## 2 Multi-Level Interactions between TA Systems
### TA Systems Sharing the Same Replicon  
#### 2.1.1 Cross-Interactions between Non-cognate Antitoxin and Toxin Proteins  
Several studies describe analyses of interactions between components of type II systems located in the same chromosome [6, 34, 41, 42]. The case of homologous systems has been well documented, and it appears that interactions between cognate toxins and antioxins are highly specific despite a good conservation at the amino sequence level. This has been shown notably for the _E. coli__mazEF_ and _chpB_ systems [6] and for 2 _yefM-yoeB_ systems of _Staphylococcus equorum_[41], as well as for seven and three RelE/ParE toxins in _Caulobacter crescentus_ and _E. coli_ O157:H7, respectively [34, 42]. The most remarkable example is certainly the case of the 30 functional VapBC system of _Mycobacterium tuberculosis_[8, 43, 44]. Despite their large number, no cross-interactions were detected between toxins and antioxins from different systems [8, 43]. Cross-interactions between toxins and antitoxins of different systems could result in a strong negative effect. Since a balanced toxin:antitoxin ratio is crucial for survival, perturbation of this ratio by, e.g., the interaction of a given antitoxin with several toxins from different systems could lead to an excess of free toxin and inhibition of cell growth and/or cell death. Proof of principle was obtained since expression of chimera MazF toxins in _E. coli_ led to endogenous MazF\({}_{\text{K-12}}\) activation, most likely by competition of these chimera with the endogenous MazF\({}_{\text{K-12}}\) toxin for MazE\({}_{\text{K-12}}\) antitoxin binding [45]. In addition, expression of inactive toxin mutants was used to titrate endogenous toxins and activate the endogenous systems of both chromosomally- and plasmid-encoded TA loci [46,47]. Interestingly, cross-interactions between homologous systems might potentially lead to TA system degeneration. We have detected two homologous _relB.parE\({}_{\text{Sme}}\)_ systems on the pSymA megaplasmid of _Sinorhizobium meliloti_[48]. The first system appears to be a _bona fide_ TA system, _i.e.,_ toxin expression is toxic for _E. coli_ and co-expression of the antitoxin restores normal growth. However, in the second system, the predicted antitoxin-encoding gene, _relB\({}_{\text{Sme2}}\)_, appears to be truncated of its 5'-end, although it shares 93% nucleic acid sequence identity with the 3' region of _relB\({}_{\text{Sme1}}\)_ antitoxin from the first system. The predicted toxin-encoding gene _parE\({}_{\text{Sme2}}\)_ toxin shares 91% nucleic acid sequence identity with the 5' region of _parE\({}_{\text{Sme1}}\)_. Cross-talks between these systems were investigated. Surprisingly, ParE2\({}_{\text{Sme2}}\) is not toxic for _E. coli_, but the predicted RelB2\({}_{\text{Sme2}}\) retained antitoxin activity against the functional ParE\({}_{\text{Sme1}}\). Thus, multiple copies of the same system might perturb the subtle balance of TA systems and lead to the decay of one of them. On the other hand, cross-interactions between homologous or non-homologous systems have been described in _M. tuberculosis_ (Figure 2) [49,50]. The first network involves direct cross-interactions between homologous _relBE_ systems. Non-cognate complexes of toxins and antitoxins are able to bind promoter regions, suggesting possible expression cross-regulation between these systems [49]. The second network includes direct cross-interactions between non-homologous _mazE-vapC_ and _mazEF_ systems [50]. Non-cognate toxin-antitoxin interactions were detected upon overexpression in _E. coli_. It was proposed that such well-regulated cross-interactions between homologous or non-homologous systems could evolve into networks acting cooperatively to facilitate _M. tuberculosis_' adaptation to its environment during infections [49, 50, 51]. However, further experiments are needed to assess the _in vivo_ relevance of these potential networks, since deletion of the _relE_ genes did not affect _M.