## 1 Introduction
The opposite seems to also be possible for some toxins depending on their mechanism of action, as [PERSON] et al. have shown that in _Bacillus subtilis_, the type II toxin \(E\), an ATPase, hydrolyzed the ATP, converted the GTP to (p)ppGpp, and raised the levels of the second messenger, the cyclic-di-adenosine monophosphate (c-di-AMP) [24]. Quorum-sensing signal molecules may also be involved in activating the type II toxins, as discussed further below. Studies of TA systems have focused on mesophiles, while thermophilic bacteria have not been given the same attention. Apart from what has been reported in the TA database (TADB) [25], there is only one study that was conducted on a thermophilic bacterial strain, _Thermus thermophilus_, to analyze the VapBC TA module [26]. Given the stress conditions that thermophilic bacteria encounter in their ecological niches, studying the roles and mechanisms of their TA systems may broaden our knowledge on these entities that help bacteria cope with stress. It may also further elucidate the adaptation strategies employed by thermophilic bacteria. Moreover, studying the TA systems of thermophilic bacteria could shed some light on their potential biotechnological applications. Genome analysis aiming for mining genomes of thermophilic strains for TAs will facilitate the future experimental work, as it will help in defining the TAs of interest, and thus will set a path for experimental design. Due to our interest in the identification of the antibacterial potential of _Geobacillus_ spp. in general, and _Geobacillus_ spp. strain ZGt-1 in particular [27; 28], we have mined the genomes of certain _Geobacillus_ strains that we are interested in for type II TA system families, as a first step towards its experimental investigation. For the TA screening, we selected _Geobacillus_ spp. strain ZGt-1, which was isolated from Zara hot spring in Jordan [27; 29], and we also selected the type strains of species that showed the closest similarity to strain ZGt-1, as indicated in [27]. The strains we selected were isolated from different ecological systems; _G. kaustophilus_ strain HTA426 was isolated from the deep-sea sediment of the Mariana Trench [30] and _G. thermolevorans_ strain CCB_US3_UF5 (hereafter referred to as \"Gts\" (for_G. thermolevovans_ strain), for simplicity) was isolated from a hot spring in Malaysia [31]. We selected _G. thermodentirificans_ NG80-2 as well, since it was isolated from an oil reservoir in China, which is a non-aquatic ecological niche [32]. The present study aims for the identification of TA-coding genes, excluding the ones residing within the genome region of the integrated prophage. Studying the TA systems of phylogenetically related strains isolated from different ecological systems will give indications about the diversity of these systems. Furthermore, mining genome sequences for TA genes will reveal their presence, since a number of them have been overlooked during the genome annotation or annotated as coding for hypothetical proteins [33]. Various analysis tools were used (Figure 1) and resulted in the identification of 28 putative TA pairs on the chromosomal genomes of the strains. Among the identified TAs, 15 represent putatively novel ones. The term \"novel\" in this context refers to toxin and antitoxin proteins that have not been identified in the literature as putative toxins/antitoxins of the respective _Geobacillus_ strain, and have either been overlooked or annotated as hypothetical in both genome records, the original and the RefSeq, of the analyzed _Geobacillus_ type strains. For the non-type strain, _Geobacillus_ sp. ZGt-1, the protein is described as novel if it has been annotated by the NCBI as hypothetical or if the NCBI blastp result showed a hypothetical protein as the top hit. The identified TAs are discussed below.