Formation of MgF3−-dependent complexes between an AAA+ ATPase and σ54

AbstractThe widely distributed bacterial σ54-dependent transcription regulates pathogenicity and numerous adaptive responses in diverse bacteria. Formation of the σ54-dependent open promoter complex is a multi-step process driven by AAA+ ATPases. Non-hydrolysable nucleotide analogues are particularly suitable for studying such complexity by capturing various intermediate states along the energy coupling pathway. Here we report a novel ATP analogue, ADP–MgF3−, which traps an AAA+ ATPase with its target σ54. The MgF3−-dependent complex is highly homogeneous and functional assays suggest it may represent an early transcription intermediate state valuable for structural studies

Tools and Principles for Microbial Gene Circuit Engineering

AbstractSynthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics

Creating the Dance and Dancing Creatively: Exploring the Liminal Space of Choreography for Emergence

In this paper, three dance scholars explore the tensions and bliss inherent in curriculum delivery through dance integration. It meets the call for a curriculum attuned to provoking encounters (Pinar & Grumet, 2015) through philosophical narration that interweaves experiences as dancers, dance educators, dance scholars and dance integrators. Personal vignettes unveil the sense-making of creative artists tasked with the duty to “deliver” curriculum, and as arts integration specialists tasked with the duty to share knowledge, with teachers, for designing learning through dance. The authors liken the inherent tensions to those of a tight rope walker balancing between forces pulled in opposite directions. They share their own encounters of pedagogical balance and counterbalance, of choreography and emergence, and of leading and following, as each relates to learning design. They also explore the duality of meeting curricular ends and unfolding endless possibilities (Aoki, 2005; Roth, 2014). Together, the authors find that their collective experience leads to three charges for curricular reform: 1) embed dance integration in teacher preparation; 2) infuse dance integration in K-12 curriculum; and 3) provide time for pedagogical experimentation through dance-based inquiry

Schlagseparierung als Ansatz zur Prävention von Möhrenfliegenschäden

Die Möhrenfliege (Psila rosae) gehört zu den wichtigsten tierischen Schaderregern im Ökologischen Möhrenanbau. In dreijährigen Erhebungen auf Praxisbetrieben wurden verschiedene beteiligte Risikofakoren für den Befall mit geo-referenzierten Daten erfasst und im Hinblick auf Verbesserungsmöglichkeiten zur Schadensprävention analysiert. Erste Ergebnisse zu räumlichen Beziehungen von Schlaggrößen und der jew. Entfernung zu Vorjahresflächen auf das Befallsergebnis ergaben, dass die Einhaltung von Distanzen um 1 km einen nutzbaren, anbauplanerischen Regulierungsbaustein darstellen kann. Dieser empirische Befund sollte aber nicht als starre Ausbreitungsstrecke aufgefasst werden, weil zusätzlich gezeigt werden konnte, dass dem Vorjahr nächstgelegene Möhrenfelder offenbar das Befallsrisiko für weitere, entfernter liegende Möhrenschläge senkend beeinflussten. Fehlen andererseits engbenachbarte Schläge, ist im Umkehrschluß damit zu rechnen, dass entsprechend einer grob geschätzten täglichen Ausbreitungskapazität auch Distanzen > 1 km überwunden werden können

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. IMPORTANCE All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response

Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in Escherichia coli

All cell types must maintain the integrity of their membranes. The conserved bacterial membrane-associated protein PspA is a major effector acting upon extracytoplasmic stress and is implicated in protection of the inner membrane of pathogens, formation of biofilms and multi-drug-resistant persister cells. PspA and its homologues in Gram-positive bacteria and archaea protect the cell envelope whilst also supporting thylakoid biogenesis in cyanobacteria and higher plants. In enterobacteria, PspA is a dual function protein negatively regulating the Psp system in the absence of stress and acting as an effector of membrane integrity upon stress. We show that in Escherichia coli the low-order oligomeric PspA regulatory complex associates with cardiolipinrich, curved polar inner membrane regions. There, cardiolipin and the flotillin 1 homologue YqiK support the PspBC sensors in transducing a membrane stress signal to the PspA-PspF inhibitory complex. After stress perception, PspA high-order oligomeric effector complexes initially assemble in polar membrane regions. Subsequently, the discrete spatial distribution and dynamics of PspA effector(s) in lateral membrane regions depend on the actin homologue MreB and the peptidoglycan machinery protein RodZ. The consequences of loss of cytoplasmic membrane anionic lipids, MreB, RodZ and/or YqiK suggest that the mode of action of the PspA effector is closely associated with cell envelope organization

Single-step method for beta-galactosidase assays in Escherichia coli using a 96-well microplate reader

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ss-D-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene). Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%

Vermeidung und Reduktion von Möhrenfliegenschäden im Ökolandbau

The carrot fly Psila rosae is a major pest in organic carrots. Damage strongly depends on local factors and is often found when cropping intensity is high. However, limited field site availability may require growing carrots close to crop sites of preceding years. Here, results of the first two years show carrots may be protected from infestation using trap crops (also carrots) to bind and actively reduce the local P. rosae population. To protect the main carrot field, pair wise arrangements of carrot strips were sown 1) directly at the previous year carrot field and 2) close to the main field to protect the present carrots. During the first generation a mean sum of 36 and 62 flies/ trap in 2007 and 2008 respectively were monitored with yellow sticky traps (Rebell orange®) leading to high infestation pressure. Monitoring results show the flies are effectively attracted and bound by strip 1) but still negotiate distances of 130 and 180 metres between previous and present carrot fields. Carrot strip 1) was to be removed in time to prevent the second generation of P. rosae to develop. Based on the simulation model SWAT monitoring data and damage assessment was used for timing concerns. Hence, critical issues about removing the trap crops are discussed. In 2008 eclector traps were established onto the sites of removed trap crops to further verify the effectiveness of trap crop handling. First results positively support applied techniques. All trials are to be repeated during field season 2009