Biomedical subjects

Gerrit Voordouw

Publications and source records attributed to Gerrit Voordouw.

At least 19 recordsLinked to original sources

PubMed2006

Synergistic inhibition of microbial sulfide production by combinations of the metabolic inhibitor nitrite and biocides.

MICs of six broad-spectrum biocides and two specific metabolic inhibitors and fractional inhibitory concentration indexes (FICIs) for controlling a sulfide-producing consortium were determined. Nitrite was synergistic (FICI<1) with all but one biocide due to its specific inhibition of dissimilatory sulfite reductase. Hence, combining nitrite with biocides allows more efficient and cost-effective control of sulfate-reducing bacteria.

Benzalkonium Compounds↗

PubMed2006

Sequential and structural analysis of [NiFe]-hydrogenase-maturation proteins from Desulfovibrio vulgaris Miyazaki F.

The complete primary structure of the hyn-region in the genome of Desulfovibrio vulgaris Miyazaki F (DvMF), encoding the [NiFe]-hydrogenase and two maturation proteins has been identified. Besides the formerly reported genes for the large and small subunits, this region comprises genes encoding an endopeptidase (HynC) and a putative chaperone (HynD). The complete genomic region covers 4086 nucleotides including the previously published upstream located promoter region and the sequences of the structural genes. A phylogenetic tree for both maturation proteins shows strongest sequential relationship to the orthologous proteins of Desulfovibrio vulgaris Hildenborough (DvH). Secondary structure prediction for HynC (168 aa, corresponding to a molecular weight of 17.9 kDa) revealed a practically identical arrangement of alpha-helical and beta-strand elements between the orthologous protein HybD from E. coli and allowed a three-dimensional modelling of HynC on the basis of the formerly published structure of HybD. The putative chaperone HynD consists of 83 aa (molecular weight of 9 kDa) and shows 76% homology to DvH HynD. Preliminary experiments demonstrate that the operon is expressed under the control of its own promoter in Escherichia coli, although no further processing could be observed, providing evidence that additional proteins have to be involved in the maturation process. Accession numbers: DQ072852, HynC protein ID AAY90127, HynD protein ID AAY90128.

Amino Acid Sequence↗

PubMed2006

Effect of nitrite on a thermophilic, methanogenic consortium from an oil storage tank.

Samples from an oil storage tank (resident temperature 40 to 60 degrees C), which experienced unwanted periodic odorous gas emissions, contained up to 2,400/ml of thermophilic, lactate-utilizing, sulfate-reducing bacteria. Significant methane production was also evident. Enrichments on acetate gave sheathed filaments characteristic of the acetotrophic methanogen Methanosaeta thermophila of which the presence was confirmed by determining the PCR-amplified 16S rDNA sequence. 16S rDNA analysis of enrichments, grown on lactate- and sulfate-containing media, indicated the presence of bacteria related to Garciella nitratireducens, Clostridium sp. and Acinetobacter sp. These sulfidogenic enrichments typically produced sulfide to a maximum concentration of 5-7 mM in media containing excess lactate and 10 mM sulfate or thiosulfate. Both the production of sulfide and the consumption of acetate by the enrichment cultures were inhibited by low concentrations of nitrite (0.5-1.0 mM). Hence, addition of nitrite may be an effective way to prevent odorous gas emissions from the storage tank.

Anti-Infective Agents↗

PubMed2006

Freeze-thaw tolerance and clues to the winter survival of a soil community.

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.

Adaptation, Physiological↗

PubMed2006

Rubredoxin:oxygen oxidoreductase enhances survival of Desulfovibrio vulgaris hildenborough under microaerophilic conditions.

Genes for superoxide reductase (Sor), rubredoxin (Rub), and rubredoxin:oxygen oxidoreductase (Roo) are located in close proximity in the chromosome of Desulfovibrio vulgaris Hildenborough. Protein blots confirmed the absence of Roo from roo mutant and sor-rub-roo (srr) mutant cells and its presence in sor mutant and wild-type cells grown under anaerobic conditions. Oxygen reduction rates of the roo and srr mutants were 20 to 40% lower than those of the wild type and the sor mutant, indicating that Roo functions as an O2 reductase in vivo. Survival of single cells incubated for 5 days on agar plates under microaerophilic conditions (1% air) was 85% for the sor, 4% for the roo, and 0.7% for the srr mutant relative to that of the wild type (100%). The similar survival rates of sor mutant and wild-type cells suggest that O2 reduction by Roo prevents the formation of reactive oxygen species (ROS) under these conditions; i.e., the ROS-reducing enzyme Sor is only needed for survival when Roo is missing. In contrast, the sor mutant was inactivated much more rapidly than the roo mutant when liquid cultures were incubated in 100% air, indicating that O2 reduction by Roo and other terminal oxidases did not prevent ROS formation under these conditions. Competition of Sor and Roo for limited reduced Rub was suggested by the observation that the roo mutant survived better than the wild type under fully aerobic conditions. The roo mutant was more strongly inhibited than the wild type by the nitric oxide (NO)-generating compound S-nitrosoglutathione, indicating that Roo may also serve as an NO reductase in vivo.

Aerobiosis↗

PubMed2005

Corrosion risk associated with microbial souring control using nitrate or nitrite.

Souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB) in oil reservoirs, can be controlled through nitrate or nitrite addition. To assess the effects of this containment approach on corrosion, metal coupons were installed in up-flow packed-bed bioreactors fed with medium containing 8 mM sulfate and 25 mM lactate. Following inoculation with produced water to establish biogenic H(2)S production, some bioreactors were treated with 17.5 mM nitrate or up to 20 mM nitrite, eliminating souring. Corrosion rates were highest near the outlet of untreated bioreactors (up to 0.4 mm year(-1)). Nitrate (17.5 mM) eliminated sulfide but gave pitting corrosion near the inlet of the bioreactor, whereas a high nitrite dose (20 mM) completely eliminated microbial activity and associated corrosion. More gradual, step-wise addition of nitrite up to 20 mM resulted in the retention of microbial activity and localized pitting corrosion, especially near the bioreactor inlet. We conclude that: (1) SRB control by nitrate or nitrite reduction shifts the corrosion risk from the bioreactor outlet to the inlet (i.e. from production to injection wells) and (2) souring treatment by continuous addition of a high inhibitory nitrite dose is preferable from a corrosion-prevention point of view.

Bacteria↗

PubMed2005

Changes in soil microbial community composition induced by cometabolism of toluene and trichloroethylene.

The effects of trichloroethylene (TCE) on microbial community composition were analyzed by reverse sample genome probing. Soil enrichments were incubated in dessicators containing an organic phase of either 1 or 10% (w/w) toluene in vacuum pump oil, delivering constant equilibrium aqueous concentrations of 16 and 143 mg/l, respectively. Increasing the equilibrium aqueous concentration of TCE from 0 to 10 mg/l led to shifts in community composition at 16, but not at 143 mg/l of toluene. In closed system co-degradation studies, TCE at an aqueous concentration of 1 mg/l was effectively degraded by toluene-degrading soil enrichments once the aqueous toluene concentration dropped below 25 mg/l. Little TCE degradation was observed at higher toluene concentrations (50-250 mg/l). The results indicate that TCE changes microbial community composition under conditions where it is being actively metabolized.

Biodegradation, Environmental↗

PubMed2005

Gene expression analysis of the mechanism of inhibition of Desulfovibrio vulgaris Hildenborough by nitrate-reducing, sulfide-oxidizing bacteria.

Sulfate-reducing bacteria (SRB) are inhibited by nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) in the presence of nitrate. This inhibition has been attributed either to an increase in redox potential or to production of nitrite by the NR-SOB. Nitrite specifically inhibits the final step in the sulfate reduction pathway. When the NR-SOB Thiomicrospira sp. strain CVO was added to mid-log phase cultures of the SRB Desulfovibrio vulgaris Hildenborough in the presence of nitrate, sulfate reduction was inhibited. Strain CVO reduced nitrate and oxidized sulfide, with transient production of nitrite. Sulfate reduction by D. vulgaris resumed once nitrite was depleted. A DNA macroarray with open reading frames encoding enzymes involved in energy metabolism of D. vulgaris was used to study the effects of NR-SOB on gene expression. Shortly following addition of strain CVO, D. vulgaris genes for cytochrome c nitrite reductase and hybrid cluster proteins Hcp1 and Hcp2 were upregulated. Genes for sulfate reduction enzymes, except those for dissimilatory sulfite reductase, were downregulated. Genes for the membrane-bound electron transferring complexes QmoABC and DsrMKJOP were downregulated and unaffected, respectively, whereas direct addition of nitrite downregulated both operons. Overall the gene expression response of D. vulgaris upon exposure to strain CVO and nitrate resembled that observed upon direct addition of nitrite, indicating that inhibition of SRB is primarily due to nitrite production by NR-SOB.

Desulfovibrio vulgaris↗

PubMed2004

The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encoding enzymes for energy transduction, together with inferred cellular location of the enzymes, provides a basis for proposing an expansion to the 'hydrogen-cycling' model for increasing energy efficiency in this bacterium. Plasmid-encoded functions include modification of cell surface components, nitrogen fixation and a type-III protein secretion system. This genome sequence represents a substantial step toward the elucidation of pathways for reduction (and bioremediation) of pollutants such as uranium and chromium and offers a new starting point for defining this organism's complex anaerobic respiration.

Desulfovibrio vulgaris↗

PubMed2004

Physiological and gene expression analysis of inhibition of Desulfovibrio vulgaris hildenborough by nitrite.

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 K(m) for nitrite = 0.03 mM), and in which NrfHA (K(m) 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.

Cytochromes a1↗

PubMed2003

Hydration structures in proteins and neutron diffraction experiment on dissimilatory sul te reductase D (DsrD).

Neutron crystallography can provide a substantial amount of information about the hydration of proteins. The hydration patterns of three proteins, whose structures have been solved at 1.5 or 1.6 A resolution using our BIX-type diffractometers, show interesting features. The water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen-bond formation and dynamics of hydration. In addition, the neutron diffraction study of a DNA-binding protein, dissimilatory sulfite reductase D (DsrD) is briefly described, and some preliminary results are presented. This topic is of interest because it is well known that hydrogen bonds play important roles in DNA-protein recognition.

Binding Sites↗

PubMed2003

Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris.

Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study. The initial crystals obtained were too small for the neutron experiment. In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction. Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique. A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal.

Crystallization↗

PubMed2003

Analysis of environmental microbial communities by reverse sample genome probing.

Development of fast and accurate methods for monitoring environmental microbial diversity is one of the great challenges in microbiology today. Oligonucleotide probes based on 16S rRNA sequences are widely used to identify bacteria in the environment. However, the successful development of a chip of immobilized 16S rRNA probes for identification of large numbers of species in a single hybridization step has not yet been reported. In reverse sample genome probing (RSGP), labelled total community DNA is hybridized to arrays in which genomes of cultured microorganisms are spotted on a solid support in denatured form. This method has provided useful information on changes in composition of the cultured component of microbial communities in oil fields, the soil rhizhosphere, hydrocarbon-contaminated soils and acid mine drainage sites. Applications and limitations of the method, as well as the prospects of extending RSGP to cover also the as yet uncultured component of microbial communities, are evaluated.

Bacteria↗

PubMed2003

Crystal structure of dissimilatory sulfite reductase D (DsrD) protein--possible interaction with B- and Z-DNA by its winged-helix motif.

The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.

Amino Acid Sequence↗

PubMed2003

Function of oxygen resistance proteins in the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough.

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H(2)O(2)) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H(2)O(2) resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H(2)O(2), but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.

Anaerobiosis↗

PubMed2003

Gene expression analysis of energy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol dehydrogenase.

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 H(2)-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.

Alcohol Dehydrogenase↗

PubMed2002

Crystallographic investigation of the role of aspartate 95 in the modulation of the redox potentials of Desulfovibrio vulgaris flavodoxin.

The side chain of aspartate 95 in flavodoxin from Desulfovibrio vulgaris provides the closest negative charge to N(1) of the bound FMN in the protein. Site-directed mutagenesis was used to substitute alanine, asparagine, or glutamate for this amino acid to assess the effect of this charge on the semiquinone/hydroquinone redox potential (E(1)) of the FMN cofactor. The D95A mutation shifts the E(1) redox potential positively by 16 mV, while a negative shift of 23 mV occurs in the oxidized/semiquinone midpoint redox potential (E(2)). The crystal structures of the oxidized and semiquinone forms of this mutant are similar to the corresponding states of the wild-type protein. In contrast to the wild-type protein, a further change in structure occurs in the D95A mutant in the hydroquinone form. The side chain of Y98 flips into an energetically more favorable edge-to-face interaction with the bound FMN. Analysis of the structural changes in the D95A mutant, taking into account electrostatic interactions at the FMN binding site, suggests that the pi-pi electrostatic repulsions have only a minor contribution to the very low E(1) redox potential of the FMN cofactor when bound to apoflavodoxin. Substitution of D95 with glutamate causes only a slight perturbation of the two one-electron redox potentials of the FMN cofactor. The structure of the D95E mutant reveals a large movement of the 60-loop (residues 60-64) away from the flavin in the oxidized structure. Reduction of this mutant to the hydroquinone causes the conformation of the 60-loop to revert back to that occurring in the structures of the wild-type protein. The crystal structures of the D95E mutant imply that electrostatic repulsion between a carboxylate on the side chain at position 95 and the phenol ring of Y98 prevents rotation of the Y98 side chain to a more energetically favorable conformation as occurs in the D95A mutant. Replacement of D95 with asparagine has no effect on E(2) but causes E(1) to change by 45 mV. The D95N mutant failed to crystallize. The K(d) values of the protein FMN complex in all three oxidation-reduction states differ from those of the wild-type complexes. Molecular modeling showed that the conformational energy of the protein changes with the redox state, in qualitative agreement with the observed changes in K(d), and allowed the electrostatic interactions between the FMN and the surrounding groups on the protein to be quantified.

Alanine↗

PubMed2002

Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough.

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.

Carbon Monoxide↗