## 5 Ultrastructural, Molecular, and Transcriptomic Changes in _L. monocytogenes_ VBNC Cells
The use of a ratiometric probe that detects local membrane fluidity changes suggested a decrease in membrane fluidity during the first 14 days. Diffusion of the fluorogenic dye Nile red detected by total internal reflection fluorescence correlation spectroscopy (TIR-FCS) showed a reduction in the diffusion coefficient in rod-shaped cells between 7 and 14 days, the period of most severe cell wall damage, while coccoid cells showed a similar diffusion coefficient after 7 and 14 days, indicating an adaptation to a wall-less status [21]. Transcriptome analysis showed the downregulation of genes associated with nucleotide and coenzyme biosynthesis, transcription regulation, uptake of phosphate and some carbohydrates, cell envelope biosynthesis and maintenance, cell division, energy production, and protein secretion at day 7. Upresulated genes included those encoding amino acid metabolism; uptake of glucose, mannose, and metal ions; protein translation and folding; transport of carnitine/glycine betaine compatible solutes, which protect from osmotic stress; and glutathione metabolism, which protects from oxidative stress. Moreover, nearly half of the regulon controlled by the stress-response sigma factor SigB was induced. However, a SigB-deficient _L. monocytogenes_ EGDe mutant became VBNC faster than the wild-type, indicating for SigB a modulation rather than an essential role for survival during starvation. The genes encoding the synthases _relA_, _relP_, and _relQ_ of the alarmone guanosine pentaphosphate ((fp)ppGpp) activator of the stringent response were not upregulated but a _\(\Delta\)relAPQ_ strain switched to the VBNC status faster than the wild-type, indicating that the stringent response plays a role in the early phase of VBNC formation [21]. In another study on VBNC cells generated by SAEW treatment, global proteome analysis with tandem mass tags (TMT) labeling showed that RpsZ, RpmI, Lmo1306, BetL, Lmo2564, RplX, RpmD, RpIT, RplO, RnpA, and Lmo0208 associated with ribosomal functions were significantly upregulated in VBNC cells, while proteins Lmo1608, FloA, TrpD, GadB, and GlyS related to aminoacyl-tRNA biosynthesis were significantly downregulated. Therefore, it was suggested that protein synthesis was initiated but peptide chain elongation was impeded. A down-regulation of LuxS in VBNC _L. monocytogenes_ was observed that might indicate the inactivation of autoinducer-2 (AI-2) mediated quorum sensing (QS) and consequent inhibition of processes such as biofilm formation, production of virulence factors and toxins, motility, and drug resistance [32]. The comparative transcriptome profile showed that upregulated genes in FeSO\({}_{4}\)-induced VBNC cells were significantly more numerous than in the control group. Significantly enhanced processes included stress response and ribosome binding with upregulation of transcription factors _rpoC_, _rpoB_, and _rpoD_; genes associated with the VBNC state formation _lmo0668_, _trxA_, _trxB_, and _lmo0593_; and stress response genes _lisK_, lmo0539, lmo1502, lmo0521, lmo0913, rsbX, spxA, metK, tig, and _htrA_[19]. Table 3 summarizes the upregulated and downregulated gene clusters and proteins in VBNC cells of _L. monocytogenes_ induced in different conditions.