## 2 Results and Discussion
### XRE-COG2856
#### 2.8.1 XRE-COG2856 of _G. kaustophilus_ HTA426 and _G. thermolevorans_ Gts  
The genes with the locus tags GK3184 and *_-35620 in strains HTA426 and Gts, respectively, code for a putative COG2856 domain-harboring toxin (Table 1). The aa sequences of the putative toxins are >99% identical between these two strains (global alignment Figure S10a). The sequence of each putative toxin contains the HEXXH motif as HEYH. The NCBI annotation supports our results, as it shows that each of these putative proteins is identical to a member of the ImmA/IrrE metallo-endopeptidase family (Table S1), which consists of Zn-dependent proteases harboring the COG2856 domain mentioned above [99; 100]. However, the current study further clarified that the two proteins are putative toxins of the type II TA system. The genes with the locus tags GK3185 and *_-35630, which are adjacent to and upstream of the putative toxin-coding gene, code for a putative protein that does not harbor a conserved domain, as indicated by the NCBI CDD and InterPro domain analysis tools (Table 1). However, the protein motif presented in the KEGG database showed that the protein products of GK3185 and *_35630 harbor the HTH domain (e-value \(<\) 0.02). Therefore, they might represent putative antitoxins of the XRE-protein family. The aa sequences of the two putative antitoxins are \(\sim\)98% identical between these two strains (Figure S10b). They have been annotated as hypothetical proteins in the genome records of each strain (Table S1). The current study, however, highlighted the identity of these hypothetical proteins as putative antitoxins (Table 2). In each strain, the intergenic region between the putative TA pair-coding genes is 4 nt long. This unfused pattern of COG2856-XRE does not seem to be common among prokaryotes, as indicated in [75], or it could simply be harbored by yet to be studied strains. The putative coding genes potentially share the operon with a third gene, locus tags GK3183 in strain HTA426, and *_35610 in strain Gts, positioned downstream of the TA-coding genes. This third putative gene codes for a hypothetical protein in each strain (Table S3). Analysis of the aa sequence using the NCBI CDD and InterPro domain analysis tools did not retrieve a protein domain (Table 1). However, the protein motif presented in the KEGG database showed that the protein products of GK3183 and *_35610 harbor the protein domain 7 TMR-HDED (e-value \(<\) 0.26). The 7 TMR-HDED stands for 7 transmembrane helices receptors-HD hydrolase; a hydrolase with a catalytic His-Asp (HD) motif, and ED stands for extracellular domain [81; 101]. The pfam of the 7 TMR-HDED protein family is PF07697, as indicated by the NCBI CDD and InterPro domain analysis tools. The 7 TMR-HDED domains are expected to be involved in signal detection and transmission to the cellular machinery, in order to stimulate a response to the environmental conditions [81; 101]. The presence of the 7 TMR-HDED receptor suggests that the protein product of GK3183 is regulated by a second messenger, which is likely to be the c-di-AMP, since the 7 TMR domains transmit the c-di-AMP [97], and in turn, it regulates other proteins. We analyzed the genome context to have a further indication of the kind of second messenger that the two \"hypothetical\" proteins transmit, and thus could affect the TA pair, since it has been reported that genes within the neighboring context have provided information on novel signaling nucleotides [81]. We found that a gene with the locus tag GK3182 in strain HTA426, and another with the locus tag *_35570 in strain Gts are involved in the adenine metabolism, as shown in the KEGG pathway maps of the purine metabolism in each of the strains, and have been annotated in the RefSeq genome record of the respective strain, as well as in the KEGG database, as \"bifunctional 2',3'-cyclic-nucleotide 2'-phosphodiesterase/3'-nucleotidase (EC:3.1.4.16 3.1.3.6)\".