Abstract
Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction.
Highlights
Microbial bioreduction of Cr(VI) to Cr(III) is a promising strategy for detoxification of chromate, a prevalent anthropogenic pollutant
Flow Cytometry—Fluorescence-activated cell sorter (FACS) cytometry analysis was performed using the H2O2-activated green fluorescent dye H2O2-activated green fluorescent dye dihydrodichlorofluorescein diacetate (H2DCFDA) (Molecular Probes). chrRϩ, wild type, and chrR mutant cells were grown in LB at 37 °C to mid-exponential phase (A660 0.4 – 0.7), and each was split into two cultures and incubated as described above for 1 h
The mechanism by which ChrR reduces quinones is of interest, as simultaneous two-electron transfer from the subunit flavin mononucleotide cofactor might suggest a protective role for the enzyme in guarding against formation of reactive semiquinone intermediates
Summary
Wild type genome sequenced strain Isogenic with wild type Overexpression strain; wild type containing pMMB(chrR). PET28aϩ expressing the KT2440 chrR gene (NdeI/EcoRI); Kmr Broad host range tac expression vector; Cbr pMMB67EH with Bam HI/HindIII His-tagged chrR insert
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