Experimental evolution of Pseudomonas stutzeri at low nitrite reactivity (pH 7.5) and at high nitrite reactivity (pH 6.5)

The production of reactive metabolites has shaped the spatial and temporal arrangement of metabolic processes within cells. While diverse solutions to mitigate or control metabolite reactivity have evolved, less is known about how evolution itself is affected by reactivity. We tested this by experimentally evolving the bacterium Pseudomonas stutzeri under denitrifying conditions. The reactive metabolite nitrite accumulates during denitrification and has pH-dependent negative effects on growth. This allowed us to evolve P. stutzeri at different magnitudes of nitrite reactivity (pH 7.5, low nitrite reactivity and pH 6.5, high nitrite reactivity). We used whole genome sequencing of the ancestors and randomly picked evolved clones from each population (8 populations were evolved at each pH and one random clone was picked for sequencing from each population) to identify novel mutations that have fixed during the experimental evolution.