# Targeting Type II Toxin-Antitoxin Systems as Antibacterial Strategies
## 5 Role of Type II TA Modules in Biofilm Formation and Bacterial Persistence  
Two potentially undesirable effects of artificial activation of TAs are bacterial persistence and biofilm formation. Persister cells are a subpopulation of slow-growing or growth-arrested bacterial cells that have a decreased susceptibility to killing by bactericidal antibiotics within an otherwise susceptible clonal population [90]. It has been shown that increased tolerance of biofilms to antibiotics is due to the higher amounts of persister cells within the biofilm community [91; 92]. While antibiotics kill the majority of biofilm cells, persisters remain viable and repopulate biofilms when the level of antibiotics drops [93]. Thus, persister cells appear to play a central role in the recalcitrance of chronic and biofilm-related infections [94]. One of the first items of evidence linking type II TA modules to biofilm formation comes from a well-characterized chromosomal TA system _mqsRA_ in _E. coli_[95; 96]. The _mqsRA_ is a unique type II TA system where the toxin gene _msqR_ precedes the antitoxin _msqA_[97]. It has been shown that a Tn5 insertion mutant of the toxin _mqsR_ formed less adherent biomass [96]. On the contrary, later reanalysis of the _mqsRA_ revealed that this TA module does not affect biofilm formation in nutrient-rich conditions [98]. The authors identified two new promoters located in the toxin coding sequence that allow the constitutive expression of _mqsA_, thereby allowing a constant and steady level of the MqsA antitoxin compared to the MqsR toxin. This work, quite understandably, has opened up the debate about the role of TA systems in persistence and biofilm formation [78; 99]. This result disproves the role of _mqsRA_ (and nine others) TA modules in spontaneous biofilm formation, but does not address the question of a role of TA systems in growth control and biofilm formation under stress conditions. The first persistence-related gene to be identified was _hipA_, encoding the toxin of the _E. coli__hipBA_ TA system--a serine/threonine kinase that inhibits cell growth by inactivating the glutamyl-tRNA synthetase GltX [100]. This discovery linked TA modules and bacterial persistence for the first time. Since then, correlations between antibiotic persistence and TA systems have been extensively studied. Evidence both for and against the participation of TA systems in persister cell formation has been accumulated, making their contribution to this phenomenon unclear [99; 101; 102; 103; 104]. For example, the deletion of 10 ribonuclease-encoding TA systems from the _E. coli_ genome was found to decrease the number of persisters. However, it was subsequently shown that this result was influenced by the presence of \(\varphi\)80 bacteriophage contamination [105]. Moreover, reconstruction of this mutant strain demonstrated that deletion of the 10 TA systems did not affect susceptibility to ofloxacin or ampicillin [106]. These contradictory findings have given rise to considerable debate [78; 107; 108; 109], and the relevance of TA systems to bacterial persistence remains unclear. In contrast, the results of several other studies support the contribution of TA systems to bacterial persistence. For example, overexpression of the toxins RelE or MazF was shown to increase the survival of _E. coli_ under antibiotic exposure [9; 110]. In another study, the _dinfl/yafQ_ TA module was found to be involved in tolerance to cephalospororin and aminoglycoso antibiotics [111]. Further evidence linking type II TA modules and bacterial persistence was obtained in a study on uropathogenic isolates of _E. coli_[112]. TA systems have also been linked to bacterial persistence in _Salmonella_, where the _shpB1_ allele, carrying a mutation in the antitoxin of the _shpAB_ TA module, was associated with the salmonella high persister phenotype [113]. In addition to the _shpAB_ system, 13 other type II TA modules were shown to contribute to persistence in _Salmonella_ triggered by stresses encountered during macrophage infection [114].