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Title: Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult

Abstract

Shewanella oneidensis MR-1 is a gram-negative, facultatively anaerobic bacterium originally isolated from a freshwater lake. S. oneidensis MR-1 has the ability to reduce toxic metal ions [e.g., Cr(VI) and U(VI)] found in industrial and governmental waste sites. Cells were grown and exposed to three different metal concentrations in order to probe the dosage response of S. oneidensis MR-1 to Cr(VI) in the form of chromate. Protein fractions were digested with trypsin and analyzed with a multidimensional HPLC-NanoESIMS/MS protocol. The goal of this work is to identify protein components of pathways/mechanisms responsible for both detoxification and reduction of chromate.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN; Purdue University, West Lafayette, IN
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
894627
Report Number(s):
CONF-ERSP2006-44
TRN: US200702%%136
DOE Contract Number:
AC05-00OR22725
Resource Type:
Conference
Resource Relation:
Conference: Annual Environmental Remediation Sciences Program PI Meeting, April 3-5, 2006, Warrenton, VA
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; CHROMATES; DETOXIFICATION; PROBES; PROTEINS; TRYPSIN; WASTES

Citation Formats

Thompson, Melissa R., VerBerkmoes, Nathan C., Chourey, Karuna, Brown, Steven D., Hettich, Robert L., and Thompson, Dorothea K.. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult. United States: N. p., 2006. Web.
Thompson, Melissa R., VerBerkmoes, Nathan C., Chourey, Karuna, Brown, Steven D., Hettich, Robert L., & Thompson, Dorothea K.. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult. United States.
Thompson, Melissa R., VerBerkmoes, Nathan C., Chourey, Karuna, Brown, Steven D., Hettich, Robert L., and Thompson, Dorothea K.. Wed . "Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult". United States. doi:. https://www.osti.gov/servlets/purl/894627.
@article{osti_894627,
title = {Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Chromate Insult},
author = {Thompson, Melissa R. and VerBerkmoes, Nathan C. and Chourey, Karuna and Brown, Steven D. and Hettich, Robert L. and Thompson, Dorothea K.},
abstractNote = {Shewanella oneidensis MR-1 is a gram-negative, facultatively anaerobic bacterium originally isolated from a freshwater lake. S. oneidensis MR-1 has the ability to reduce toxic metal ions [e.g., Cr(VI) and U(VI)] found in industrial and governmental waste sites. Cells were grown and exposed to three different metal concentrations in order to probe the dosage response of S. oneidensis MR-1 to Cr(VI) in the form of chromate. Protein fractions were digested with trypsin and analyzed with a multidimensional HPLC-NanoESIMS/MS protocol. The goal of this work is to identify protein components of pathways/mechanisms responsible for both detoxification and reduction of chromate.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Apr 05 00:00:00 EDT 2006},
month = {Wed Apr 05 00:00:00 EDT 2006}
}

Conference:
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  • Proteome alterations in the metal-reducing bacterium Shewanella oneidensis MR-1 in response to different acute dose challenges (0.3, 0.5, or 1 mM) of the toxic metal chromate [Cr(VI)] were characterized with multidimensional HPLC-MS/MS on a linear trapping quadrupole MS. A total of 2,406 functionally diverse proteins were identified, with a subset demonstrating dosage-dependent up- and down-regulated expression, such as proteins involved in detoxification and iron binding and transport.
  • 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
  • Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was alsomore » observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.« less
  • Predicted orphan response regulators encoded in the Shewanella oneidensis MR-1 genome are poorly understood from a cellular function perspective. Our previous transcriptomic and proteomic analyses demonstrated that an annotated DNA-binding response regulator, SO2426, was significantly up-regulated in wild-type S. oneidensis cells at both themRNAand protein levels in response to acute chromate [Cr(VI)] challenge, suggesting a potential regulatory role for this protein in metal stress pathways. To investigate the impact of SO2426 activity on chromate stress response at a genome-wide scale, we describe here comparative and temporal proteome characterizations using multidimensional HPLC-MS/MS and statistical analysis to identify differentially expressed proteins inmore » biological replicates of wild-type S. oneidensis MR-1 and a so2426 deletion ( so2426) strain, which exhibited an impaired Cr(VI) transformation rate compared to that of the parental strain. Global protein profiles were examined at different time intervals (0, 1, 3, 4 h) following exogenous chromate challenge. Results indicated that deletion of the so2426 gene negatively affected expression of a small protein subset (27 proteins) including those with annotated functions in siderophore biosynthesis (SO3032), Fe uptake (SO4743), intracellular Fe storage (Bfr1), and other transport processes. Cr(VI) exposure and subsequent ransformation dramatically increased the number of differentially expressed proteins detected,with up-regulated bundance patterns observed largely for proteins involved in general stress protection and detoxification trategies, cell motility, and protein fate. In addition, the proteome data sets were mined for amino acids with otential post-translational modifications (PTMs) indicative of a level of gene expression regulation extending eyond the transcriptional control imposed by SO2426.« less