## 2 Results and Discussion
### Mnt-Hepn
#### 2.3.1 Mnt-Hepn (I)  
COG1669-COG2361 Composite  
MNT-HEPN (I) of _Geobacillus_ sp. ZGt-1 and _G. thermolevorans_ Gts  
The gene encoded on contig 12_20 in ZGt-1 codes for a HEPN-containing protein, as does the gene with the locus tag *_10720 annotated in both genome records of strain Gts. The aa sequences of these proteins are identical (global alignment in Figure S4a) and harbor the domain COG2361 (Clusters of Orthologous Group 2361) which belongs to the DUF86 \"domain of unknown function 86\" protein family, both of which are associated with HEPN toxin [7,76]. The putative HEPN toxin is labeled here as HEPN (I) in each of the strains (Table 1).  
Most HEPN domains harbor a conserved Rx4-6H catalytic motif (R stands for arginine, H stands for histidine, and x stands for 4-6 of aa residues between R and H), where the residue immediately after the R is a polar aa [77]. This motif is considered the most conserved characteristic of HEPN domains [77]. It is thought to be responsible for the mRNase activity of the HEPN toxins [77]. Both of the above-mentioned HEPN-containing proteins harbor the domain R(75)DMLIH(80), where D is aspartic acid (Asp) which is a polar aa. The presence of this motif further supports the probability that contig 12_20 and *_10720 are part of the putative TA system in those two _Geobacillus_ strains.  
The gene encoded on contig 12_19, and that with the locus tag *_10710, each of which is adjacent to and upstream of the toxin-coding gene, code for a putative COG1669 domain-harboring protein (Table 1). COG1669 is an MNT antitoxin-associated domain [7]. MNT antitoxin is labeled here as MNT (I) in both strains (Table 1). In strain Gts, there is a slight difference in the aa sequence of the putative antitoxin between the original and RefSeq genome annotations. The putative MNT antitoxin annotated in the RefSeq genome record is perfectly aligned with that of ZGt-1 (Figure S4b). Therefore, we used it for our analysis (Table S1). Nevertheless, in both records, the protein harbors the domain that identifies it as a putative MNT antitoxin. We noticed that the two genes of the TA pair in each strain overlap by 17 nt.  
In addition to the MNT and HEPN-coding genes which are adjacent, our analysis indicated that there is another MNT-coding gene potentially sharing the operon with the mentioned putative MNT-HEPN TA pair in each of the strains (Table 1). This second MNT-coding gene in strain ZGt-1 is encoded on contig 12_18, upstream of contig 12_19 and both putative genes are 23 nt apart. This putative MNT (II) also has COG1669 domain (Table 1), and blastp showed that it is 90% identical to nucleotidyltransferase (Table S2). Interestingly, the gene coding for MNT (II) has neither been annotated in the original nor in the RefSeq annotations of the genome records of strain Gts. Therefore, our analysis has identified a gene that has been overlooked and this gene seems to be part of the putative MNT-HEPN TA family of strain Gts. Accordingly, we recommend annotating this MNT-coding gene at the position presented in Table S2. The aa sequences of MNT (II) protein in both strains are identical (Table S1; global alignment in Figure S4c). On the other hand, the aa sequences of the two adjacent MNT (I) and (II) proteins are 19 % identical between the strains ZGt-1 and Gts, respectively. This low matching identity is not unexpected, since nucleotidyltransferase domain-harboring proteins comprise a large and highly diverse protein superfamily [78]. The global alignment confirms that the putative MNT antioxins of _Geobacillus_ strains are diverse (Figure S4b,c). Moreover, our results indicated that _Geobacillus_ sp. ZGt-1 and _G. thermolevorans_ Gts represent examples of strains that have putative three-component TA systems among their various TA systems.