# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 6 Proteomics, Transcriptomics, and Metabolomics of Bacterial Cells in VBNC State  
When bacteria enter the VBNC state, various cellular changes may occur. Studying these changes at the post-transcriptional and post-translational levels could elucidate which genes are important for bacteria to perform this phenomenon; however, knowledge is still limited under the VBNC state. It has been observed that under various types of stress inducing the VBNC state, different genes may be expressed--for instance, more alkaline phosphatase and \(\alpha\)-ketoglutate oxidoreductasetase [120]; an increase in OmpWexternal membrane proteins [121]; the high expression of glutathione S-transferase [122]; the overexpression of several proteins related to transcription, translation, ATP synthesis, and gluconeogenesis; and antioxidants [123]. The expression of the RpoS sigma factor in the VBNC state has been demonstrated in several bacterial species, with guanosine 3\({}^{\prime}\), 5\({}^{\prime}\)-bispirophosphate (ppGpp) acting as a positive regulator during the synthesis and function of this sigma factor [124]. Interestingly, it has been observed that RpoS mutant strains quickly lose cultivability and cannot return to the culturable state with existing resuscitation methods [124; 125]. Considering that the absence of this protein can mean imminent cell death, it could be one of the key genes involved in the entrance and exit from the VBNC state in bacteria. The activity in the ribosomes is one of the most important processes in all living cells. A low expression level for genes encoding ribosome-associated inhibitor A (RaiA) proteins has been observed in the VBNC state for several bacterial species [125]. This could mean that bacteria decrease their translational activity during the latency status, thereby saving energy in order to face stress conditions. It is likely that the entrance into this state begins with metabolic pathways that control stimulus response mechanisms (two-component systems) and bacterial movement (chemotaxis), determined by the presence of chemicals in the environment where the cells are located [119]. Furthermore, the VBNC state in some bacteria could also be regulated by the proteins leucine-responsive regulatory protein/asparagine synthase C products (Lrp/AsnC) and MarR [119]. The MarR protein controls genes involved in the degradation of toxic compounds (including phenols), virulence, the export of harmful chemicals, and resistance to oxidative stress [126]. Within the scant literature on beneficial bacteria, there are studies on _P. putida_ KT2440 and _C. metallidurans_. Transcriptomic studies of _P. putida_ KT2440 in the VBNC state caused by desiccation stress [127] showed that six genes related to transmembrane transport and oxidation-reduction processes were upregulated. The ethylene glycol porin (PP_2662) and substrate-binding protein (PP_2676) genes could transport and degrade polyhydric alcohols, which are accumulated during desiccation stress as compatible solutes. The upregulation of the TonB-dependent receptor (PP_1446) gene could be a strategy used by _P. putida_ KT2440 to provide the ferrous iron required for vital functions during the VBNC state. _P. putida_ KT2440 cells return to a cultivable state upon 24 h of rehydration; after this return, 148 genes related to transport, oxidation-reduction, the regulation of transcription, and biosynthetic processes were upregulated, while 42 genes related to translation, oxidation-reduction, and the regulation of transcription were downregulated. During the prolonged rehydration of _P. putida_ KT2440 cells, the catabolism of phenylalanine/tyrosine is activated, possibly to provide energy and a carbon source for ubiquinone biosynthesis, while maintaining reduced protein synthesis [127]. Studies in _C.