Q-300. Impact of Growth Phase and Copper Toxicity on Elemental Composition of Nitrosomonas europaea

R. Yu1, B. Lai2, S. Vogt2, I. Noyan1, K. Chandran1;
1Columbia Univ., New York, NY, 2Advanced Photon Source, Argonne Natl. Lab., Argonne, IL.

Background: The guiding hypothesis of this study was that the elemental make-up of a cell can be significantly influenced by its physiology and exposure to stress. The elemental composition of Nitrosomonas europaea (an autotrophic ammonia oxidizing bacterium) was studied via synchrotron microbeam X-ray fluorescence (SMXRF) during batch growth and copper (Cu) intoxication. SMXRF is a novel and sensitive technique for determining a cell’s elemental composition, concentration and spatial distribution. Copper was chosen since it is a widespread environmental pollutant and a cofactor in several essential enzymes in N. europaea. Methods: Total cell concentration, activity, gene expression and elemental composition of N. europaea cultures were measured during lag, exponential and stationary phases. Exponential cultures were exposed to 0.1, 0.5 and 1 mM copper sulfate for 4 h. Results: Cu limitation (inferred from progressively lower intracellular Cu levels) was observed as the cells transitioned from lag phase to exponential and stationary phases and is in line with the supposed high cellular Cu requirement of N. europaea. Cu exposure resulted in significant, saturation-type intracellular Cu accumulation accompanied by reduced total cell abundance, membrane integrity, specific oxygen uptake rate, (sOUR) and ammonia monooxygenase subunit A (amoA) expression. Notably, the toxicity coefficients for sOUR and amoA expression were in close correspondence. Cu toxicity resulted in significant efflux of potassium (K+) indicating that the glutathione gated potassium efflux (GGKE) stress response was involved. Cellular concentrations of several other elements increased unexpectedly at the 0.5mM Cu dose before decreasing uniformly at a dose of 1mM Cu, due to eventual membrane damage (confirmed experimentally). Conclusions: SMXRF afforded unique insights into the elemental composition of N. europaea during batch growth and Cu intoxication. Cu-limitation occurred during batch growth of N. europaea and Cu-induced toxic responses included selective K+ efflux, transient accumulation of several metals and ultimately, membrane damage.