## 4 Conclusions  
In this study, we conducted an in-silico genome analysis for the identification of bacterial TAs in 4 _Geobacillus_ strains; _G. kaustophilus_ HT426, _Geobacillus_ sp. ZGt-1, _G. thermodenitrificans_ NG80-2, and _G. thermolevorans_ Gts. The analysis was carried out by employing the TA finder software and the resulting sequences were manually curated using the NCBI CDD and InterPro domain analysis tools, as well as inspecting both genome records of each strain (Figure 1). We identified 28 putative TA pairs, distributed over eight TA families (Table 1), potentially targeting various cellular processes, in the 4 strains. Out of the identified putative TAs, 15 represent putatively novel TAs (Table 2). We found that the number and families of type II TA pairs varied among the four strains. While _G. thermoleovorans_ Gts has the eight TA families that were found in all the four strains with a total number of 10 putative TA pairs and one apparently solo putative antitoxin, _G. thermolevitrificans_ NS60-2 has only two TA families, with two TA pairs, and two and one apparently solo putative toxins and antitoxin, respectively (Table 1). The reason for the presence of a variety of TA families or the presence of more than one pair of the same TA family per strain is unknown, but there could be crosstalk among them to coordinate the cellular response to various stress conditions [8]. Furthermore, we suggested a putative TA family, GaTA that has not been reported previously in _Geobacillus_. We also identified a putatively new TA composite of the ParDE TA family, AbrB-ParE in three strains. Moreover, our analysis indicated that the XRE-COG2856 TA family, which has not been studied experimentally yet, might be regulated by second messengers, c-di-AMP and (p)ppGpp, and we proposed a hypothesis on the roles of these two messengers in regulating both the gene expression of _xre-cog2856_ TA pair and the toxin mechanism of action. Additionally, we suggested an approach to abolish the contamination caused by the food spoiling _G. stearothermophilus_ in food factories via targeting its TA genes. Our results indicated that the putative TA families of _Geobacillus_ seem to have special characteristics. For example, the putative GaTA TA family has a reverse gene order that has not been reported for the GNAT-HTH TA family, where the toxin gene precedes that of the antitoxin. This gene order renders this putative GaCTA family another potentially \"unique\" TA family. Additionally, we identified putative TA families that seem to feature three components instead of two, in three of the strains. The putative GaTA family of _G. thermolevitrificans_ NS60-2 has a putative antitoxin and two adjacent putative toxins, a case that has not been described for the GNAT-HTH TA family. The genes coding for the putative 3-component TA system in this strain seem to be encoded on opposite DNA strands, as explained in the Results and Discussion section. We also found that the genes coding for the XRE-COG2856 family are not fused, which does not seem to be a common pattern for this TA family in prokaryotes [75]. All these characteristics make experimental investigation of the type II TA families of _Geobacillus_ of significant importance, especially since our knowledge about TAs in thermophilic bacteria in general is very limited. Supplementary materials can be found at [[http://www.mdpi.com/1422-0067/20/23/5869/s1](http://www.mdpi.com/1422-0067/20/23/5869/s1)]([http://www.mdpi.com/1422-0067/20/23/5869/s1](http://www.mdpi.com/1422-0067/20/23/5869/s1)). Conceptualization, R.N.A. and B.C. ; methodology, R.N.A. and J.W. ; validation, R.N.A., J.W. and B.C. ; formal analysis, R.N.A. and J.W. ; investigation, R.N.A. and J.W. ; data curation, R.N.A. and J.W. ; writing--original draft preparation, R.N.A. ([PERSON] contributed to the \"Materials and Methods\" section); writing--review and editing, R.N.A., J.W. and B.C. ; visualization, R.N.A. and J.W. ; supervision, B.C. ; project administration, B.C.