deduced to be a transcriptional activator controlling the production of FHL. It is proposed that formate interacts with the FhlA protein and that this active complex initiates transcription of the fhlB gene. The FhlA and FhlB proteins act as a cascade in regulating the production of FDH-H and the FHL-linked hydrogenase and ultimately the production of FHL and fermentative hydrogen.
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PMID:Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB. 211 3

We have sequenced downstream of the last previously sequenced gene of the glucitol operon (gutABDMRQ) in E. coli and have found that gutQ is the last gene of this operon. Downstream of the gutQ gene is found a palindromic unit (PU or REP sequence), followed by a large open reading frame of 1515 (or possibly 1590) bps transcribed in the direction opposite to that of the gut operon. This open reading frame encodes a protein of 504 (or possibly 529) amino acids with a tripartite structure. The N-terminal "receiver" domain of 187 (or possibly 212) residues is homologous to the FhlA protein of E. coli, a transcriptional activator of formate hydrogen lyase. It may possess a short domain at its extreme N-terminus exhibiting sequence similarity to carbohydrate binding proteins. The central ATPase domain (236 residues) exhibits greatest sequence similarity to the HydG protein of E. coli, a transcriptional activator of labile hydrogenase. The C-terminal DNA binding domain (81 residues) is homologous to NtrX of Azorhizobium caulinodans, a protein involved in transcriptional regulation of nitrogen fixation. Sequence comparisons with well-characterized transcription factors suggest that ORF504 encodes a protein that hydrolyzes ATP to generate the open transcriptional initiation complex of sigma 54-dependent promoters, possibly in response to redox conditions and/or ligand binding. We propose that this tripartite transcription factor arose by fusion of gene fragments encoding its three constituent modules.
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PMID:DNA sequence of a gene in Escherichia coli encoding a putative tripartite transcription factor with receiver, ATPase and DNA binding domains. 789 55

Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.
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PMID:The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation. 802 Dec 24

In the photosynthetic bacterium Rhodospirillum rubrum, the presence of carbon monoxide (CO) induces expression of several proteins. These include carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. Together these enzymes catalyze the following conversion: CO + H2O --> CO2 + H2. This system enables R. rubrum to grow in the dark on CO as the sole energy source. Expression of this system has been shown previously to be regulated at the transcriptional level by CO. We have now identified the remainder of the CO-regulated genes encoded in a contiguous region of the R. rubrum genome. These genes, cooMKLXU, apparently encode proteins related to the function of the CO-induced hydrogenase. As seen before with the gene for the large subunit of the CO-induced hydrogenase (cooH), most of the proteins predicted by these additional genes show significant sequence similarity to subunits of Escherichia coli hydrogenase 3. In addition, all of the newly identified coo gene products show similarity to subunits of NADH-quinone oxidoreductase (energy-conserving NADH dehydrogenase I) from various eukaryotic and prokaryotic organisms. We have found that dicyclohexylcarbodiimide, an inhibitor of mitochondrial NADH dehydrogenase I (also called complex I), inhibits the CO-induced hydrogenase as well. We also show that expression of the cooMKLXUH operon is regulated by CO and the transcriptional activator CooA in a manner similar to that of the cooFSCTJ operon that encodes the subunits of CODH and related proteins.
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PMID:Characterization of the region encoding the CO-induced hydrogenase of Rhodospirillum rubrum. 889 19

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.
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PMID:A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species. 904 26

The transcriptional regulation of two energy metabolism operons, hya and cbdAB-appA, has been investigated during carbon and phosphate starvation. The hya operon encodes hydrogenase 1, and the cbdAB-appA operon encodes cytochrome bd-II oxidase and acid phosphatase, pH 2.5. Both operons are targets for the transcriptional activator AppY. In exponential growth, expression of the hya and cbd operons was reduced in an rpoS mutant lacking the RNA polymerase sigmaS factor, and the induction of the two operons by entry into stationary phase in rich medium was strongly dependent on sigmaS. Both operons were induced by carbon starvation, but only induction of the hya operon was dependent on sigmaS, whereas that of the cbd promoter was dependent on AppY. The appY gene also showed sigmaS-dependent induction by carbon starvation. The cbd and hya operons were also found to exhibit a sigmaS-dependent transient twofold induction by osmotic upshift. Like the cbd operon, the hya operon was highly induced by phosphate starvation. For both operons the induction was strongly dependent on AppY. The induction ratio of the two operons was the same in rpoS+ and rpoS mutant strains, indicating that the phosphate starvation-induced increase in sigmaS concentration is not involved in the phosphate regulation of these operons.
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PMID:Effects of sigmaS and the transcriptional activator AppY on induction of the Escherichia coli hya and cbdAB-appA operons in response to carbon and phosphate starvation. 907 97

In-frame deletion mutagenesis was used to study the roles of two Bradyrhizobium japonicum proteins, HoxX and HoxA, in hydrogenase biosynthesis; based on their sequences, these proteins were previously proposed to be sensor and regulator proteins, respectively, of a two-component regulatory system necessary for hydrogenase transcription. Deletion of the hoxX gene resulted in a strain that expressed only 30 to 40% of wild-type hydrogenase activity. The inactive unprocessed form of the hydrogenase large subunit accumulated in this strain, indicating a role for HoxX in posttranslational processing of the hydrogenase enzyme but not in transcriptional regulation. Strains containing a deletion of the hoxA gene or a double mutation (hoxX and hoxA) did not exhibit any hydrogenase activity under free-living conditions, and extracts from these strains were inactive in gel retardation assays with a 158-bp fragment of the DNA region upstream of the hupSL operon. However, bacteroids from root nodules formed by all three mutant types (hoxX, hoxA, and hoxX hoxA) exhibited hydrogenase activity comparable to that of wild-type bacteroids. Bacteroid extracts from all of these strains, including the wild type, failed to cause a shift of the hydrogenase upstream region used in our assay. It was shown that HoxA is a DNA-binding transcriptional activator of hydrogenase structural gene expression under free-living conditions but not under symbiotic conditions. Although symbiotic hydrogenase expression is still sigma54 dependent, a transcriptional activator other than HoxA functions presumably upstream of the HoxA binding site.
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PMID:Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum. 917 16

Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a -24/-12 type promoter (hupSp). A regulatory region located in the -173 to -88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential -173 to -88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN sigma-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.
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PMID:Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA. 917 61

Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins. Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules. In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.
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PMID:FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791. 928 75

The nucleotide sequence has been determined for a twelve-gene operon of Escherichia coli designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon is located at 55.8-56.0 min and encodes a putative nine-subunit hydrogenase complex (hydrogenase four or Hyf), a potential formate- and sigma 54-dependent transcriptional activator, HyfR (related to FhlA), and a possible formate transporter, FocB (related to FocA). Five of the nine Hyf-complex subunits are related to subunits of both the E. coli hydrogenase-3 complex (Hyc) and the proton-translocating NADH:quinone oxidoreductases (complex I and Nuo), whereas two Hyf subunits are related solely to NADH:quinone oxidoreductase subunits. The Hyf components include a predicted 523 residue [Ni-Fe] hydrogenase (large subunit) with an N-terminus (residues 1-170) homologous to the 30 kDa or NuoC subunit of complex I. It is proposed that Hyf, in conjunction with formate dehydrogenase H (Fdh-H), forms a hitherto unrecognized respiration-linked proton-translocating formate hydrogenlyase (FHL-2). It is likely that HyfR acts as a formate-dependent regulator of the hyf operon and that FocB provides the Hyf complex with external formate as substrate.
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PMID:A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. 938 41


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