Graduate Thesis Or Dissertation
 

Physiological, proteomic, and whole-genome transcriptional responses of Nitrosomonas europaea to heavy metals (Zn²⁺,Cd²⁺, Cu²⁺, and Hg²⁺) and cyanide : identification of heavy metals and cyanide stress response genes

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  • Nitrosomonas europaea (ATCC19718) participates in the biological removal of nitrogen from wastewater by oxidizing ammonia to nitrite, the first step in nitrification. This bacterium is sensitive to heavy metals (Zn²⁺, Cd²⁺, Cu²⁺, and Hg²⁺) and cyanide, compounds commonly encountered in wastewater treatment plants. Therefore, we characterized physiological and transcriptional responses of N. europaea cells exposed to these inhibitors in batch reactors to identify stress-induced genes. N. europaea exposed to 3.4 uM ZnCl₂ showed about 61% inhibition in nitrification within 30 minutes. In microarrays of N. europaea exposed to 3.4 uM ZnCl₂ for 60 minutes, 27 genes were observed to be up regulated more than 2-fold, of the total of 2460 genes, while 30 genes were seen to be down regulated. Up-regulated genes included mercury resistance genes (merTPCAD), inorganic ion transport genes, oxidative stress genes, toxin-antitoxin genes (TA). merTPCAD was the highest up regulated operon (46-fold). Down- regulated genes included RubisCO (cbbO), biosynthesis (mrsA), and amino acid transporters. N. europaea exposed to 1 uM CdCl₂, 6 uM HgCl₂, or 8 uM CuCl₂ showed about 90% inhibition in nitrification within 30 minutes. In microarrays of N. europaea exposed to 1 uM CdCl₂ for 60 minutes, 66 genes were up regulated while 50 genes were down-regulated. merTPCADE showed the highest up regulation (277-fold) under cadmium stress. Interestingly, merA also showed 250-fold up regulation in N. europaea cells exposed to 6 uM HgCl₂. Therefore, merTPCADE appear to be common stress response genes to zinc, cadmium, and mercury exposure. N. europaea cells were very sensitive to low concentrations of cyanide; nitrification decreased about 50% in 30 minutes after exposure to 1 uM NaCN. In microarrays of N. europaea exposed to 1 uM NaCN for 60 minutes, 35 genes showed up regulation while 29 genes showed down regulation. A gene cluster that included moeZ (NE2353), encoding a rhodanese homologue, thought to be involved in detoxification of cyanide, showed the highest up regulation (7-fold). The down-regulated genes included genes encoding proteins involved in the sulfate reduction pathway, signal transduction mechanism, carbohydrate transport, energy production, and coenzyme metabolism.
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