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Abstract
Chromium and uranium are highly toxic metals that contaminate many natural environments. We investigated their mechanisms of toxicity under anaerobic conditions using nitrate-reducing Pseudomonas stutzeri RCH2, which was originally isolated from a chromium-contaminated aquifer. A random barcode transposon site sequencing library of RCH2 was grown in the presence of the chromate oxyanion (Cr[VI]) or uranyl oxycation (U[VI]). Strains lacking genes required for a functional nitrate reductase had decreased fitness as both metals interacted with heme-containing enzymes required for the later steps in the denitrification pathway after nitrate is reduced to nitrite. Cr[VI]-resistance also required genes in the homologous recombination and nucleotide excision DNA repair pathways, showing that DNA is a target of Cr[VI] even under anaerobic conditions. The reduced thiol pool was also identified as a target of Cr[VI] toxicity and psest_2088, a gene of previously unknown function, was shown to have a role in the reduction of sulfite to sulfide. U[VI] resistance mechanisms involved exopolysaccharide synthesis and the universal stress protein UspA. As the first genome-wide fitness analysis of Cr[VI] and U[VI] toxicity under anaerobic conditions, this study provides new insight into the impact of Cr[VI] and U[VI] on an environmental isolate from a chromium contaminated site, as well as into the role of a ubiquitous protein, Psest_2088.
Highlights
The industrial use of chromium for metallic plating, industrial catalysts, and pesticides has led to wide scale environmental contamination (Ayres, 1992)
We propose a similar model for the effect of U[VI] on nitrate reduction gene fitness values as we did for Cr[VI], in that U[VI] likely interacts with cytochromes required for the steps in the denitrification pathway after nitrate is reduced to nitrite (Figure 1)
Many new insights were gained through the course of this genome-wide fitness analysis on the targets of Cr[VI] and U[VI] toxicity in RCH2 grown under denitrifying conditions, as well as the defense mechanisms RCH2 uses to defend itself against these metals
Summary
The industrial use of chromium for metallic plating, industrial catalysts, and pesticides has led to wide scale environmental contamination (Ayres, 1992). Chromium exists in several different oxidation states from −4 to +6 with the oxyanion hexavalent state (Cr[VI]) and the oxyanion trivalent state (Cr[III]) being the most stable (Cervantes et al, 2001; Cheung and Gu, 2007) Of these two oxidation states, Cr[VI] is over 1,000-fold more toxic as it is highly soluble and can be transported across membranes via sulfate transport channels (Ohtake and Silver, 1994; Cervantes et al, 2001; Costa, 2003). Cr[VI] is reduced to Cr[V] and Cr[III] by compounds such as glutathione and ascorbic acid, a process that generates reactive oxygen species (ROS) (Arslan et al, 1987; Costa, 2003; Xu et al, 2004). While there have been many studies on the toxic effects of Cr[VI] and U[VI] using microorganisms grown under aerobic conditions, this is the first in depth look at Cr[VI] and U[VI] toxicity in an anaerobic denitrifying system on a genome wide scale
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