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Title: Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1

Abstract

The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on ShewanellaoneidensisMR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic charac terization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (pepti doglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressedmore » under conditions of 24-h chromate treatment.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [2];  [1]
  1. ORNL
  2. {Bob} L [ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
930903
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 72; Journal Issue: 9
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BIOSYNTHESIS; CELL DIVISION; CHROMATES; DNA; GENES; INDUCTION; MEMBRANES; METABOLISM; MORPHOLOGY; PROTEINS

Citation Formats

Chourey, Karuna, Thompson, Melissa R, Morrell-Falvey, Jennifer L, Verberkmoes, Nathan C, Brown, Steven D, Shah, Manesh B, Zhou, Jizhong, Doktycz, Mitchel John, Hettich, Robert, and Thompson, Dorothea K. Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1. United States: N. p., 2006. Web. doi:10.1128/AEM.00813-06.
Chourey, Karuna, Thompson, Melissa R, Morrell-Falvey, Jennifer L, Verberkmoes, Nathan C, Brown, Steven D, Shah, Manesh B, Zhou, Jizhong, Doktycz, Mitchel John, Hettich, Robert, & Thompson, Dorothea K. Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1. United States. doi:10.1128/AEM.00813-06.
Chourey, Karuna, Thompson, Melissa R, Morrell-Falvey, Jennifer L, Verberkmoes, Nathan C, Brown, Steven D, Shah, Manesh B, Zhou, Jizhong, Doktycz, Mitchel John, Hettich, Robert, and Thompson, Dorothea K. Sun . "Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1". United States. doi:10.1128/AEM.00813-06.
@article{osti_930903,
title = {Global Molecular and Morphological Effects of 24-Hour Chromium(VI)Exposure on Shewanella oneidensis MR-1},
author = {Chourey, Karuna and Thompson, Melissa R and Morrell-Falvey, Jennifer L and Verberkmoes, Nathan C and Brown, Steven D and Shah, Manesh B and Zhou, Jizhong and Doktycz, Mitchel John and Hettich, Robert and Thompson, Dorothea K},
abstractNote = {The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on ShewanellaoneidensisMR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic charac terization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (pepti doglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.},
doi = {10.1128/AEM.00813-06},
journal = {Applied and Environmental Microbiology},
number = 9,
volume = 72,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • The growth and Cr(VI) reduction by Shewanella oneidensisMR-1 was examined using a mini-bioreactor system that independentlymonitors and controls pH, dissolved oxygen, and temperature for each ofits 24, 10-mL reactors. Independent monitoring and control of eachreactor in the cassette allows the exploration of a matrix ofenvironmental conditions known to influence S. oneidensis chromiumreduction. S. oneidensis MR-1 grew in minimal medium without amino acidor vitamin supplementation under aerobic conditions but required serineand glycine supplementation under anaerobic conditions. Growth wasinhibited by dissolved oxygen concentrations>80 percent. Lactatetransformation to acetate was enhanced by low concentration of dissolvedoxygen during the logarithmic growth phase. Between 11 andmore » 35oC, thegrowth rate obeyed the Arrhenius reaction rate-temperature relationship,with a maximum growth rate occurring at 35oC. S. oneidensis MR-1 was ableto grow over a wide range of pH (6-9). At neutral pH and temperaturesranging from 30-35oC, S. oneidensis MR-1 reduced 100 mu M Cr(VI) toCr(III) within 20 minutes in the exponential growth phase, and the growthrate was not affected by the addition of chromate; it reduced chromateeven faster at temperatures between 35 and 39oC. At low temperatures(<25oC), acidic (pH<6.5), or alkaline (pH>8.5) conditions, 100mu M Cr(VI) strongly inhibited growth and chromate reduction. Themini-bioreactor system enabled the rapid determination of theseparameters reproducibly and easily by performing very few experiments.Besides its use for examining parameters of interest to environmentalremediation, the device will also allow one to quickly assess parametersfor optimal production of recombinant proteins or secondarymetabolites« less
  • The mediation of metal reduction by microorganisms has been investigated intensively from physiological and biochemical perspectives; however, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform or immobilize a wide array of heavy metals contaminating DOE field sites. Chromium(VI), for example, is one of several risk-driving contaminants at DOE sites and has been targeted by the DOE for bioremediation research. The bacterium Shewanella oneidensis MR-1 can potentially be used to immobilize chromium, a toxic and mutagenic metal, by reducing soluble Cr(VI) to the insoluble and less bioavailable form of Cr(III), thusmore » facilitating its removal from contained-storage and natural sites. The overall goal of this study is to integrate targeted biochemical and proteomic analyses with genome-wide gene expression profiling to examine the molecular basis and regulation of chromium(VI) reduction by Shewanella oneidensis MR-1. Towards this goal, we will (1) isolate and identify the terminal chromium(VI) reductase and the gene(s) encoding this activity using whole-genome sequence information for MR-1 and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with conventional protein purification and characterization techniques; (2) verify the function of the gene(s) encoding the terminal Cr(VI) reductase and compare whole transcriptome data with whole proteome data in order to understand the regulation of chromium reduction; and (3) investigate the molecular stress response and adaptation of S. oneidensis to toxic levels of soluble Cr(VI) and other heavy metals. This research will provide important information on the functional components and regulatory mechanisms of microbial metal reduction, which should prove valuable in developing effective assessment strategies for in situ bioremediation and genetically engineering desired bacteria for enhanced bioremediation.« less
  • Although microbial metal reduction has been investigated intensively from physiological and biochemical perspectives, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform, detoxify, or immobilize a wide array of heavy metals contaminating DOE-relevant environments. The major goal of this work is to elucidate the molecular components comprising the chromium(VI) response pathway, with an emphasis on components involved in Cr(VI) detoxification and the enzyme complex catalyzing the terminal step in Cr(VI) reduction by Shewanella oneidensis MR-1. We have identified and characterized (in the case of DNA-binding response regulator [SO2426] and a putativemore » azoreductase [SO3585]) the genes and gene products involved in the molecular response of MR-1 to chromium(VI) stress using whole-genome sequence information for MR-1 and recently developed proteomic technology, in particular liquid chromatographymass spectrometry (LC-MS), in conjunction with conventional protein purification and characterization techniques. The proteome datasets were integrated with information from whole-genome expression arrays for S. oneidensis MR-1 (as illustrated in Figure 1). The genes and their encoded products identified in this study are of value in understanding metal reduction and bacterial resistance to metal toxicity and in developing effective metal immobilization strategies.« less
  • Shewanella oneidensis MR-1 is a model environmental organism that possesses diverse respiratory capacities, including the ability to reduce soluble Cr(VI) to sparingly soluble, less toxic Cr(III). Chromate is a serious anthropogenic pollutant found in subsurface sediment and groundwater environments due to its widespread use in defense and industrial applications. Effective bioremediation of chromate-contaminated sites requires knowledge of the molecular mechanisms and regulation of heavy metal resistance and biotransformation by dissimilatory metal-reducing bacteria. Towards this goal, our ERSP-funded work was focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Our work utilizedmore » temporal transcriptomic profiling and whole-cell proteomic analyses to characterize the dynamic molecular response of MR-1 to an acute chromate shock (up to 90 min) as well as to a 24-h, low-dose exposure. In addition, we have examined the transcriptome of MR-1 cells actively engaged in chromate reduction. These studies implicated the involvement of a functionally undefined DNA-binding response regulator (SO2426) and a putative azoreductase (SO3585) in the chromate stress response of MR-1.« less
  • Shewanella oneidensis MR-1 is a model environmental organism that possesses diverse respiratory capacities, including the ability to reduce soluble Cr(VI) to sparingly soluble, less toxic Cr(III). Effective bioremediation of Cr-contaminated sites requires knowledge of the molecular mechanisms and regulation of heavy metal resistance and biotransformation by dissimilatory metal-reducing bacteria. Towards this goal, our ERSP-funded work is focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Previous transcriptomic profiling and whole-cell proteomic analyses implicated the involvement of a functionally undefined DNA-binding response regulator (SO2426) and a putative azoreductase (SO3585) in the chromatemore » stress response of MR-1. Here we describe a detailed functional analysis of SO2426 and SO3585 in order to begin to understand the role of these proteins in the cellular response to chromate. The protein products encoded by genes so2426 and so3585 were expressed and detected only in chromate-shocked samples as determined by multidimensional high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Both genes were also highly induced (>46-fold) in MR-1 cells actively reducing chromate based on whole-genome microarray analysis. We have created in-frame deletions of the so2426 and so3585 loci in the MR-1 chromosome and have characterized the phenotype of the resulting mutants in the presence of varying concentrations of Cr, Cu, Co, Sr, and H{sub 2}O{sub 2} under aerobic respiratory conditions. Growth studies indicated that the so2426 deletion mutant was more sensitive to heavy metals compared to the WT reference, and chromate reduction by the so2426 mutant was impaired significantly. The growth response of the mutant to H{sub 2}O{sub 2} was similar to that of MR-1. To gain insight into the regulon of this response regulator, MR-1 microarrays were used to explore dynamic changes in the WT and so2426 mutant transcriptomes during chromate stress and reduction. The so3585 deletion mutant resembled the WT in terms of growth; however, this mutant was able to reduce chromate at a substantially faster rate compared to the WT strain. Based on its genomic proximity and coregulated expression profile, we predict that SO3585 functions in a complex together with the proteins SO3586 (glyoxalase family) and membrane-associated SO3587 (hypothetical protein). Future studies will include purifying SO3585 to determine whether it can reduce Cr(VI) and whether it interacts with SO3586 and SO3587.« less