Characterization of acetic acid-detoxifying Escherichia coli evolved under phosphate starvation conditions

Patrice L. Moreau, Laurent Loiseau

During prolonged incubation of Escherichia coli K-12 in batch culture under aerobic, phosphate (Pi) starvation conditions, excess glucose is converted into acetic acid, which may trigger cell death. Following serial cultures, we isolated five evolved strains in two populations that survived prolonged incubation. We sequenced the genomes of the ancestral and evolved strains, and determined the effects of the genetic changes, tested alone and in combination, on characteristic phenotypes in pure and in mixed cultures. Evolved strains used two main strategies: (1) the constitutive expression of the Trk- and Kdp-dependent K(+) transport systems, and (2) the inactivation of the ArcA global regulator. Both processes helped to maintain a residual activity of the tricarboxylic acid cycle, which decreased the production of acetic acid and eventually allowed its re-consumption. Evolved strains acquired a few additional genetic changes besides the trkH, kdpD and arcA mutations, which might increase the scavenging of organophosphates (phnE (+), lapB, and rseP) and the resistance to oxidative (rsxC) and acetic acid stresses (e14(-)/icd (+)). Evolved strains rapidly acquired mutations (phnE (+) lapB rpoS trkH and phnE (+) rseP kdpD) that were globally beneficial to growth on glucose and organophosphates, but detrimental to long-term viability. The spread of these mutant strains might give the ancestral strain time to accumulate up to five genetic changes (phnE (+) arcA rsxC crfC e14(-)/icd (+)), which allowed growth on glucose and organophosphates, and provided a long-term survival. The latter strain, which expressed several mechanisms of protection against endogenous and exogenous stresses, might provide a platform for producing toxic recombinant proteins and chemicals during prolonged incubation under aerobic, Pi starvation conditions.