The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5\'-GC-3\' sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity. We show that MqsR preferentially binds substrates that contain purines in the -2 and -1 position relative to the MqsR consensus cleavage sequence and that two residues of MqsR, Tyr81, and Lys56 are strictly required for mRNA cleavage. We also show that MqsA inhibits MqsR activity by sterically blocking mRNA substrates from binding while leaving the active site fully accessible to mononucleotides. Together, these data identify the residues of MqsR that mediate RNA cleavage and reveal a novel mechanism that regulates MqsR substrate specificity.

Toxin-antitoxin (TA) systems are broadly distributed modules whose biological roles remain mostly unknown. The mqsRA system is a noncanonical TA system in which the toxin and antitoxins genes are organized in operon but with the particularity that the toxin gene precedes that of the antitoxin. This system was shown to regulate global processes such as resistance to bile salts, motility, and biofilm formation. In addition, the MqsA antitoxin was shown to be a master regulator that represses the transcription of the csgD, cspD, and rpoS global regulator genes, thereby displaying a pleiotropic regulatory role. Here, we identified two promoters located in the toxin sequence driving the constitutive expression of mqsA, allowing thereby excess production of the MqsA antitoxin compared to the MqsR toxin. Our results show that both antitoxin-specific and operon promoters are not regulated by stresses such as amino acid starvation, oxidative shock, or bile salts. Moreover, we show that the MqsA antitoxin is not a global regulator as suggested, since the expression of csgD, cspD and rpoS is similar in wild-type and ΔmqsRA mutant strains. Moreover, these two strains behave similarly in terms of biofilm formation and sensitivity to oxidative stress or bile salts.IMPORTANCE There is growing controversy regarding the role of chromosomal toxin-antitoxin systems in bacterial physiology. mqsRA is a peculiar toxin-antitoxin system, as the gene encoding the toxin precedes that of the antitoxin. This system was previously shown to play a role in stress response and biofilm formation. In this work, we identified two promoters specifically driving the constitutive expression of the antitoxin, thereby decoupling the expression of antitoxin from the toxin. We also showed that mqsRA contributes neither to the regulation of biofilm formation nor to the sensitivity to oxidative stress and bile salts. Finally, we were unable to confirm that the MqsA antitoxin is a global regulator. Altogether, our data are ruling out the involvement of the mqsRA system in Escherichia coli regulatory networks.

MazEF and MqsRA are toxin-antitoxin systems, where the toxins MazF and MqsR sequence-specifically cleave single-stranded RNA, thereby shutting down protein synthesis and cell growth. However, it has been proposed that MazF functions in a highly specific pathway, where it truncates the 5\' ends of a set of E. coli transcripts (the MazF regulon), which are then translated under stress conditions by specialized ribosomes. We mapped the cleavage sites of MazF and MqsR throughout the E. coli transcriptome. Our results show that both toxins cleave mRNA independently of the recognition site position and MazF freely cleaves transcripts of the proposed MazF regulon within coding sequences. Proteome analysis indicated that MazF expression leads to overall inhibition of protein synthesis and the putative MazF regulon proteins are not selectively synthesized in response to the toxin. Our results support a simpler role for endoribonuclease TA systems as indifferent destroyers of unstructured RNA.

The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of luxR and aphA from a library of 285 TFs encoded by the fish pathogen Vibrio alginolyticus A total of 7 TFs were identified to bind to the promoters of both luxR and aphA Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of vqsA expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of luxR and aphA as well as the binding sites of LuxR, AphA, and VqsA in the vqsA gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and in vivo virulence of V. alginolyticus Collectively, our data showed that VqsA is an important QS regulator in V. alginolyticusIMPORTANCE Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of vqsA disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5\'- and 3\'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

Toxin/antitoxin (TA) systems are the means by which bacterial cells become persistent; that is, those cells that are tolerant to multiple environmental stresses such as antibiotics by becoming metabolically dormant. These persister cells are responsible for recalcitrant infections. Once toxins are activated by the inactivation of antitoxins (e.g., stress-triggered Lon degradation of the antitoxin), many toxins reduce metabolism by inhibiting translation (e.g., cleaving mRNA, reducing ATP). The MqsR/MqsA TA system of Escherichia coli cleaves mRNA to help the cell withstand oxidative and bile acid stress. Here, we investigated the role of secondary structure and 5\' mRNA processing on MqsR degradation of mRNA and found that MqsR cleaves only single-stranded RNA at 5\'-GCU sites and that MqsR is equally active against RNA with 5\'-triphosphate, 5\'-monophosphate, and 5\'-hydroxyl groups.

Bacterial persisters represent a small number of slow-growing antibiotic-tolerant cells among populations of rapidly growing cells, and are the main cause of frequent recurrent infections. MqsR-MqsA, the toxin-antitoxin (TA) pair, is the most frequently induced TA system associated with antibiotic persistence in Escherichia coli. In this study, we show that the cyclic AMP receptor protein (CRP) indirectly upregulates mqsRA transcription. We also show that CRP plays an important role in antibiotic persistence, which seems to be partially mediated through MqsRA. Overall, this study highlights the role of CRP as an important regulator of antibiotic persistence in E. coli.