Prior observations of chromium (Cr[VI]) and uranium (U[VI]) toxicity under aerobic conditions, where metal toxicity was caused not only directly by the metal, but also indirectly due to redox reactions of the metal with oxygen and the resulting reactive oxygen species. ENIGMA researchers from University of Georgia, University of Missouri, and Lawrence Berkeley Lab report the results of random barcode transposon site sequencing experiments performed on the chromium-contaminated environmental isolate, Pseudomonas stutzeri RCH2, grown under anaerobic denitrifying conditions. The mechanisms by which uranium and chromium cause toxicity under anaerobic denitrifying conditions and the defense mechanisms Pseudomonas stutzeri RCH2 uses to defend against these metals were examined using RB-TnSeq technology combined with physiology, biochemistry, and genetics. Novel insights include characterization of a gene of previously unknown function (Psest_2088), involved in sulfite reduction and a key to Cr resistance.
This comprehensive analysis of (U[VI]) and (Cr[VI]) toxicity under anaerobic denitrifying conditions was possible only by leveraging all of the resources available in ENIGMA; which allowed Michael Thorgersen et al to elucidate on a genome wide scale the anaerobic toxicity targets of Cr[VI] and U[VI] and the mechanisms used by RCH2 to defend against these metals. For Cr[VI], DNA is a toxicity target even under anaerobic conditions. Cr[VI]-dependent fitness defects were seen under anaerobic conditions for strains lacking proteins involved in homologous recombination and nucleotide excision DNA repair. Fitness data together with physiological growth studies on wild-type RCH2 and the Δ2088 mutant strain were used to develop a model in which the reduced thiol pool is an additional target of Cr[VI] toxicity. In this model, Psest_2088, a protein of previously unknown function, is a key protein involved in sulfur assimilation at the step of sulfite reduction. Both Cr[VI] and U[VI] toxicity have large fitness effects on RCH2 strains with defects in nitrate reduction. We propose that both metals interfere with cytochrome components of the remainder of the denitrification pathway, which is critical to respiration and survival when nitrate reduction is hindered. This could hinder the remediation of sites contaminated with both nitrate and heavy metals such as Cr[VI] and U[VI]. Finally, exopolysaccharide biosynthesis and the universal stress protein UspA were identified as possible defenses mechanisms against U[VI] toxicity. Cr[VI] and U[VI] damage living organisms in diverse ways, and RB-TnSeq technology is a powerful tool that can be used to study these processes, and identify the metabolic pathways involved.
RCH2 data can be found in the Fitness Browser:http://fit.genomics.lbl.gov
Thorgersen MP, Lancaster WA, Ge X, Zane GM, Wetmore KM, Vaccaro BJ, Poole FL 2nd, Younkin AD, Deutschbauer AM, Arkin AP, Wall JD, Adams MWW. (2017) Mechanisms of Chromium and Uranium Toxicity in Pseudomonas stutzeri RCH2 Grown under Anaerobic Nitrate-Reducing Conditions. Front Microbiol. 8:1529 10.3389/fmicb.2017.01529