(281e) The Role of Toxin/Antitoxin Systems in the General Stress Response, Biofilm Formation, and Persister Cell Formation

Almost all bacteria grow on solid surfaces or at air/liquid interfaces in complex communities known as biofilms. These communities are useful for engineering applications like the remediation of toxic chemicals and are important for medical problems such as chronic infections. We investigated the genetic basis of biofilm formation using a whole-transcriptome approach and the best-studied bacterium, Escherichia coli, and discovered that toxin/antitoxin pairs are related to biofilm formation (Appl. Micro. Biotechnol. 64:515, 2004). Upon further investigation, it was found that the uncharacterized protein YgiU (renamed MqsR) is involved in the regulation of motility through cell-cell signaling that utilizes autoinducer-2 (J. Bacteriol. 188: 305, 2006). We also showed that MqsR functions as a toxin (Nature ISMEJ 2:615, 2008), and the three-dimensional structure of this protein revealed that MqsR functions as an RNase (PLoS Pathogens 5: e1000706, 2009). The antidote to this toxin, MqsA, is an elongated dimer that neutralizes MqsR toxicity as an antitoxin. MqsR adopts an alpha/beta fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. Critically, we have found that MqsA binds promoters of genes important for physiology including mcbR and spy as well as its own promoter through its C-terminal domain. Furthermore, using three sets of whole-transcriptome studies, two nickel-enrichment DNA binding microarrays, and cell survival studies in which MqsR was overproduced in isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB, relE, and hokA) that are involved in MqsR toxicity in addition to its RNase activity (Environ. Micro, in press). Quantitative real-time polymerase chain reaction showed that (i) the MqsR/MqsA complex (and MqsA alone) represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii) stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are overproduced or when mqsRA is deleted. Hence, upon stress, cells down-regulate their metabolism via toxin MqsR as well as via other toxins controlled by antitoxin MqsA. In addition, we found that deletion of the mqsRA locus decreases persister cell formation (i.e., cells that are resistant to antibiotics without genetic change) and, consistently, overexpression of MqsR increases persister cell formation (BBRC 39:209, 2010); this is the first set of toxin/antitoxin genes found to control persister cell formation. Furthermore, we have found antitoxin MqsA directly controls the transcription of the master regulator of stress, RpoS. Upon stress, MqsA is degraded by proteases which derepresses rpoS. Conversely, production of MqsA represses rpoS and reduces concentrations of the second messenger 3,5-cyclic diguanylic acid due to repression of diguanylate cyclases that are controlled by RpoS. Repression of rpoS by MqsA leads to increased motility and a reduction in the cell adhesins curli and cellulose as well as a reduction in stress resistance and biofilm formation. Hence, external stress alters gene regulation via toxin/antitoxin systems and leads to a switch from the planktonic state (high motility) to the biofilm state (low motility). Therefore, using several systems biology approaches, we have determined the primary roles of toxin/antitoxin systems in cell physiology.