## 2 Results and Discussion  
In this study, the strategic approach plotted for the in-silico TA identification involved several steps (Figure 1). The workflow started with predicting the TAs of the four _Geobacillus_ strains using the TA finder software [25]. This was followed by inspecting the genome context of the putative TA-coding genes. TAs whose genes were located within prophage islands, as indicated from the neighboring genes, were excluded. The included putative TAs were then manually curated. The protein domains of each toxin and antitoxin were identified using the CDD (Conservation Domain Database) and InterPro  
Figure 1: Workflow chart summarizing the analysis approach carried out in this study for the identification of type II TA families.  
prediction tools. The KEGG Genes database and the KEGG KOALA BLAST [34] were used in cases where the CDD and InterPro tools did not predict a protein domain, as explained in the text. At this stage, proteins that did not harbor toxin-/antitoxin-related domains were excluded. The proteins that harbored related domains were further analyzed using various tools. The NCBI blastp showed the annotation of the putative toxin/antitoxin. Those annotated as hypothetical proteins in the RefSeq and original genome records, or their genes had not been annotated have been described here as putative novel toxins/antitoxins, as mentioned above. The organization of the putative TA-coding genes on the genome was also inspected, to see if the putative antitoxin-coding gene was upstream of the toxin-coding gene, or if they had a reverse gene order. The Operon Mapper tool [35] was used for predicting whether the putative TA-coding genes were potentially sharing the same operon or not. The Prokaryotic Operon Database (ProOpDb) [36] was used when the Operon Mapper did not predict the operon for the type strains. Worth mentioning here is that the Operon Mapper helped in identifying additional TAs, since some TA-coding genes identified by the TA finder software were shown, using the Operon Mapper, to share the operon with a third gene. Protein domain analysis of the third gene sometimes showed a toxin-/antitoxin-related domain. Therefore, these proteins were subjected to the same manual curation steps mentioned above, as shown in Figure 1. The resulting TAs were aligned based on their protein families; i.e., their type II TA families (Figures S1-S10). In parallel to these analysis steps, we mined the literature for the features of the TA families of the identified putative TAs and checked whether these TAs had already been reported or not yet.  
Employing this approach, we identified 28 putative TA pairs on the chromosome of each of the four _Geobacillus_ strains analyzed here, whereas there were no type II TA genes detected on the plasmids of any of the strains. The predicted TA pairs are distributed over eight TA families (Table 1). Additionally, in three of the strains, we identified two and three apparently solo putative toxins and antioxins, respectively, that could be acting together with the predicted TA pairs (Table 1). Out of the identified putative TAs, 15 represent putatively novel ones as they were not recognized previously as part of the TA system of the given _Geobacillus_ strain (Table 2). Below, follows a description of the identified TA families.