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ABSTRACT Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress (“primed cells”) resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation. IMPORTANCEAntibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information (“memory”) to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics. 

Abstract Antibiotic treatment kills a large portion of a population, while a small, tolerant subpopulation survives. Tolerant bacteria disrupt antibiotic efficacy and increase the likelihood that a population gains antibiotic resistance, a growing health concern. We examined howE. colitranscriptional networks changed in response to lethal ampicillin concentrations. We are the first to apply transcriptional regulatory network (TRN) analysis to antibiotic tolerance by leveraging existing knowledge and our transcriptional data. TRN analysis shows that gene expression changes specific to ampicillin treatment are likely caused by specific sigma and transcription factors typically regulated by proteolysis. These results demonstrate that to survive lethal concentration of ampicillin specific regulatory proteins change activity and cause a coordinated transcriptional response that leverages multiple gene systems.