## 4 Discussion  
In this study, we detected decreases in the duration of growth arrest when HipA was reinduced after the initial growth arrest. Shortening of the growth arrest phase at the subsequent round(s) of induction was observed under different conditions. Several factors were also found to affect the length of arrest, such as the growth environment, age of cells used to prepare the culture and the presence or absence of an inducer removal step. Our results showed that the loss of sensitivity to HipA was not caused by the emergence of a HipA-resistant subpopulation. Alteration of the plasmids that abolish the expression of HipA was not detected. This strongly supports the notion that the differences in the length of growth arrest are not due to a genetic change but due to phenotypic differences that reflect their past growth conditions. An earlier single-cell analysis of an HipA-induced population revealed that the HipA expression level was positively correlated with the length of the growth arrest [23]. Our  
Figure 6: Effects of the growth phase of a starter culture on HipA-induced growth arrest. _E. coli_ BW25113 cells carrying _pBAD24_hipA_ were grown on a microplate with constant agitation. Pink and blue points represent data for the cultures with and without a 0.2% arabinose addition, respectively. T = 0 in each figure indicates the time when 0.2% arabinose was added. Arabinose was not removed. Cells were grown from the early stationary phase (8 h post-inoculation) starter culture, and HipA was induced one (**a**), two (**b**) and three (**c**) times. (**d**) Summary of the growth arrest phase in each round of induction. (**e**) Comparison of the growth arrest after the first induction of HipA using the starter culture at the early (8 h) or late (16 h) stationary phase. The * symbol indicates when the cells were removed to initiate the culture for the next round. The value above the graph indicates the \(p\)-value from the \(t\)-test. experiments that compared the effects of the wash step after induction corroborated this observation. The unwashed cells exhibited a longer growth arrest than when the inducers were washed after 15 min of induction (Figure 5). The absence of the wash step would allow for a higher and/or longer expression of HipA, which perhaps, in turn, could increase the length of the growth arrest phase. Regardless of the presence or absence of the inducer removal step, we observed the reduction in the growth arrest phase in the successive rounds of inductions (Figures 4 and 6). This effect lasted longer than one cell cycle. This is reminiscent of other phenomena associated with memory in bacteria (i.e., different phenotypes based on past histories and effects that can last for several generations). [PERSON] et al. reported that the cells in a biofilm contained a higher fraction of persisters than in a liquid culture [31]. The fraction of persisters remained high even after a prolonged period following the transfer from the biofilm to the liquid culture. Various mechanisms for non-genetic memory have been described for single-cellular organisms. Cells in a biofilm exhibit membrane potential-based memory when transiently exposed to light [36]. Potassium ion channels are involved in changing the membrane potential. A faster response to recurring phosphate limitations is mediated by two-component regulatory systems [26]. Signal transduction systems are also implicated in a positive effect of memory in chemotaxis and nitrogen starvation [27; 37]. Our group is currently investigating the molecular mechanisms of how cells have \"memories\" of past HipA-induced growth arrests. Our study also revealed that growth conditions impact the length of HipA-induced growth arrest. When grown in microplates, the duration of the growth arrest was shorter than when grown in a larger volume in flasks (Figure 3). While we used the same medium, initial cell density and incubation temperature, the mode of agitation and the sample's volume were different for the Erlenmeyer flasks and microplates.