## 2 Results and Discussion
### TAs of Geobacillus Strains--Conserved, Yet Diverse  
TA families of every _Geobacillus_ strain analyzed here are highly diverse (Figures S1-S10); it was not possible to get a consensus when a global alignment of all sequences was tried. Moreover, in a strain that has more than one toxin/antitoxin molecule belonging to the same TA family, diversity is observed among these molecules, such as the observations reported here about the MazEF and MNT-HEPN TApairs and the MNT solo antitoxins as well (Figures S2-S5). However, for each TA family, the TA pairs are highly conserved among the four strains, but TAs of _G. thermodentiriftcans_ NG80-2 are much lower in number and are relatively less conserved compared to the other three strains (Figures S1 and S3). One of the reasons could be related to the different ecological niches where the strains were isolated from. Strain NG80-2 was isolated from an oil field [32], while the other three strains were isolated from aquatic environments [27; 30; 31].  
The nature of the environment may also have an important impact on the number of encoded TAs, as has been suggested for archaea [129; 130]. Terrestrial hot springs (such as Ulu Slim, where _G. thermoleevorans_ Gts was isolated from, and Zara, where _Geobacillus_ sp. ZGt-1 was isolated from) represent challenging environments for the cells due to continuous fluctuations in temperature over a wide range, along with changes in nutrient levels and pH [129]. This may explain the reason why strain Gts harbors the highest number of TA loci, 10 putative TA pairs and one apparently solo putative antitoxin, among the four strains. Strain ZGt-1 has eight putative TA pairs and one apparently solo putative antitoxin, but since its genome sequence is incomplete, it cannot be determined whether these are all the type II TA loci that strain ZGt-1 harbors, or it does have more.  
On the other hand, there does not seem to be a correlation between the temperature of the ecological niche of the strain and the number of TA loci. A known example showing the lack of this correlation is illustrated by _Mycobacterium tuberculosis_, which is a mesophilic species and its strains harbor up to 67 type II TA pairs [131]. Regarding the _Geobacillus_ strains analyzed here, while the temperature of the Dagang oil field (strain NG80-2) at the time of isolating the strain was 73 \({}^{\circ}\)C [32], the strain has only two putative TA pairs. Contrarily, the temperature of the Mariana Trench and Zara hot spring were 55 \({}^{\circ}\)C [30] and 46 \({}^{\circ}\)C [27], respectively, and strains HTA426 and ZGt-1 have eight putative TA pairs each. Moreover, the temperature of Ulu Slim at the time of isolating strain Gts was around 92 \({}^{\circ}\)C and the strain has 10 TA pairs, as mentioned above [31]. Experimental analysis is needed to conclude the impact of different environmental factors [129].  
It is worth noting that in an exceptional case of a TA pair of the type I TA system in _E. coli_, the toxin SymE (SOS-induced _yjiW_ gene with similarity to _MazE_) of the SymE-SymR TA pair showed homology to the MazE antitoxin, as it harbors an AbrB domain [132]. However, experimental results indicated that SymE is actually a toxin that seems to have evolved from the AbrB-domain protein superfamily [132]. Accordingly, such exceptions should be kept in mind when carrying out experiments, especially when studying solo toxins and antitoxins, since an antitoxin domain-harboring protein could be a toxin that evolved from an antitoxin protein family, or vice versa. As is the case with any protein and as mentioned above, only experiments may confirm the function of a predicted toxin/antitoxin.