## 2 Results and Discussion
### RelBe-XRE-ReIE Composite
#### 2.6.1 RelBE of G. kaustophilus HTA426 and Geobacillus  
The gene with the locus tag GK3104, annotated in both genome records of strain HTA426, and the gene encoded on contig 25_195 in strain ZGt-1 code for a putative RelE domain-harboring protein (Table 1). Therefore, it is likely that each of these two genes codes for a putative RelE toxin. The aa sequences of these two putative toxins are 99% identical (global alignment in Figure S8a).  
The gene with the locus tag GK3105 in strain HTA426, and the one encoded on contig 25_196 in strain ZGt-1, each of which is adjacent to and upstream of the putative RelE toxin-coding gene, seem to code for a putative antitoxin. The two genes coding for each of the putative TA pairs in each of the strains overlap by 32 nt. In strain HTA426, the two putative genes potentially share the operon (Table 1). Although the Operon-Mapper did not annotate the genes encoded on contigs 25_195 and 25_196 in strain ZGt-1, it could be easily inferred that the two putative genes potentially share the operon (Table 1).  
The aa sequences of the protein products of GK3105 and contig 25_196 are 98% identical (global alignment in Figure S8b). Neither the NCBI CDD nor the InterPro domain analysis tools identified a conserved domain in the protein products of GK3105 or contig 25_196. GK3105 has been annotated as a hypothetical protein in its genome records (Table S1), and contig 25_196 is 99% identical to a hypothetical protein (Table S2). However, when checking the orthologs of GK3105 presented in the KEGG database, we found that GK3105 is orthologous to a _G. genomosp_. strain 3 protein, AGT33452, and showed 100% identity over its entire length. AGT33452 is annotated as an XRE family transcriptional regulator in the genome of _G. genomosp_. strain 3 [93]. Moreover, among the other orthologs that are annotated as XRE family transcriptional regulators are ALA70040, AMQ22632, and AMX82334, all of which are geobacillus proteins that showed \(\sim\)99%, \(\sim\)98%, and \(\sim\)94% identity to GK3105, respectively. The protein encoded on contig 25_196 in strain ZGt-1 is also orthologous to the same proteins, ALA70040, AGT33452, AMQ22632, and AMX82334, which showed \(\sim\)98%, \(\sim\)97%, \(\sim\)97%, and \(\sim\)96% identity, respectively. However, each of these four proteins has been annotated as hypothetical in its RefSeq version. On the other hand, when searching for proteins homologous to GK3105 and the protein encoded on contig 25_196 within _Bacillus_ species, using blastp, the best hit was \"XRE family transcriptional regulator\" (WP_066367164) of _B. fumarioli_ NBRC 102428, which showed 56% identity to the two geobacillus proteins, with an e-value of 9e-27 and query coverage of 97% (GK3105), and an e-value of 6e-25 with a query coverage of 98% (contig 25_196). It is worth noting that, as is the case with GK3105 and the protein encoded on contig 25_196, this \"XRE family transcriptional regulator\" of _Bacillus_ has no conserved domain, as indicated by the NCBI CDD and InterPro domain analysis tools. Moreover, the secondary structure prediction of the two geobacillus XRE proteins showed that they have repeated alternation between coils and helices, similar to WP_066367164 (Figure S11a-c).  
Taken together, these results indicate that the protein products of GK3105 and contig 25_196 could represent putative XRE family transcriptional regulators acting as antiotoxins (Table 2). Experimental studies are required to confirm the identity of these geobacillus proteins.  
In summary, protein products of GK3105 and GK3104, and those of contigs 25_196 and 25_195 represent putative XRE-ReLE TA pairs (Table 1).