Toxin-mediated depletion of NAD and NADP drives persister formation in a human pathogen.

Toxin-antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR-NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa, generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides.

A deterministic, c-di-GMP-dependent program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis.

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum.

A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution.

Monitoring changes of signaling molecules and metabolites with high temporal resolution is key to understanding dynamic biological systems. Here, we use directed evolution to develop a genetically encoded ratiometric biosensor for c-di-GMP, a ubiquitous bacterial second messenger regulating important biological processes like motility, surface attachment, virulence and persistence. The resulting biosensor, cdGreen2, faithfully tracks c-di-GMP in single cells and with high temporal resolution over extended imaging times, making it possible to resolve regulatory networks driving bimodal developmental programs in different bacterial model organisms.

Complete genome sequence of phage Knedl.

Bacteriophage Knedl is the first reported phage that targets the Psl exopolysaccharide as receptor. Here, we report the genome of Knedl, demonstrating that it belongs to the genus of the subfamily. Future studies on the infection mechanism of Knedl could inform phage-based approaches to eradicate biofilms.

Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication.

Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates ("hibernation").

A genetic switch controls Pseudomonas aeruginosa surface colonization.

Efficient colonization of mucosal surfaces is essential for opportunistic pathogens like Pseudomonas aeruginosa, but how bacteria collectively and individually adapt to optimize adherence, virulence and dispersal is largely unclear. Here we identified a stochastic genetic switch, hecR-hecE, which is expressed bimodally and generates functionally distinct bacterial subpopulations to balance P. aeruginosa growth and dispersal on surfaces.

A Synthetic Cumate-Inducible Promoter for Graded and Homogenous Gene Expression in Pseudomonas aeruginosa.

Inducible gene expression systems are powerful genetic tools to study bacterial physiology, probing essential and toxic gene functions, gene dosage effects, and overexpression phenotypes. For the opportunistic human pathogen Pseudomonas aeruginosa, dedicated inducible gene expression systems are scarce. In the current study, we developed a minimal synthetic 4-isopropylbenzoic acid (cumate)-inducible promoter, called P, that is tunable over several orders of magnitude.

Mutant structure of metabolic switch protein in complex with monomeric c-di-GMP reveals a potential mechanism of protein-mediated ligand dimerization.

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of c-di-GMP inducing a conformational change that involves loop 7 of the protein that leads to downstream signaling.

The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB.

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia.

Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation.

Surface attachment of bacteria is the first step of biofilm formation and is often mediated and coordinated by the extracellular appendages, flagellum and pili. The model organism Caulobacter crescentus undergoes an asymmetric division cycle, giving rise to a motile "swarmer cell" and a sessile "stalked cell", which is attached to the surface. In the highly polarized predivisional cell, pili and flagellum, which are assembled at the pole opposite the stalk, are both activated before and during the process of cell separation.

Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds.

Combining CRISPRi and metabolomics for functional annotation of compound libraries.

Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli.

A New Sugar for an Old Phage: a c-di-GMP-Dependent Polysaccharide Pathway Sensitizes for Bacteriophage Infection.

Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli.

The Use of Experimental Evolution to Study the Response of Pseudomonas aeruginosa to Single or Double Antibiotic Treatment.

The widespread use of antibiotics promotes the evolution and dissemination of drug resistance and tolerance. Both mechanisms promote survival during antibiotic exposure and their role and development can be studied in vitro with different assays to document the gradual adaptation through the selective enrichment of resistant or tolerant mutant variants. Here, we describe the use of experimental evolution in combination with time-resolved genome analysis as a powerful tool to study the interaction of antibiotic tolerance and resistance in the human pathogen Pseudomonas aeruginosa .

Defining Proteomic Signatures to Predict Multidrug Persistence in Pseudomonas aeruginosa.

Bacterial persisters are difficult to eradicate because of their ability to survive prolonged exposure to a range of different antibiotics. Because they often represent small subpopulations of otherwise drug-sensitive bacterial populations, studying their physiological state and antibiotic stress response remains challenging. Sorting and enrichment procedures of persister fractions introduce experimental biases limiting the significance of follow-up molecular analyses.

Pareto optimality between growth-rate and lag-time couples metabolic noise to phenotypic heterogeneity in Escherichia coli.

Despite mounting evidence that in clonal bacterial populations, phenotypic variability originates from stochasticity in gene expression, little is known about noise-shaping evolutionary forces and how expression noise translates to phenotypic differences. Here we developed a high-throughput assay that uses a redox-sensitive dye to couple growth of thousands of bacterial colonies to their respiratory activity and show that in Escherichia coli, noisy regulation of lower glycolysis and citric acid cycle is responsible for large variations in respiratory metabolism. We found that these variations are Pareto optimal to maximization of growth rate and minimization of lag time, two objectives competing between fermentative and respiratory metabolism.

Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections.

The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used evolution and analyzed the inpatient microevolution of , an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance , and we present evidence that similar processes shape antibiotic exposure in human patients.

Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus.

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA.

Surface Sensing and Adaptation in Bacteria.

Bacteria thrive both in liquids and attached to surfaces. The concentration of bacteria on surfaces is generally much higher than in the surrounding environment, offering bacteria ample opportunity for mutualistic, symbiotic, and pathogenic interactions. To efficiently populate surfaces, they have evolved mechanisms to sense mechanical or chemical cues upon contact with solid substrata.

Intercepting second-messenger signaling by rationally designed peptides sequestering c-di-GMP.

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger.

Regulation of Bacterial Cell Cycle Progression by Redundant Phosphatases.

In the model organism , a network of two-component systems involving the response regulators CtrA, DivK, and PleD coordinates cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G-S transition, phosphorylated DivK (DivK∼P) and PleD (PleD∼P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation.