Toxin–antitoxin (TA) systems are small genetic elements found on plasmids or chromosomes of countless bacteria, archaea, and possibly also unicellula

Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas -secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of P seudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hem B mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.

Summary: Inducible gene expression based upon Tet repressor ( tet regulation) is a broadly applied tool in molecular genetics. In its original environment, Tet repressor (TetR) negatively controls tetracycline (tc) resistance in bacteria. In the presence of tc, TetR is induced and detaches from its cognate DNA sequence tetO , so that a tc antiporter protein is expressed. In this article, we provide a comprehensive overview about tet regulation in bacteria and illustrate the parameters of different regulatory architectures. While some of these set‐ups rely on natural tet ‐control regions like those found on transposon Tn 10 , highly efficient variations of this system have recently been adapted to different Gram‐negative and Gram‐positive bacteria. Novel tet ‐controllable artificial or hybrid promoters were employed for target gene expression. They are controlled by regulators expressed at different levels either in a constitutive or in an autoregulated manner. The resulting tet systems have been used for various purposes. We discuss integrative elements vested with tc‐sensitive promoters, as well as tet regulation in Gram‐negative and Gram‐positive bacteria for analytical purposes and for protein overproduction. Also the use of TetR as an in vivo biosensor for tetracyclines or as a regulatory device in synthetic biology constructs is outlined. Technical specifications underlying different regulatory set‐ups are highlighted, and finally recent developments concerning variations of TetR are presented, which may expand the use of prokaryotic tet systems in the future.

Bacterial persister cells are non- or slow-growing reversible phenotypic variants of the wild type, tolerant to bactericidal antibiotics. We analyzed here Staphylococcus aureus persister levels by monitoring colony-forming unit counts of planktonically grown cells treated with six different antimicrobials over time. The model laboratory strains HG001–HG003, SA113 and the small colony variant (SCV) strains hemB and menD were challenged by the compounds at different logs of minimal inhibitory concentration (MIC) in exponential or stationary growth phase. Antibiotic tolerance was usually elevated in SCV strains compared to normally growing cells and in stationary versus exponential phase cultures. Biphasic killing kinetics, typical for persister cell enrichment, were observed in both growth phases under different selective conditions. Treatment of exponential phase cultures of HG001–HG003 with 10-fold MIC of tobramycin resulted in the isolation of persisters which upon cultivation on plates formed either normal or phenotypically stable small colonies. Trajectories of different killing curves indicated physiological heterogeneity within persister subpopulations. Daptomycin added at 100-fold MIC to stationary phase SA113 cells rapidly isolated very robust persisters. Fractions of antibiotic-tolerant cells were observed with all S. aureus strains and mutants tested. Our results refute the hypothesis that S. aureus stationary phase cells are equivalent to persisters, as not all of these cells showed antibiotic tolerance. Isolation of S. aureus persisters of different robustness seems to depend on the kind and concentration of the antibiotic, as well as on the strain used.

Persister cells form a multi-drug tolerant subpopulation within an isogenic culture of bacteria that are genetically susceptible to antibiotics. Studies with different Gram negative and Gram positive bacteria have identified a large number of genes associated with the persister state. In contrast, the revelation of persister metabolism has only been addressed recently. We here summarize metabolic aspects of persisters, which includes an overview about the bifunctional role of selected carbohydrates as both triggers for the exit from the drug tolerant state and metabolites which persisters feed on. Also alarmones as indicators for starvation have been shown to influence persister levels via different signaling cascades involving the activation of toxin-antitoxin systems and other regulatory factors. Finally, recent data obtained by 13C-isotopologue profiling demonstrated an active amino acid anabolism in Staphylococcus aureus cultures challenged with high drug concentrations. Understanding the metabolism of persister cells poses challenges but also paves the way for the development of anti-persister compounds.

Recalcitrance of genetically susceptible bacteria to antibiotic killing is a hallmark of bacterial drug tolerance. This phenomenon is prevalent in biofilms, persisters, and also planktonic cells and is associated with chronic or relapsing infections with pathogens such as Staphylococcus aureus . Here we report the in vitro evolution of an S. aureus strain that exhibits a high degree of nonsusceptibility to daptomycin as a result of cyclic challenges with bactericidal concentrations of the drug. This phenotype was attributed to stationary growth phase-dependent drug tolerance and was clearly distinguished from resistance. The underlying genetic basis was revealed to be an adaptive point mutation in the putative inorganic phosphate (P _i ) transporter gene pitA . Drug tolerance caused by this allele, termed pitA6 , was abrogated when the upstream gene pitR was inactivated. Enhanced tolerance toward daptomycin, as well as the acyldepsipeptide antibiotic ADEP4 and various combinations of other drugs, was accompanied by elevated intracellular concentrations of P _i and polyphosphate, which may reversibly interfere with critical cellular functions. The evolved strain displayed increased rates of survival within human endothelial cells, demonstrating the correlation of intracellular persistence and drug tolerance. These findings will be useful for further investigations of S. aureus drug tolerance, toward the development of additional antipersister compounds and strategies.