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Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome …