## 2 Results and Discussion
### XRE-COG2856
#### 2.8.2 Hypothesis--Regulation of the _xre-cog2856_ Expression
The possibility of regulating the TA expression via quorum sensing has also been reported in other studies [5; 119]. Expanding on this, we could assume that the regulation of _xre-cog2856_ expression by the second messenger, c-di-AMP, via quorum sensing is possible. The high level of c-di-AMP molecules activates the (p)ppGpp-synthesizing enzyme via an unknown mechanism, causing an increase in the level of (p)ppGpp [108; 110]. On the other hand, independently of c-di-AMP, the (p)ppGpp level also increases due to environmental stresses [112]. The increase in the level of (p)ppGpp affects different cellular processes in order to control the growth rate, including the inhibition of the ribosomal RNA (rRNA) synthesis, regulation of nucleotide synthesis,and differential gene transcription to help bacteria adapt to stress [112]. The high level of (p)ppGpp strongly inhibits GTP synthesis, which in turn inhibits protein biosynthesis [112; 120; 121]. Moreover, the high level of (p)ppGpp could inhibit the expression of TA promoters, leading to toxin activation as a measure to help the bacterial population survive the stress, such as the case reported for the MazEF promoter in _E. coli_[111; 12]. Furthermore, high levels of (p)ppGpp prevent the degradation of c-di-AMP, leading to the accumulation of the latter, which thus becomes toxic to the cell, as was shown for _B. subtilis_[104; 122] (for details on the effects of (p)ppGpp on cellular processes and gene transcription in bacteria, the reader is referred to [112; 123; 124]). Conclusively, regardless of which one of the two signaling messengers increases first, the level of the other one will increase as well. Consequently, the cells will be left under high levels of both messengers, and their elevated levels constitute a stress signal that the cells respond to in the ways mentioned above. In addition to the potential role of the high levels of these two messengers in regulating the expression of the TA promoter, they could act as mediators that enhance the toxin-induced effects in order to create a rapid response, due to their actions mentioned above. However, their role in enhancing the growth inhibition will be limited. Since (p)ppGpp is synthesized from GTP/GDP and since the high level of (p)ppGpp inhibits salvaging and de novo synthesis of these nucleotides [120; 121; 125; 126], the synthesis of further (p)ppGpp will cease. The same applies to c-di-AMP since metabolically inactive cells have a reduced proton motive force (PMF) (i.e., reduced ATP level) [24; 127]. The cessation of further formation of the two messengers could protect the cell from \"early\" death, with the hope that the conditions may improve. When the stress is released, (p)ppGpp and c-di-AMP levels will be reduced to basal levels; therefore, the TA promoter will be unblocked. If the toxin has had only a bacteriostatic effect, or if the cells have not reached the \"point of no return\" mentioned above, the cells will recover and resume their growth. The regulation of a TA pair by a third protein encoded by a gene downstream of the TA-coding genes and potentially sharing the same operon has been reported previously for the MazEF pair in _E. coli_[111]. The principle of delaying cell death has also been reported in the same study [111]. Additionally, the existence of an interconnection between a type II toxin and second messengers, the (p)ppGpp and the c-di-AMP has been reported previously in [24]. [PERSON] et al. have reported an interrelationship between a type II toxin (\(\xi\)), the (p)ppGpp, the c-di-AMP, and other nucleotides [24]. However, the effects of these two messengers on inducing the TA expression were not studied since the expression was controlled by external inducers [24]. Taken together, the conclusions of the mentioned studies support the basis of our hypothesis. In summary, we identified an uncommon pattern of unfused genes coding for the XRE-COG2856 TA pair in _G. kaustophilus_ HTA426 and _G. thermolevorans_ *_ 35610.