# Emerging Roles of Toxin-Antitoxin Modules in Bacterial Pathogenesis
## 4 Biofilm Formation is Influenced by the Action of Multiple TA Systems  
A biofilm describes populations of microbial cells growing on a wide variety of solid surfaces, either biotic or abiotic, and that exhibit multicellular-like behavior. These cells are enclosed in an extracellular matrix primarily composed of polysaccharides, glycoproteins and DNA, which enables their adhesion and protection from the surrounding environment [56; 57]. It is estimated that biofilms are involved in at least 80% of human bacterial chronic inflammatory and infectious diseases [58]and cause serious therapeutic problems due to their multidrug tolerance as well as resistance to host immune system actions [59]. Biofilm development is a dynamic process, which involves the attachment of planktonic cells to the solid surface, microcolony formation followed by cell immobilization, maturation of biofilm architecture into a three dimensional structure, and finally detachment and release of cells back into the planktonic state [57]. The transition between planktonic and biofilm phases requires precise and well-coordinated regulation of expression of many genes at different stages of biofilm development [60]. Cells within biofilms show reduced growth rates, as well as differences in the expression of specific genes compared to suspension cultures [61]. Biofilm development is strongly stimulated by environmental conditions and stress genes are involved in biofilm formation [60]. Moreover, biofilm production is influenced by multiple TA systems [57; 62; 63; 64]. The most penetrating insights into the role of TAs in biofilm organization come from research on _E. coli_ biofilms. At least 34 TA modules have been identified on the _E. coli_ K-12 genome (Figure 2). Cellular proteases, including Lon, ClpXP and ClpPA, seem to be the key players that mediate the environmental stress response by activating multiple TA systems via degradation of the antitoxins (Figure 3). With one known exception, TA modules implicated to date in biofilm formation in _E. coli_ belong to type II in which both toxin and antitoxin are proteins. The MqsR-MqsA type II pair was first documented to be engaged in a complex regulatory gene network, which promotes biofilm formation. The antitoxin MqsA directly inhibits the expression of a master regulator of stress, RpoS [65]. This stationary-phase sigma factor controls the expression of up to 10% genes involved in the response to different stresses, such as temperature shock, starvation and growth shift from exponential to stationary phase [66]. MqsA also decreases the production of CsgD, the master regulator for biofilm formation [67] (Figure 3). CsgD transcriptionally activates curli gene expression, as well as the gene for diguanylate cyclase (AdrA) which synthesizes cyclic diguanylate (c-di-GMP) that is implicated in cellulose production [68]. Both curli and cellulose are important components of biofilms. Moreover, the _csgD_ gene is transcribed by RNA polymerase which contains the stationary phase sigma subunit, RpoS, which in turn represses the expression of _flhD_ which encodes a transcriptional regulator of flagella biogenesis. Thus, in the absence of stress MqsA increases motility by activating _flhD_ through _rpoS_ as well as _csgD_ inhibition (Figure 3). Under stressful conditions, MqsA degradation by Lon protease leads to derepression of _rpoS_ and _csgD_ and inhibition of _flhD_[65]. Activation of _rpoS_ also increases concentrations of the internal messenger c-di-GMP. This regulatory network leads to decreased motility and increased biofilm formation. Therefore, the role of the MqsA antitoxin seems to be the inhibition of biofilm formation in the absence of stress and activation of this mode of growth under unfavorable conditions (Figure 3) [65]. The MqsR toxic component of the MqsRA system also participates in the regulation of biofilm production (Figure 3).