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

Abstract

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.

Authors:
 [1];  [1];  [1];  [1];  [1];  [2]
  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:
931468
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Proteome Research; Journal Volume: 6; Journal Issue: 5
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CHROMATES; DETOXIFICATION; IRON; PROTEINS; QUADRUPOLES; TRANSPORT; TRAPPING; Mass Spectrometry; multi-dimensional liquid chromatography; shotgun proteomics; Shewanella oneidensis; Linear Trapping Quadrupole; chromate stress

Citation Formats

Thompson, Melissa R, Verberkmoes, Nathan C, Chourey, Karuna, Shah, Manesh B, Thompson, Dorothea K, and Hettich, Robert. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge. United States: N. p., 2007. Web. doi:10.1021/pr060502x.
Thompson, Melissa R, Verberkmoes, Nathan C, Chourey, Karuna, Shah, Manesh B, Thompson, Dorothea K, & Hettich, Robert. Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge. United States. doi:10.1021/pr060502x.
Thompson, Melissa R, Verberkmoes, Nathan C, Chourey, Karuna, Shah, Manesh B, Thompson, Dorothea K, and Hettich, Robert. Mon . "Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge". United States. doi:10.1021/pr060502x.
@article{osti_931468,
title = {Dosage-Dependent Proteome Response of Shewanella oneidensis MR-1 to Acute Chromate Challenge},
author = {Thompson, Melissa R and Verberkmoes, Nathan C and Chourey, Karuna and Shah, Manesh B and Thompson, Dorothea K and Hettich, Robert},
abstractNote = {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.},
doi = {10.1021/pr060502x},
journal = {Journal of Proteome Research},
number = 5,
volume = 6,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • 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.
  • Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI)] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426more » regulon.« 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
  • A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, andmore » density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.« less