scholarly journals Gene Expression Analysis of Energy Metabolism Mutants of Desulfovibrio vulgaris Hildenborough Indicates an Important Role for Alcohol Dehydrogenase

2003 ◽  
Vol 185 (15) ◽  
pp. 4345-4353 ◽  
Author(s):  
Shelley A. Haveman ◽  
Véronique Brunelle ◽  
Johanna K. Voordouw ◽  
Gerrit Voordouw ◽  
John F. Heidelberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Energy Metabolism ◽  
Carbon Source ◽  
Alcohol Dehydrogenase ◽  
Sulfate Reduction ◽  
Electron Donor ◽  
Desulfovibrio Vulgaris ◽  
Wild Type ◽  
Reading Frame ◽  
Metabolic Conditions

ABSTRACT Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of Desulfovibrio vulgaris Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the hyd mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified D. vulgaris genes encoding proteins active in energy metabolism indicated that the adh gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the adh gene was strongly downregulated in the hyd mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An adh mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The hyd mutant also grew poorly on ethanol, in agreement with its low level of adh gene expression. The adh mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of adh in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of D. vulgaris under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H2-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.

scholarly journals Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

2002 ◽  
Vol 184 (3) ◽  
pp. 679-686 ◽  
Author(s):  
Brant K. J. Pohorelic ◽  
Johanna K. Voordouw ◽  
Elisabeth Lojou ◽  
Alain Dolla ◽  
Jens Harder ◽  
...  
Keyword(s):  
Sulfate Reduction ◽  
Electron Donor ◽  
Type Strain ◽  
Wild Type Strain ◽  
Physiological Role ◽  
Hydrogen Uptake ◽  
Desulfovibrio Vulgaris ◽  
Lactate Metabolism ◽  
Wild Type ◽  
Genes Encoding

ABSTRACT The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H2 as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H2 was also present, growth in lactate- and sulfate-containing media lacking H2 was less efficient. The hyd mutant produced, transiently, significant amounts of H2 under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688–3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.

Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: the role of carbon metabolites and the response regulators DorR and RegA

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 605-614 ◽  
Author(s):  
Ulrike Kappler ◽  
Wilhelmina M Huston ◽  
Alastair G McEwan
Keyword(s):  
Gene Expression ◽  
Carbon Source ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Reductase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Dmso Reductase ◽  
Dimethylsulfoxide Reductase ◽  
Operon Expression

Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m−2) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb 3 oxidase. A cco mutant lacking cytochrome cbb 3 exhibited significantly higher levels of Φ[dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.

scholarly journals Uranium Reduction by Desulfovibrio desulfuricans Strain G20 and a Cytochrome c3 Mutant

2002 ◽  
Vol 68 (6) ◽  
pp. 3129-3132 ◽  
Author(s):  
Rayford B. Payne ◽  
Darren M. Gentry ◽  
Barbara J. Rapp-Giles ◽  
Laurence Casalot ◽  
Judy D. Wall
Keyword(s):  
Electron Donor ◽  
Desulfovibrio Desulfuricans ◽  
Desulfovibrio Vulgaris ◽  
In Vitro Experiments ◽  
Wild Type ◽  
Cytochrome C3 ◽  
Uranium Reduction

ABSTRACT Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c 3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c 3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c 3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.

scholarly journals Physiological and Gene Expression Analysis of Inhibition of Desulfovibrio vulgaris Hildenborough by Nitrite

2004 ◽  
Vol 186 (23) ◽  
pp. 7944-7950 ◽  
Author(s):  
Shelley A. Haveman ◽  
E. Anne Greene ◽  
Claire P. Stilwell ◽  
Johanna K. Voordouw ◽  
Gerrit Voordouw
Keyword(s):  
Gene Expression ◽  
Nitrite Reduction ◽  
Desulfovibrio Vulgaris ◽  
Wild Type ◽  
Apparent Dissociation Constant ◽  
Dissimilatory Sulfite Reductase ◽  
Membrane Bound ◽  
Electron Transport Complexes ◽  
Adenosine Phosphosulfate Reductase ◽  
Bound Electron

ABSTRACT A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant Km for nitrite = 0.03 mM), and in which NrfHA (Km for nitrite = 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.

scholarly journals Gene Expression by the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough Grown on an Iron Electrode under Cathodic Protection Conditions

2008 ◽  
Vol 74 (8) ◽  
pp. 2404-2413 ◽  
Author(s):  
Sean M. Caffrey ◽  
Hyung Soo Park ◽  
Jenny Been ◽  
Paul Gordon ◽  
Christoph W. Sensen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Cathodic Protection ◽  
Gaseous Hydrogen ◽  
Iron Electrode ◽  
Desulfovibrio Vulgaris ◽  
Wild Type ◽  
Sulfate Reducing ◽  
Genes Encoding

ABSTRACT The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of −1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion.

scholarly journals The Pseudomonas aeruginosa devB/SOL Homolog,pgl, Is a Member of the hex Regulon and Encodes 6-Phosphogluconolactonase

2000 ◽  
Vol 182 (14) ◽  
pp. 3934-3941 ◽  
Author(s):  
Paul W. Hager ◽  
M. Worth Calfee ◽  
Paul V. Phibbs
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Carbon Source ◽  
Normal Growth ◽  
Wild Type ◽  
Reading Frame ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Type Rate ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Wild Type Rate

ABSTRACT A cyclic version of the Entner-Doudoroff pathway is used byPseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form thehex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOLfamily of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5′ end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgland propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.

scholarly journals Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression

2008 ◽  
Vol 190 (10) ◽  
pp. 3538-3545 ◽  
Author(s):  
Nikolai V. Ravin ◽  
Jérôme Rech ◽  
David Lane
Keyword(s):  
Gene Expression ◽  
Late Gene ◽  
Northern Hybridization ◽  
Partition Functions ◽  
Wild Type ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Partition System ◽  
Plasmid Partition

ABSTRACT The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3 −-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3 + fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3 +-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product

1990 ◽  
Vol 10 (6) ◽  
pp. 2638-2644
Author(s):  
S Marathe ◽  
I F Connerton ◽  
J R Fincham
Keyword(s):  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Protein Product ◽  
Predicted Amino Acid Sequence ◽  
Wild Type ◽  
Reading Frame ◽  
Acetate Utilization ◽  
Group 2

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.

scholarly journals Insertion of the retroposable element, jockey, near the Adh gene of Drosophila melanogaster is associated with altered gene expression

1996 ◽  
Vol 68 (3) ◽  
pp. 203-209 ◽  
Author(s):  
Lisa D. White ◽  
James W. Jacobson
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Insertion Mutation ◽  
Activity Levels ◽  
Mrna Levels ◽  
Wild Type ◽  
Reporter System ◽  
Wild Type Counterpart ◽  
Altered Gene

SummaryThe alcohol dehydrogenase (Adh) gene of Drosophila melanogaster is well suited to be a gene expression reporter system. Adh produces a measurable phenotype at both the enzyme and mRNA levels. We recovered a spontaneous transposable element (TE) insertion mutation near the Adh gene. The insertion is a truncated retroposable element, jockey, inserted upstream of the adult Adh enhancer region. Comparisons between the Adhjockey allele and its direct wild-type ancestral allele were made in an isogenic background (i.e. identical cis and trans factors). Differences in Adhjockey expression compared with the wild-type can be attributed solely to the presence of the jockey element. This jockey insertion results in a decrease in adult mRNA transcript levels in the Adhjockey homozygous lines relative to the wild-type counterpart and accounts for a correlated decrease in alcohol dehydrogenase (ADH) enzyme activity. The larval ADH activity levels are not detectably different.

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