## 2 Results and Discussion
### MazEF
#### 2.2.2 MazEF (II)-RHH-MazF Composite  
MazEF (II) of _G. kaustophilus_ HTA426, _Geobacillus_ sp. ZGt-1, and _G. thermolevorans_ Gts  
The genes with the locus tags GK0233 and *_2500 in strains HTA426 and Gts, respectively, code for a putative MazF domain-harboring toxin, MazF (II) (Table 1) belonging to the PemK toxin superfamily. The gene encoded on contig 4_61 in ZGt-1 also codes for MazF (II) (Table 1). In strain Gts, there is a difference in the aa sequence of the putative toxin between the original and RefSeq genome annotations. Nevertheless, in both records, the protein harbors the same domain that identifies it as a putative MazF toxin. For our analysis, we considered the RefSeq annotation, since the MazF annotated in the RefSeq record is better aligned with the toxins of the other analyzed _Geobacillus_ strains (Figure S3a). Each of the putative toxin-coding genes has been annotated as coding for a \"type II toxin-antitoxin system endoribonuclease\" in the genome records of the three strains (Table S1). Furthermore, our results specified the type of the endoribonuclease to be MazF. The analysis also indicated that in the three strains, the genes with the locus tags GK0232 and *_2490, and the gene encoded on contig 4_60 code for an RHH-domain harboring antitoxin (Table 1). The antitoxin-coding gene is adjacent to and upstream of its cognate toxin-coding one in all three strains. The two genes coding for each of the putative TA pairs share the same putative operon in each of the strains (Table 1), and there is no overlap between the two genes of any TA pair. In each of the three strains, the putative antitoxin-coding gene has been annotated by NCBI as coding for a hypothetical protein (Table S1). However, using the results of our analysis, we could identify each of these three hypothetical proteins as a putative RHH domain-harboring antitoxin (Table 2). The aa sequences of the three putative toxins are identical among the three strains, and the same applies to the putative antiotoxins (Table S1; global alignment Figure S3). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Strain** & **Genome Accession Number** & **Putative T/AT 1** & **Locus Tag** & **Protein ID 2** \\ \hline _Gd_1 & NC\_009328 & RHH & GTNG\_0206 & WP\_008881474 3 \\ \hline \multirow{6}{*}{_Gt_1 NC\_006510_} & AbrB & GK2355 & WP\_015375348 3 \\ \cline{2-4}  & & HTH & GK3185 & WP\_011232655 3 \\ \cline{2-4}  & & ParE & GK2354 & WP\_020278248 3 \\ \cline{2-4}  & & RHH & GK0232 & WP\_011229742 3 \\ \cline{2-4}  & & XRE & GK3105 & WP\_011232575 3 \\ \hline \multirow{6}{*}{_Gt_1 NC\_016593_} & AbrB & *\_26570 & WP\_014196297 3 \\ \cline{2-4}  & & HTH & *\_35630 & WP\_014196828 3 \\ \cline{2-4}  & & MNT solo antitoxin & Unannotated & WP\_013146011 4 \\ \cline{2-4}  & ParE & *\_26560 & WP\_014196296 3 \\ \cline{2-4}  & & Xre & *\_34820 & WP\_014196753 3 \\ \cline{2-4}  & & AbrB & Contig 23\_243 & WP\_015375348 4 \\ \cline{2-4}  & & ParE & Contig 23\_242 & WP\_020278248 3 \\ \cline{2-4}  & & RHH & Contig 4\_60 & WP\_011229742 3 \\ \cline{2-4}  & & XRE & Contig 25\_196 & WP\_082218538 4 \\ \hline \end{tabular} \({}^{\dagger}\)_G. thermodentirificans_ NG80-2; \({}^{\ddagger}\)_G. kaustophilus_ HTA426. \({}^{\lx@sectionsign}\)_G. thermodeworans_ CCB_US_IF5, \({}^{\ddagger}\)_Geobacillus_ sp. ZGt-1; \({}^{\ast}\)_Stands_ for “GTCBCBUSUS” that is part of the locus tags in _G. thermolevorans_ CCB_US_IF5, \({}^{\ddagger}\)_Tooxin/Antitoxin_;_\({}^{\ddagger}\)_Represents the RefSeq accession number of the putative toxin/antitoxin protein;_\({}^{\ddagger}\)_Accession number belongs to the putative toxin/antitoxin protein as annotated in the RefSeq genome record of the type strain/draft genome sequence of strain ZGt-1; \({}^{\ddagger}\)_Accession number belongs to the NCBI blast top hit, e-value \(<10^{-20}\).  
\end{table} 
Table 2: Previously unrecognized toxins and antiotoxins that have been identified in the current study as putatively novel ones. These TAs have either been annotated as hypothetical proteins or have not been annotated.