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Study of Nitrate Stress in Desulfovibrio vulgaris Hildenborough Using iTRAQ Proteomics

Description: The response of Desulfovibrio vulgaris Hildenborough (DvH),a sulphate-reducing bacterium, to nitrate stress was examined usingquantitative proteomic analysis. DvH was stressed with 105 m M sodiumnitrate(NaNO3), a level that caused a 50 percent inhibition in growth.The protein profile of stressed cells was compared with that of cellsgrown in the absence of nitrate using the iTRAQ peptide labellingstrategy and tandem liquid chromatography separation coupled with massspectrometry (quadrupoletime-of-flight) detection. A total of 737 uniqueproteins were identified by two or more peptides, representing 22 percentof the total DvH proteome and spanning every functional category. Theresults indicate that this was a mild stress, as proteins involved incentral metabolism and the sulphate reduction pathway were unperturbed.Proteins involved in the nitrate reduction pathway increased. Increasesseen in transport systems for proline, glycine^ betaineandglutamateindicate that the NaNO3 exposure led to both salt stress and nitratestress.Up-regulation observed in oxidative stress response proteins (Rbr,RbO, etc.) and a large number of ABC transport systems as well as in iron^ sulphur -cluster-containing proteins, however, appear to be specific tonitrate exposure. Finally, a number of hypothetical proteins were amongthe most significant changers, indicating that there may be unknownmechanisms initiated upon nitrate stress in DvH.
Date: October 12, 2006
Creator: Redding, A.M.; Mukhopadhyay, A.; Joyner, D.; Hazen, T.C. & Keasling, J.D.
Partner: UNT Libraries Government Documents Department

Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples

Description: Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field.
Date: April 15, 2011
Creator: Chakraborty, R.; Hazen, T.C.; Joyner, D.C.; Kusel, K.; Singer, M.E.; Sitte, J. et al.
Partner: UNT Libraries Government Documents Department

Response of Desulfovibrio vulgaris to Alkaline Stress

Description: The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).
Date: November 30, 2007
Creator: Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D. et al.
Partner: UNT Libraries Government Documents Department

Cell wide responses to low oxygen exposure in Desulfovibriovulgaris Hildenborough

Description: The responses of the anaerobic, sulfate-reducing Desulfovibrio vulgaris Hildenborough to low oxygen exposure (0.1% O{sub 2}) were monitored via transcriptomics and proteomics. Exposure to 0.1% O{sub 2} caused a decrease in growth rate without affecting viability. A concerted up regulation in the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O{sub 2} exposure. Several of these candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the up regulation of the predicted transmembrane tetraheme cytochrome c3 complex. Other known oxidative stress response candidates remained unchanged during this low O{sub 2} exposure. To fully understand the results of the 0.1% O{sub 2} exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O{sub 2} exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were down regulated during air exposure. Our results highlight the differences in the cell wide response to low and high O{sub 2} levels of in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O{sub 2} levels in its environment.
Date: March 11, 2007
Creator: Mukhopadhyay, A.; Redding, A.; Joachimiak, M.; Arkin, A.; Borglin, S.; Dehal, P. et al.
Partner: UNT Libraries Government Documents Department

Evaluation of sustained release polylactate electron donors for removal of hexavalent chromium from contaminated groundwater

Description: To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.
Date: February 15, 2011
Creator: Brodie, E.L.; Joyner, D. C.; Faybishenko, B.; Conrad, M. E.; Rios-Velazquez, C.; Mork, B. et al.
Partner: UNT Libraries Government Documents Department

Toward a rigorous network of protein-protein interactions of the model sulfate reducer Desulfovibrio vulgaris Hildenborough

Description: Protein–protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study E. coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio 5 vulgaris Hildenborough, a model anaerobe and sulfate reducer. In this paper we present the first attempt to identify protein-protein interactions in an obligate anaerobic bacterium. We used suicide vector-assisted chromosomal modification of 12 open reading frames encoded by this sulfate reducer to append an eight amino acid affinity tag to the carboxy-terminus of the chosen proteins. Three biological replicates of the 10 ‘pulled-down’ proteins were separated and analyzed using liquid chromatography-mass spectrometry. Replicate agreement ranged between 35% and 69%. An interaction network among 12 bait and 90 prey proteins was reconstructed based on 134 bait-prey interactions computationally identified to be of high confidence. We discuss the biological significance of several unique metabolic features of D. vulgaris revealed by this protein-protein interaction data 15 and protein modifications that were observed. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.
Date: May 1, 2011
Creator: Chhabra, S.R.; Joachimiak, M.P.; Petzold, C.J.; Zane, G.M.; Price, M.N.; Gaucher, S. et al.
Partner: UNT Libraries Government Documents Department

A Conceptual model of coupled biogeochemical and hydrogeologicalprocesses affected by in situ Cr(VI) bioreduction in groundwater atHanford 100H Site

Description: The overall objective of this presentation is to demonstratea conceptual multiscale, multidomain model of coupling of biogeochemicaland hydrogeological processes during bioremediation of Cr(VI)contaminated groundwater at Hanford 100H site. A slow releasepolylactate, Hydrogen Release Compound (HRCTM), was injected in Hanfordsediments to stimulate immobilization of Cr(VI). The HRC injectioninduced a 2-order-of-magnitude increase in biomass and the onset ofreducing biogeochemical conditions [e.g., redox potential decreased from+240 to -130 mV and dissolved oxygen (DO) was completely removed]. Athree-well system, comprised of an injection well and upgradient anddowngradient monitoring wells, was used for conducting the in situbiostimulation, one regional flow (no-pumping) tracer test, and fivepumping tests along with the Br-tracer injection. Field measurements wereconducted using a Br ion-selective electrode and a multiparameter flowcell to collect hourly data on temperature, pH, redox potential,electrical conductivity, and DO. Groundwater sampling was conducted bypumping through specially designed borehole water samplers.Cross-borehole radar tomography and seismic measurements were carried outto assess the site background lithological heterogeneity and themigration pathways of HRC byproducts through groundwater after the HRCinjection.
Date: September 6, 2006
Creator: Faybishenko, B.; Long, P.E.; Hazen, T.C.; Hubbard, S.S.; Williams, K.H.; Peterson, J.E. et al.
Partner: UNT Libraries Government Documents Department

Impact of elevated nitrate on sulfate-reducing bacteria: A comparative study of Desulfovibrio vulgaris

Description: Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70 mM NaNO{sub 3} but not by 70 mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level.
Date: July 15, 2010
Creator: He, Q.; He, Z.; Joyner, D.C.; Joachimiak, M.; Price, M.N.; Yang, Z.K. et al.
Partner: UNT Libraries Government Documents Department

Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) at Hanford 100H

Description: The overall objective of this paper is to carry out field investigations to assess the potential for immobilizing and detoxifying chromium-contaminated groundwater using lactate-stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site.
Date: April 20, 2005
Creator: Hazen, T. C.; Faybishenko, B.; Joyner, D.; Borglin, S.; Brodie, E.; Hubbard, S. et al.
Partner: UNT Libraries Government Documents Department

Deep-sea oil plume enriches psychrophilic oil-degrading bacteria

Description: The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous {gamma}-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5 C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.
Date: September 1, 2010
Creator: Hazen, T.C.; Dubinsky, E.A.; DeSantis, T.Z.; Andersen, G.L.; Piceno, Y.M.; Singh, N. et al.
Partner: UNT Libraries Government Documents Department