Social Evolution and Regulatory Architecture of Pseudomonas aeruginosa Quorum Sensing

Cell-cell communication in bacteria is understood to facilitate the coordination of population-wide cooperative behavior in the form of concerted gene expression. The opportunistic pathogen Pseudomonas aeruginosa uses such a communication mechanism to regulate a large group of genes important to virulence strategies in this bacterium. This general mechanism of communication is termed quorum sensing (QS) and restricts activation of target genes to high cell density when cooperation is beneficial. QS in P. aeruginosa, like many Gram-negative Proteobacteria, is mediated through the synthesis of diffusible N-acyl-homoserine lactone (AHL) signals by LuxI-type synthases, and recognition by LuxR-type receptors that function as transcriptional regulators. P. aeruginosa harbors two complete AHL QS synthase receptor pairs termed LasI R and RhlI R. Here we use P. aeruginosa QS as a model system to investigate mechanisms that help maintain cooperative, QS-dependent secretion in the face of non-cooperating cheater mutants, and that define the cell density threshold that triggers the activation of QS target gene expression.We begin with analysis of an in vitro evolution system in which P. aeruginosa must express QS-controlled extracellular proteases in order to grow. In this system, QS-deficient cheater mutants evolve over time. They take advantage of protease production by the QS-proficient wild-type. Curiously, QS-deficient cheaters onlyreach a frequency of about 25% during the duration of the experiment. They do not enrich to levels that would cause a collapse of the population, generally referred to as a “tragedy of the commons”. Genomic sequence analysis revealed a previously unknown mutation in this system in the transcriptional regulator PsdR. Mutations in the gene coding for PsdR derepress growth rate limiting nutrient uptake and metabolism, a non-social adaptation. Combining mutational analysis with phenotypic assays and measurements of relative fitness, we show that rapid fixation of PsdR mutation in evolving populations serves to preserve cooperation and prevent a tragedy of the commons.Next, we focus on the mechanisms that determine the threshold of QS induction in P. aeruginosa. We constructed a set of isogenic mutant strains deficient in one, two, or three anti-activator proteins that serve to delay QS activation: QteE, QscR, and QslA. While these anti-activator proteins are understood to bind LasR and RhlR QS receptors, it is yet unclear why multiple anti-activators are needed, and how they work in concert to achieve the QS threshold. Using phenotypic assays, QS gene activation kinetics, and transcriptomic profiling, we found additive effects in the deletion of multiple anti-activator genes with largely overlapping sets of anti-activator-affected genes. Progressive deletion of anti-activators advances the induction threshold and increases expression levels. Our results suggest some anti-activators may even co-associate with R-proteins in exerting their effect.Together, these studies contribute new mechanistic understanding of how P. aeruginosa uses QS to coordinate cooperative behaviors to specific conditions, and how this cooperative communication system may be safeguarded against social exploitation.Keywords: social interactions, evolution, gene regulation, social evolution, quorum sensin

Design and development of an automatic tool changer for an articulated robot arm

In the creative industries, the length of time between the ideation stage and the making of physical objects is decreasing due to the use of CAD/CAM systems and adicitive manufacturing. Natural anisotropic materials, such as solid wood can also be transformed using CAD/CAM systems, but only with subtractive processes such as machining with CNC routers. Whilst some 3 axis CNC routing machines are affordable to buy and widely available, more flexible 5 axis routing machines still present themselves as a too big investment for small companies. Small refurbished articulated robots can be a cheaper alternative but they require a light end-effector. This paper presents a new lightweight tool changer that converts a small 3kg payload 6 DOF robot into a robot apprentice able to machine wood and similar soft materials

RhIR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy and activates the transcription of dozens of genes, including that encoding the QS regulator RhIR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rh1R are rare. We have recently shown that in CF isolates, the QS circuitry is often rewired such that RhIR acts in a LasR-independent manner. To begin understanding how QS activity differs in this rewired background, we characterized QS activation and RhIR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhIR-active chronic infection isolate. In this isolate, RhIR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhIR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations as PAO1, and in E90, RhIR plays a significant role in mediating cytotoxicity in a three-dimensional lung epithelium cell model. These data illuminate a rewired LasR-independent RhIR regulon in chronic infection isolates and suggest further investigation of RhIR as a possible target for therapeutic development in chronic infections. IMPORTANCE Pseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Many isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhIR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhIR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhIR QS and highlight the centrality of RhIR in chronic P. aeruginosa infections

Role of Two Cell Wall Amidases in Septal Junction and Nanopore Formation in the Multicellular Cyanobacterium Anabaena sp PCC 7120

Work in the Tübingen lab was supported by Deutsche Forschungsgemeinschaft (SFB766). RP was supported by Deutsche Forschungsgemeinschaft (GRK1708). Work in Seville was supported from Plan Nacional de Investigación, Spain, co-financed by the European Regional Development Fund (grant no. BFU2014-56757-P)

Bacterial competition and quorum-sensing signalling shapes the eco-evolutionary outcomes of model in vitro phage therapy

The rapid rise of antibiotic resistance has renewed interest in phage therapy - the use of bacteria-specific viruses (phages) to treat bacterial infections. Even though phages are often pathogen-specific, little is known about the efficiency and eco-evolutionary outcomes of phage therapy in polymicrobial infections. We studied this experimentally by exposing both quorum-sensing (QS) signalling PAO1 and QS-deficient lasR Pseudomonas aeruginosa genotypes (differing in their ability to signal intraspecifically) to lytic PT7 phage in the presence and absence of two bacterial competitors: Staphylococcus aureus and Stenotrophomonas maltophilia-two bacteria commonly associated with P. aeruginosa in polymicrobial cystic fibrosis lung infections. Both the P. aeruginosa genotype and the presence of competitors had profound effects on bacteria and phage densities and bacterial resistance evolution. In general, competition reduced the P. aeruginosa frequencies leading to a lower rate of resistance evolution. This effect was clearer with QS signalling PAO1 strain due to lower bacteria and phage densities and relatively larger pleiotropic growth cost imposed by both phages and competitors. Unexpectedly, phage selection decreased the total bacterial densities in the QS-deficient lasR pathogen communities, while an increase was observed in the QS signalling PAO1 pathogen communities. Together these results suggest that bacterial competition can shape the eco-evolutionary outcomes of phage therapy

Industrial Safety And Health Management

xiv. 464 hal. 24 c

An analysis of dramatic effectiveness in the Passion sequence of the York Corpus Christi cycle

Critical commentary on medieval drama has largely ignored both the inherent dramatic value of the English Corpus Christi plays as a product of their time and the influence of ritual upon these cyclic dramas. The idea that it is possible to evaluate the cycle plays on the basis of their intrinsic dramatic worth and to work creatively with dramatic structure and still to remain aware of the religious overtones within this structure, has led to the present study. In this study, I propose to analyze the twelve plays of the Passion sequence in the York Corpus Christi cycle as important examples of medieval religious cyclical drama which owe much of their effectiveness to emotional and structural parallels with religious ritual, specifically, with the medieval Mass and Holy Week ceremonies. At the same time, I will analyze the dramatic effectiveness of the York Passion sequence, not only in terms of dramatic technique as it relates to the development of action, conflict, and character, but also in terms of position in a cyclic structure which is both representational and ritualistic, historical and universal. [...]English, Department o

The things which are not seen are eternal

World Gospel Mission chapel.https://place.asburyseminary.edu/ecommonsatschapelservices/7000/thumbnail.jp