Polar flagellar wrapping and lateral flagella jointly contribute to Shewanella putrefaciens environmental spreading.

Flagella enable bacteria to actively spread within the environment. A number of species possess two separate flagellar systems, where in most cases a primary polar flagellar system is supported by distinct secondary lateral flagella under appropriate conditions. Using functional fluorescence tagging on one of these species, Shewanella putrefaciens, as a model system, we explored how two different flagellar systems can exhibit efficient joint function. The S. putrefaciens secondary flagellar filaments are composed as a mixture of two highly homologous non-glycosylated flagellins, FlaA2 and FlaB2 . Both are solely sufficient to form a functional filament, however, full spreading motility through soft agar requires both flagellins. During swimming, lateral flagella emerge from the cell surface at angles between 30° and 50°, and only filaments located close to the cell pole may form a bundle. Upon a directional shift from forward to backward swimming initiated by the main polar flagellum, the secondary filaments flip over and thus support propulsion into either direction. Lateral flagella do not inhibit the wrapping of the polar flagellum around the cell body at high load. Accordingly, screw thread-like motility mediated by the primary flagellum and activity of lateral flagella cumulatively supports spreading through constricted environments such as polysaccharide matrices.

Bioinformatic prediction reveals posttranscriptional regulation of the chromosomal replication initiator gene dnaA by the attenuator sRNA rnTrpL in Escherichia coli.

DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.

3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives.

Protein aggregation is a consequence of the dormancy-inducing membrane toxin TisB in Escherichia coli.

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy, and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present a moderate expression system for toxin genes based on an artificial 5' untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin-antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA-sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpAB and spy. The importance of chaperone genes suggested that TisB-producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells. IMPORTANCE: Protein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which, in turn, promotes an extended state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

A Shift in Perspective: A Role for the Type I Toxin TisB as Persistence-Stabilizing Factor.

Bacterial persistence is a phenomenon that is founded by the existence of a subpopulation of multidrug-tolerant cells. These so-called persister cells endure otherwise lethal stress situations and enable restoration of bacterial populations upon return to favorable conditions. Persisters are especially notorious for their ability to survive antibiotic treatments without conventional resistance genes and to cause infection relapse. The persister state is typically correlated with reduction or inhibition of cellular activity. Early on, chromosomal toxin-antitoxin (TA) systems were suspected to induce the persister state in response to environmental stress. However, this idea has been challenged during the last years. Especially the involvement of toxins from type II TA systems in persister formation is put into question. For toxins from type I TA systems the debate has just started. Here, we would like to summarize recent knowledge gained for the type I TA system tisB/istR-1 from Escherichia coli. TisB is a small, membrane-targeting toxin, which disrupts the proton motive force (PMF), leading to membrane depolarization. Based on experimental data, we hypothesize that TisB primarily stabilizes the persister state through depolarization and further, secondary effects. We will present a simple model that will provide a framework for future directions.

Post-transcriptional deregulation of the tisB/istR-1 toxin-antitoxin system promotes SOS-independent persister formation in Escherichia coli.

Bacterial dormancy is a valuable strategy to endure unfavourable conditions. The term 'persister' has been coined for cells that tolerate antibiotic treatments due to reduced cellular activity. The type I toxin-antitoxin system tisB/istR-1 is linked to persistence in Escherichia coli, because toxin TisB depolarizes the inner membrane and causes ATP depletion. Transcription of tisB is induced upon activation of the SOS response by DNA-damaging drugs. However, translation is repressed both by a 5' structure within the tisB mRNA and by RNA antitoxin IstR-1. This tight regulation limits TisB production to SOS conditions. Deletion of both regulatory RNA elements produced a 'high persistence' mutant, which was previously assumed to depend on stochastic SOS induction and concomitant TisB production. Here, we demonstrate that the mutant generates a subpopulation of growth-retarded cells during late stationary phase, likely due to SOS-independent TisB accumulation. Cell sorting experiments revealed that the stationary phase-derived subpopulation contains most of the persister cells. Collectively our data show that deletion of the regulatory RNA elements uncouples the persister formation process from the intended stress situation and enables the formation of TisB-dependent persisters in an SOS-independent manner.

Elevated Expression of Toxin TisB Protects Persister Cells against Ciprofloxacin but Enhances Susceptibility to Mitomycin C.

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.

Type I toxin-dependent generation of superoxide affects the persister life cycle of Escherichia coli.

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.

High-Throughput Proteomics Identifies Proteins With Importance to Postantibiotic Recovery in Depolarized Persister Cells.

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.