ORIGINAL ARTICLE Zinc toxicity and ATP production in Pseudomonas fluorescens A. Alhasawi, C. Auger, V.P. Appanna, M. Chahma and V.D. Appanna Department Chemistry & Biochemistry, Laurentian University, Sudbury, ON, Canada Keywords bioremediation, enzymology, metabolism. Correspondence Vasu D. Appanna, Department Chemistry & Biochemistry, Laurentian University, 935 Ram- sey Lake Rd, Sudbury, ON P3E 2C6, Canada. E-mail: vappanna@laurentian.ca 2013/2300: received 15 November 2013, revised 28 February 2014 and accepted 8 March 2014 doi:10.1111/jam.12497 Abstract Aims: To identify the molecular networks in Pseudomonas fluorescens that convey resistance to toxic concentrations of Zn, a common pollutant and hazard to biological systems. Methods and Results: Pseudomonas fluorescens strain ATCC 13525 was cultured in growth medium with millimolar concentrations of Zn. Enzymatic activities and metabolite levels were monitored with the aid of in-gel activity assays and high-performance liquid chromatography, respectively. As oxidative phosphorylation was rendered ineffective, the assimilation of citric acid mediated sequentially by citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) appeared to play a key role in ATP synthesis via substrate-level phosphorylation (SLP). Enzymes generating the antioxidant, reduced nicotinamide adenine dinucleotide phosphate (NADPH) were enhanced, while metabolic modules mediating the formation of the pro-oxidant, reduced nicotinamide adenine dinucleotide (NADH) were downregulated. Conclusions: Pseudomonas fluorescens reengineers its metabolic networks to generate ATP via SLP, a stratagem that allows the microbe to compensate for an ineffective electron transport chain provoked by excess Zn. Significance and Impact of the Study: The molecular insights described here are critical in devising strategies to bioremediate Zn-polluted environments. Introduction Zn is an essential micronutrient in most if not all organ- isms as it is involved in a variety of biochemical pro- cesses. It plays a pivotal catalytic, structural and regulatory role (Stefanidou et al. 2006). This metal is an important cofactor of numerous enzymes that are critical in metabolism, transcription and cell signalling (Haase and Rink 2009; Sera 2009; Bong et al. 2010). However, when present in elevated amounts, Zn interferes with numerous biological pathways. This metal can interact strongly with enzymatic imidazole and sulfhydryl groups and arrest their reactions (Duruibe et al. 2007). Further- more, it disturbs the redox potential of the cell and helps to create an oxidative environment. Indeed, oxidative stress is an important feature of Zn toxicity (Lemire et al. 2008). Hence, to survive a Zn-polluted environment, a situa- tion common due to industrialization and anthropogenic activity, organisms have evolved a plethora of intricate strategies to combat this divalent metal (Bondarenko et al. 2008). Its intracellular immobilization in phytochel- atins, metalothioneins and other cysteine-rich moieties has been observed (Blindauer et al. 2002; Di Baccio et al. 2005). Microbes living in Zn-polluted environments are known to precipitate the metal as sulfide- or phosphate- containing moieties and actively efflux Zn (Radhika et al. 2006). The synthesis of antioxidants, such as glutathione and NADPH, is also part of the arsenal at the disposal of bacteria to counter Zn (Lemire et al. 2010a; Poirier et al. 2013). Although some of the molecular pathways that mediate Zn detoxification have been reported, the involvement of metabolic networks in the adaptation to elevated levels of Zn has yet to be fully uncovered. Journal of Applied Microbiology © 2014 The Society for Applied Microbiology 1 Journal of Applied Microbiology ISSN 1364-5072