Nitric oxide (NO) is an important antimicrobial produced by the immune system to impair pathogens. Many pathogens harbor NO detoxification and repair systems that have been linked to their virulence, and thus represent attractive targets toward which novel anti-infective therapies could be identified. Unfortunately, the compounds that have been found to inhibit these systems suffer from poor transport or toxicity, warranting a search for other targets. The broad reactivity of NO and its reaction products give rise to an expansive, complex reaction network, necessitating the use of computational models to effectively study the system in its entirety. To facilitate quantitative exploration of the E. coli NO stress response, we constructed a kinetic model of the intracellular NO biochemical network, including relevant processes such as enzymatic NO detoxification, iron-sulfur cluster nitrosylation and repair, DNA deamination and repair, transcriptional regulation, and NO autoxidation. Using this model, we investigated NO stress under low O2 conditions due to their relevance to pathogenesis. The model predicted a deficiency in the overall rate of NO detoxification for O2 environments between anaerobic and the upper-end of microaerobic (50 μM O2). Experimental measurements of NO detoxification at these intermediate O2 concentrations confirmed the predicted trend; however, the extent to which NO detoxification was impaired was underestimated, and oscillations appeared in the concentration of NO at 5 and 10 μM O2. Using a model-guided approach, we determined that the NO hypersensitivity resulted from decreased substrate (O2) abundance for Hmp, strong O2-mediated suppression of NorV activity, and a general reduction in translation rate at reduced [O2]. When we dissected the oscillatory behavior, we found that it resulted from a kinetic competition for O2 between Hmp and the respiratory cytochromes. In support of this mechanism, we experimentally confirmed several model predictions, which were that the concentration of O2 would oscillate out-of-phase with NO, and that genetic deletion of either Hmp or the cytochrome oxidoreductases would eliminate oscillations. This work demonstrates the utility of modeling approaches for the quantitative investigation of bacterial NO defense networks, and identifies oscillatory behavior that could be widespread due to the coexistence of Hmp and O2-consuming cytochrome oxidoreductases in &gt;700 different species of bacteria.

Discovery and deconstruction of oscillations in the nitric oxide stress network of Escherichia coli

251st National Meeting (2016)

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