A Kinetic Platform to Determine the Fate of Nitric Oxide in Escherichia coli

Nitric oxide (NO center dot) is generated by the innate immune response to neutralize pathogens. NO center dot and its autoxidation products have an extensive biochemical reaction network that includes reactions with iron-sulfur clusters, DNA, and thiols. The fate of NO center dot inside a pathogen depends on a kinetic competition among its many targets, and is of critical importance to infection outcomes. Due to the complexity of the NO center dot biochemical network, where many intermediates are short-lived and at extremely low concentrations, several species can be measured, but stable products are non-unique, and damaged biomolecules are continually repaired or regenerated, kinetic models are required to understand and predict the outcome of NO center dot treatment. Here, we have constructed a comprehensive kinetic model that encompasses the broad reactivity of NO center dot in Escherichia coli. The incorporation of spontaneous and enzymatic reactions, as well as damage and repair of biomolecules, allowed for a detailed analysis of how NO center dot distributes in E. coli cultures. The model was informed with experimental measurements of NO center dot dynamics, and used to identify control parameters of the NON distribution. Simulations predicted that NON dioxygenase (Hmp) functions as a dominant NO center dot consumption pathway at O-2 concentrations as low as 35 mM (microaerobic), and interestingly, loses utility as the NO center dot delivery rate increases. We confirmed these predictions experimentally by measuring NO center dot dynamics in wild-type and mutant cultures at different NO center dot delivery rates and O-2 concentrations. These data suggest that the kinetics of NO center dot metabolism must be considered when assessing the importance of cellular components to NON tolerance, and that models such as the one described here are necessary to rigorously investigate NO center dot stress in microbes. This model provides a platform to identify novel strategies to potentiate the effects of NO center dot, and will serve as a template from which analogous models can be generated for other organisms.