Global Molecular Characterization of the Chromate Stress Response in Shewanella oneidensis MR-1: Identification of a Putative DNA-Binding Response Regulator and Azoreductase Involved in Cr(VI) Detoxification

Conference · Wed Apr 05 04:00:00 EDT 2006

OSTI ID:894621

Chourey, Karuna; Thompson, Melissa R; Brown, Steven D; VerBerkmoes, Nathan C; Hettich, Robert L; Thompson, Dorothea K

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 chromate 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.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN; University of Tennessee, Knoxville, TN; Purdue University, West Lafayette, IN
• Sponsoring Organization: USDOE - Office of Science (SC)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 894621
• Report Number(s): CONF-ERSP2006-40
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
54 ENVIRONMENTAL SCIENCES
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOREMEDIATION
CHROMATES
CHROMOSOMES
DETOXIFICATION
FUNCTIONAL ANALYSIS
GENES
MASS SPECTROSCOPY
MUTANTS
PHENOTYPE
PROTEINS
REGULATIONS