Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising after antibiotic treatment at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through eight sets of experiments), we used microscopy of single cells to determine that the resuscitation of dormant persisters is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found during the observation of persister cells when fresh nutrients were added: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.

Toxin/antitoxin (TA) systems are present in most prokaryote genomes. Toxins are almost exclusively proteins that reduce metabolism (but do not cause cell death), and antitoxins are either RNA or proteins that counteract the toxin or the RNA that encodes it. Although TA systems clearly stabilize mobile genetic elements, after four decades of research, the physiological roles of chromosomal TA systems are less clear. For example, recent reports have challenged the notion of TA systems as stress-response elements, including a role in creating the dormant state known as persistence. Here, we present evidence that a primary physiological role of chromosomally encoded TA systems is phage inhibition, a role that is also played by some plasmid-based TA systems. This includes results that show some CRISPR-Cas system elements are derived from TA systems and that some CRISPR-Cas systems mimic the host growth inhibition invoked by TA systems to inhibit phage propagation.

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Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients.

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Stress is ubiquitous for bacteria and converts a subpopulation of cells into a dormant state known as persistence, in which cells are tolerant to antimicrobials. These cells revive rapidly when the stress is removed and are likely the cause of many recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. However, how persister cells are formed is not understood well. Here we 5 propose the ppGpp ribosome dimerization persister (PRDP) model in which the alarmone guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) generates persister cells directly by inactivating ribosomes via the ribosome modulation factor (RMF) and the hibernation promoting factor (Hpf). We demonstrate both RMF and HpF increase persistence and reduce single-cell persister resuscitation and that ppGpp has no effect on single-cell persister resuscitation. Hence, a direct connection between ppGpp and 10 persistence is shown.