Basal level of ppGpp coordinates Escherichia coli cell heterogeneity and ampicillin resistance and persistence

The universal stringent response alarmone ppGpp (guanosine penta and tetra phosphates) plays a crucial role in various aspects of fundamental cell physiology (e.g., cell growth rate, cell size) and thus bacterial tolerance to and survival of external stresses, including antibiotics. Besides transient antibiotic tolerance (persistence), ppGpp was recently found to contribute to E. coli resistance to ampicillin. How ppGpp regulates both the persistence and resistance to antibiotics remains incompletely understood. In this study, we first clarified that the absence of ppGpp in E. coli (ppGpp⁰ strain) resulted in a decreased minimal inhibition concentration (MIC) value of ampicillin but, surprisingly, a higher persistence level to ampicillin during exponential growth in MOPS rich medium. High basal ppGpp levels, thus lower growth rate, did not produce high ampicillin persistence. Importantly, we found that the high ampicillin persistence of the ppGpp⁰ strain is not due to dormant overnight carry-over cells. Instead, the absence of ppGpp produced higher cell heterogeneity, propagating during the regrowth and the killing phases, leading to higher ampicillin persistence. Consistently, we isolated a suppressor mutation of the ppGpp⁰ strain that restored the standard MIC value of ampicillin and reduced its cell heterogeneity and the ampicillin persistence level concomitantly. Altogether, we discussed the fundamental role of basal level of ppGpp in regulating cell homogeneity and ampicillin persistence.