## 2 Results and Discussion
### Starvation as a Mode of ITC Action against V. cholerae  
It has been proposed that cytosol shrinkage might be one of the results of cell starvation [40]. According to [PERSON] and colleagues, the condensation of cytoplasm is a natural feature of cells experiencing nutrient depletion. However, the prolonged metabolic stress observed in starved cells might lead to membranes dysfunction and loss of their potential [41; 42]. In our previous studies, we showed that ITC treatment resulted in stringent response activation and a drastic decrease in nucleic acids synthesis as a consequence of the accumulation of starvation alarmome, (p)ppGpp [22; 25]. Thus, we compared SFN and PEITC effect on RNA/DNA synthesis in [3H] radiolabeled nucleotide assay. A significant reduction of stable RNA synthesis was observed (Figure 3a,b), which indicates that general downregulation of major metabolic processes occurs rapidly after SFN or PEITC treatment as a result of the global stress response. We also observed the impaired DNA synthesis in treated cultures as assessed by [3H] thymidine incorporation (Figure 3c,d). To determine whether the stringent response alarmome accumulated after the ITC treatment, we measured the levels of the [3P] labeled nucleotides in the treated cells. This examination showed that under treatment with SFN or PETC, the (p)ppGpp alarmome levels increased and were comparable to those observed in _V. cholerae_ cultures experiencing amino acid deprivation, which was induced by the addition of serine hydroxamate (SHX), the toxic analog of L-serine. Namely, the SFN or PEITC treatment for 15 min (at MIC) resulted in the accumulation of ppGpp (\(\pm\)162.5% and \(\pm\)54.5%, respectively, in relation to cells treated with SHX). What is noteworthy is that both ITCs at concentrations lower than their MICs were able to significantly elevate the alarmome synthesis. SFN at 1/2, 1/4, and 1/8 of MIC elevated ppGpp level up to 168.5%, 116.7%, and 76.3%, respectively (Figure 3e). The cells treated with PEITC at the 1/2, 1/4, and 1/8 of MIC showed different dynamics of alarmome accumulation, namely 72%, 159.7%, and 117.6%, respectively (Figure 3f). The amounts of the detected guanosine nucleotides correlated with the inhibition of nucleotides synthesis in PEITC treated cells. At higher doses of PEITC (1/2 or 1 MIC), the GTP/GDP spots visible on chromatograms gave apparently lower signals, which suggests that the process of their synthesis was disturbed (Figure 3f). Figure 2: TEM analysis of morphological changes after ITC treatment. Cells treated with MIC of SFN or PEITC after different treatment times (30, 180 min) at 37 \({}^{\circ}\)C, or none for control experiment. The white arrows indicate shape malformations and cytosol leakage. White bars indicate the scale: 0.5 μm and 2 μm for the upper panel and the bottom panel, respectively. The activation of the stringent response results in modulation of gene expression and dramatically changes metabolic pathways by recruitment of alternative sigma factors [43]. Therefore, the observed drop in DNA synthesis in our study might be connected to the indirect effect of the inhibition of gene expression and alarmome accumulation. (p)ppGpp generally acts as a signal of nutritional deprivation, but also physical, and other environmental changes can induce its synthesis. Its levels increase upon nutrient downshifts and are inversely correlated with growth rate [43; 44]. In _V. cholerae_, (p)ppGpp is synthesized by  
Figure 3: SFN and PEITC impair nucleic acid synthesis and activate stringent response and (p)ppGpp alarmome accumulation in _V. cholerae_. The effect of SFN (**a**) and PEITC (**b**) on RNA synthesis was assessed in [3H]uridine incorporation assay. The effect of SFN (**c**) and PEITC (**d**) on DNA synthesis assessed in [3H]thymidine incorporation assay. The (p)ppGpp alarmome level in cells treated (15 min) with SFN (**e**) and PEITC (**f**).