## 2 Results and Discussion
### XRE-COG2856
#### 2.8.2 Hypothesis--Regulation of the _xre-cog2856_ Expression  
As illustrated in Figure 2, when bacterial cells are experiencing stress, (p)ppGpp levels increase, and this in turn will raise the c-di-AMP levels, since they are interconnected as mentioned above. The opposite may also take place, where the c-di-AMP level could rise first and the increase in (p)ppGpp level would follow. The c-di-AMP synthesis from ATP is catalyzed by the diadenylate cyclase (EC:2.7.7.85) [114], while the (p)ppGpp synthesis from ATP and either GTP or GDP is catalyzed by the enzyme RSH (a bifunctional RelA/SpoT homologue) (EC:2.7.6.5) [112]. These two signaling messengers help the cells cope with the stress through synchronizing cellular responses [110]. The synthesized c-di-AMP molecules would be secreted into the extracellular space and would be sensed by the protein product of GK3183 in strain HTA426 and the product of *_35610 in strain Gts via the extracellular receptor domain (7 TMR-HDED) mentioned above. The GK3183/*_35610 protein might then experience conformational changes and could function as a regulatory protein that negatively regulates the expression of the adjacent TA loci. This regulation could take place via one or more quorum sensing molecules that, under stress, could lead to the inhibition of the TA gene expression. Therefore, the synthesis of the TA pair, together with GK3183/*_35610, would stop, and this would result in activating the toxin, since the unstable antitoxin would have been degraded by the Lon protease (EC:3.4.21.53), which is activated by the high level of (p)ppGpp [112]. Consequently, the cell growth would be halted. When the conditions improve, the levels of (p)ppGpp and c-di-AMP would decrease and the expectedly stable GK3183/*_35610 protein would sense that and unblock the expression of the TA-coding genes via one or more quorum sensing molecules. The cell could then resume its growth. Since the mechanism of action of the toxin COG2856 is still unknown, it cannot be confirmed whether the cells will resume their growth when the conditions improve or not. The cells could undergo the PCD after reaching the \"point of no return\" [66]. The genes coding for the enzymes needed for the synthesis of the two signaling messengers and for the Lon protease have been annotated on the genomes of the two strains. The locus tags of the gene coding for the diadenylate cyclase (EC:2.7.7.85) are GK0152 and *_1680, those coding for the RSH enzyme (EC:2.7.6.5) are GK0829 (and GK2578) and *_29020 (and *_9850), and those coding for the Lon protease are GK2650 and *_29780 in strains HTA426 and Gts, respectively. The regulation of the expression of TA-coding genes could take place at the transcription level, the translation level, or both [115]. In the case of COG2856-XRE, we expect the regulation to be at the transcription level since the (p)ppGpp is a transcription regulator [112], as is the c-di-AMP [107]. We have envisioned our hypothesis based on findings of previous studies, as discussed below. The c-di-AMP level is crucial for the cell [104]. In Gram positive bacteria, intracellular levels of c-di-AMP are expected to change in response to environmental or intracellular signals [109; 116]. Neither the causes nor the exact results of changes in c-di-AMP levels and their influence on the whole cell physiology are well understood [110]. After sensing a stimulus raising the c-di-AMP level, the latter transduces the signal by binding to a receptor or a protein and changing its conformation [117; 109; 116]. This in turn will trigger a signal cascade, resulting in the regulation of different cellular processes, including the gene expression [117; 109; 116]. Accordingly, the regulation of the TA gene expression may possibly be one of the c-di-AMP targets. The regulation of type II TA system expression by a receptor protein of a second messenger, cyclic AMP, has been reported along with speculation on the involvement of quorum sensing molecules in the regulation [118].