The consequences to human health of metal pollution (i.e., toxicity) is dependent on metal bioavailability, which in turn is dependent on the species and form of the metal. Soluble metal species, particularly free metal ions, are generally more bioavailable and toxic to cells whereas the insoluble metal forms are less toxic. Bacteria may interact with metals in a variety of ways that lead to reduced metal bioavailability and toxicity. This project will examine the mechanisms of oxidation of manganese(II) and reduction of chromium(VI) by bacteria.processes that lead to the precipitation or removal of these and other metals from solution. The long-range goals of this research are, 1) to identify the genes and proteins involved and to characterize the mechanisms of these processes, including those that are turned on by exposure to toxic metals, 2) to develop biomarkers of metal bioavailability, and 3) manipulate these systems for metal bioremediation applications. Molecular biological and biochemical approaches will be employed to identify and characterize the genes and proteins involved in Mn(II) oxidation and Cr(VI) reduction/Cr toxicity. The genes encoding Mn(II)-oxidizing proteins will be cloned, sequenced and analyzed for key amino acid residues. One organism/protein will be used as a model for large-scale native or heterologous expression and purification and detailed characterization (enzyme kinetics, localization, cofactors and sites of glycosylation and metal-binding) largely using mass spectrometry. The effect of Mn, Cr, Pb, and/or Cu on the Mn(II)-oxidizing activity of cells will be examined at the physiological and molecular (e.g., microarrays or protein profiling) level in order to develop a model of environmental controls on Mn(II) oxidation. Chromium research will focus on genes regulated in response to Cr and the uptake and toxicity of different forms of Cr. Shewanella oneidensis MR-1 mutants deficient in genes up-regulated in response to Cr(VI) will be constructed and characterized. The specificity of gene expression to Cr(VI) relative to other metals (e.g., Pb, As, Se) will be tested. Real-time RT-PCR will be used to assess the expression of the Cr(VI) specific genes in laboratory mesocosms (or a field site) and correlate the expression patterns to Cr concentration and speciation. Mutants deficient in sulfate uptake will be tested for their ability to withstand exposure to Cr(VI) physiologically and using TEM. The effect of Cr(III) on gene expression and the complexation of Cr(III) by bacterial siderophores will also be investigated.
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