Bacterial dormancy is a valuable survival strategy upon challenging environmental conditions. Dormant cells tolerate the consequences of high stress levels and may re-populate the environment upon return to favorable conditions. Antibiotic-tolerant bacteria—termed persisters—regularly cause relapsing infections, increase the likelihood of antibiotic resistance, and, therefore, earn increasing attention. Their generation often depends on toxins from chromosomal toxin–antitoxin systems. Here, we review recent insights concerning RNA-based control of toxin synthesis, and discuss possible implications for persister generation.

Induction of growth stasis by bacterial toxins from chromosomal toxin-antitoxin systems is suspected to favor formation of multidrug-tolerant cells, named persisters. Recurrent infections are often attributed to resuscitation and regrowth of persisters upon termination of antibiotic therapy. Several lines of evidence point to oxidative stress as a crucial factor during the persister life cycle. Here, we demonstrate that the membrane-depolarizing type I toxins TisB, DinQ, and HokB have the potential to provoke reactive oxygen species formation in Escherichia coli. More detailed work with TisB revealed that mainly superoxide is formed, leading to activation of the SoxRS regulon. Deletion of the genes encoding the cytoplasmic superoxide dismutases SodA and SodB caused both a decline in TisB-dependent persisters and a delay in persister recovery upon termination of antibiotic treatment. We hypothesize that expression of depolarizing toxins during the persister formation process inflicts an oxidative challenge. The ability to counteract oxidative stress might determine whether cells will survive and how much time they need to recover from dormancy.

Bacterial populations produce phenotypic variants called persisters to survive harmful conditions. Persisters are highly tolerant to antibiotics and repopulate environments after the stress has vanished. In order to resume growth, persisters have to recover from the persistent state, but the processes behind recovery remain mostly elusive. Deciphering these processes is an essential step toward understanding the persister phenomenon in its entirety. High-throughput proteomics by mass spectrometry is a valuable tool to assess persister physiology during any stage of the persister life cycle, and is expected to considerably contribute to our understanding of the recovery process. In the present study, an Escherichia coli strain, that overproduces the membrane-depolarizing toxin TisB, was established as a model for persistence by the use of high-throughput proteomics. Labeling of TisB persisters with stable isotope-containing amino acids (pulsed-SILAC) revealed an active translational response to ampicillin, including several RpoS-dependent proteins. Subsequent investigation of the persister proteome during postantibiotic recovery by label-free quantitative proteomics identified proteins with importance to the recovery process. Among them, AhpF, a component of alkyl hydroperoxide reductase, and the outer membrane porin OmpF were found to affect the persistence time of TisB persisters. Assessing the role of AhpF and OmpF in TisB-independent persisters demonstrated that the importance of a particular protein for the recovery process strongly depends on the physiological condition of a persister cell. Our study provides important insights into persister physiology and the processes behind recovery of depolarized cells.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a "plug and play" plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the β-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user's needs.

Bacterial chromosomes harbor toxin-antitoxin (TA) systems, some of which are implicated in the formation of multidrug-tolerant persister cells. In Escherichia coli, toxin TisB from the tisB/istR-1 TA system depolarizes the inner membrane and causes ATP depletion, which presumably favors persister formation. Transcription of tisB is induced upon DNA damage due to activation of the SOS response by LexA degradation. Transcriptional activation of tisB is counteracted on the post-transcriptional level by structural features of tisB mRNA and RNA antitoxin IstR-1. Deletion of the regulatory RNA elements (mutant Δ1-41 ΔistR) uncouples TisB expression from LexA-dependent SOS induction and causes a ‘high persistence’ (hip) phenotype upon treatment with different antibiotics. Here, we demonstrate by the use of fluorescent reporters that TisB overexpression in mutant Δ1-41 ΔistR inhibits cellular processes, including the expression of SOS genes. The failure in SOS gene expression does not affect the hip phenotype upon treatment with the fluoroquinolone ciprofloxacin, likely because ATP depletion avoids strong DNA damage. By contrast, Δ1-41 ΔistR cells are highly susceptible to the DNA cross-linker mitomycin C, likely because the expression of SOS-dependent repair systems is impeded. Hence, the hip phenotype of the mutant is conditional and strongly depends on the DNA-damaging agent.