== To purify the N-terminally histidine-tagged GraA proteins, nickel affinity chromatography was used

== To purify the N-terminally histidine-tagged GraA proteins, nickel affinity chromatography was used. that the degradation rate of GraA depends on the growth phase of bacteria being fastest in the transition from exponential to stationary phase. In accordance with this, higher ATP levels were shown to stabilize GraA. Differently from other antitoxins, the main cellular proteases Lon and Clp are not involved in GraA stability. Instead, GraA seems to be degraded through a unique pathway involving an endoprotease that cleaves the antitoxin into two unequal parts. We also identified the global transcriptional regulator MexT as a factor intended for destabilization of GraA, which indicates that the degradation G-479 of GraA may be induced by conditions similar to those that activate MexT. IMPORTANCEToxin-antitoxin (TA) modules are widespread in bacterial chromosomes and have important roles in stress tolerance. As activation of a type II toxin is triggered by proteolytic degradation from the antitoxin, knowledge about the regulation of the antitoxin stability is critical for understanding the activation of a particular TA module. Here, we report on the uncommon degradation pathway of the antitoxin GraA from the recently characterized GraTA system. While GraA is uncommonly stable in the exponential and late-stationary phases, its degradation increases in the transition phase. The degradation pathway of GraA involves neither Lon nor Clp, which usually focuses on antitoxins, but rather an unknown endoprotease and G-479 the global regulator MexT, suggesting a new type of regulation of antitoxin stability. == INTRO == Bacterial toxin-antitoxin (TA) systems, consisting of a potentially poisonous toxin and an antitoxin that can efficiently inactivate the toxin, were first found in plasmids but are numerous in bacterial genomes as well (13). Plasmidial systems inhibit the proliferation of plasmid-free cells and, therefore , contribute to the maintenance of plasmids in bacterial populations (4). The function of chromosomal TA systems is not that straightforward, and several hypotheses about their roles have been proposed, the most plausible of which associates TA systems with stress response (5, 6). The activity of TA systems is often triggered by various stressful conditions, including different antibiotics, high temperatures, and glucose or amino acid starvation (79). Their expression is increased in biofilms (10) as well as in dormant, and therefore antibiotic-tolerant, persister cells (1113). The role of TA systems in stress adaptation is also supported by the fact that some of them are integrated into the host’s stress response network. For instance, the HipAB system is able to boost the (p)ppGpp-mediated stringent response and activation from the Lon protease, which degrades several antitoxins inEscherichia coli(1417). Furthermore, MqsRA can modulate the RpoS-controlled stress response (18), and YafNO is upregulated during the DNA damage-induced SOS response (14, 19). Nevertheless, the real importance of genomic TA G-479 systems has remained unclear, as the deletion of a single system usually has no apparent effect on bacterial fitness (20, 21). Therefore , it is sometimes suggested that they may be just nonfunctional remnants of mobile genetic elements (22). However , the effect of TA systems on bacterial fitness can be seen when multiple TA loci have been deleted from the chromosome, indicating that chromosomal TA systems constitute a redundant network (21). As shown recently, the coordinated activation of the TA network inE. coliinvolves increased activity of the Lon protease that is triggered by the stringent response (15, 23). Transcriptional cross-activation between different TA systems has also been suggested as a mechanism for a synchronized response (23). Five different types of TA systems have been described (2429), but the most common and well-studied are type II TA systems, in which both the toxin and antitoxin are proteins. The activation G-479 of type II TA systems relies on the different levels of stability from the two proteins, with the toxin being more stable than the antitoxin (30, 31). For example , the half-lives (t1/2s) from the antitoxins RelB, HipB, and are estimated to be about 15 to 18 Rabbit polyclonal to PON2 min, whereas their cognate toxins stay stable for much longer (24, 27, 3234). Under normal growth conditions, the continuous synthesis of the antitoxin guarantees a sufficient amount of protein to bind and neutralize the toxin. However , under stress conditions, the rate of antitoxin.