## 2 Results and Discussion
### GNAT-HTH (GaCTA)
#### 2.1.3. _Ga_TA of \(G\). _thermodenitrificans_ NG80-2  
The gene with the locus tag GTNG_1349 codes for a putative GNAT domain-harboring toxin (Table 1). There is a slight difference in the aa sequence of the toxin between the original and RefSeq genome annotations. The RefSeq annotated toxin is better aligned with the GNAT toxins of the other strains (Figure S1a). Therefore, we considered it for our analysis (Table S1). Nevertheless, the putative toxin annotated in the original genome record also has a GNAT domain. The start codon of GTNG_1349 is GTG, instead of the common ATG. The adjacent gene with the locus tag GTNG_1350, annotated in the original and RefSeq genome records, codes for a wHTH-harboring putative antitoxin (Table 1). The intergenic region between the putative genes of the TA pair is 127 nt long. The aa sequences of the Ga_TA pair of this strain showed a few aa differences compared to the pairs of the other three strains (Figure S1). Furthermore, the TA finder identified GTNG_1577 and GTNG_1578 as a TA pair. However, our manual curation indicated that both genes with these locus tags code for putative GNAT toxins (Table 1 and Table S1) that did not align with the other GNAT toxins in the four strains (Figure S1). The putative genes coding for these two putative toxins are potentially regulated by the same operon (Table 1). In a trial to identify the antitoxin-coding gene(s), we analyzed the neighboring genome context. The analysis revealed the presence of a putative gene with the locus tag GTNG_1575, harboring an HTH domain (Table 1). This putative gene is encoded on the opposite DNA strand and is not sharing the operon with a toxin. It has been reported previously that the interaction within a TA pair operated by different operons, despite being uncommon, is possible [45; 46; 47]. Accordingly, it could be that those two GNAT-coding genes and the HTH-coding gene form a TA system. Although a TA system is usually a two-component system composed of a toxin and an antitoxin, cases of a three-component system have also been reported [48; 49] and they seem to be distributed in bacteria more commonly than previously thought [50]. It is interesting to note that the previously reported three-component systems have one toxin each, while the Ga_TA system reported here has two adjacent putative toxins. Carrying out experimental work will clarify the actual components of this system. On the other hand, since GNATs can acetylate the amino group of a broad variety of substrates and thus are involved in various cellular processes [51], it could be that the identified GNAT-harboring proteins of the _Geobacillus_ strains do not act as part of a TA system. Regarding the GNAT-wHTH pairs of the four _Geobacillus_ strains, our analysis showed that the putative toxin and its cognate antitoxin are potentially regulated by two separate, yet adjacent operons. We noticed that while the gene coding for the putative GNAT toxin is the only gene in its operon, the gene coding for the putative antitoxin is potentially regulated by the adjacent operon together with four to seven other genes. It could be that the operon prediction algorithm mistakenly separated the two adjacent genes coding for the putative TA pair into two operons. When we manually checked the distance between the genes of each TA pair, we found that it is within the range 128-132 nt, making it hard to deduce whether the toxin- and antitoxin-coding genes share the same operon or not. It could also be that the two putative TA genes are potentially regulated by two separate operons, as mentionedabove. However, based on functional annotations of the other genes sharing the operon with the putative antitoxin-coding one, these genes are not related to TA-coding or regulating genes (Table S3). Therefore, it is more likely that the TA-coding genes were mistakenly predicted as regulated by two separate operons.