Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholera and are stabilized by a double signaling domain receptor DosM

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae’s cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array

Climate and site management as driving factors for the atmospheric greenhouse gas exchange of a restored wetland

The atmospheric greenhouse gas (GHG) budget of a restored wetland in western Denmark was established for the years 2009–2011 from eddy covariance measurements of carbon dioxide (CO<sub>2</sub>) and methane (CH<sub>4</sub>) fluxes. The water table in the wetland, which was restored in 2002, was unregulated, and the vegetation height was limited through occasional grazing by cattle and grass cutting. The annual net CO<sub>2</sub> uptake varied between 195 and 983 g m<sup>−2</sup> and the annual net CH<sub>4</sub> release varied between 11 and 17 g m<sup>−2</sup>. In all three years the wetland was a carbon sink and removed between 42 and 259 g C m<sup>−2</sup> from the atmosphere. However, in terms of the full annual GHG budget (assuming that 1 g CH<sub>4</sub> is equivalent to 25 g CO<sub>2</sub> with respect to the greenhouse effect over a time horizon of 100 years) the wetland was a sink in 2009, a source in 2010 and neutral in 2011. Complementary observations of meteorological factors and management activities were used to explain the large inter-annual variations in the full atmospheric GHG budget of the wetland. The largest impact on the annual GHG fluxes, eventually defining their sign, came from site management through changes in grazing duration and animal stocking density. These changes accounted for half of the observed variability in the CO<sub>2</sub> fluxes and about two thirds of the variability in CH<sub>4</sub> fluxes. An unusually long period of snow cover in 2010 had the second largest effect on the annual CO<sub>2</sub> flux, whose interannual variability was larger than that of the CH<sub>4</sub> flux. Since integrated CO<sub>2</sub> and CH<sub>4</sub> flux data from restored wetlands are still very rare, it is concluded that more long-term flux measurements are needed to quantify the effects of ecosystem disturbance, in terms of management activities and exceptional weather patterns, on the atmospheric GHG budget more accurately

Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost

Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

ParAWalker ATPases form part of the machinery that promotes better-thanrandom segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts.IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATPdependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus,the NTD may modulate the polymerisation interface, facilitating polymerisation/ depolymerisation and DNA binding, to promote the cycle that drives partitioning

Repurposing a chemosensory macromolecular machine

Microbial Biotechnolog

Three dimensional three component whole heart cardiovascular magnetic resonance velocity mapping: comparison of flow measurements from 3D and 2D acquisitions

<p>Abstract</p> <p>Background</p> <p>Two-dimensional, unidirectionally encoded, cardiovascular magnetic resonance (CMR) velocity mapping is an established technique for the quantification of blood flow in large vessels. However, it requires an operator to correctly align the planes of acquisition. If all three directional components of velocity are measured for each voxel of a 3D volume through the phases of the cardiac cycle, blood flow through any chosen plane can potentially be calculated retrospectively. The initial acquisition is then more time consuming but relatively operator independent.</p> <p>Aims</p> <p>To compare the curves and volumes of flow derived from conventional 2D and comprehensive 3D flow acquisitions in a steady state flow model, and in vivo through planes transecting the ascending aorta and pulmonary trunk in 10 healthy volunteers.</p> <p>Methods</p> <p>Using a 1.5 T Phillips Intera CMR system, 3D acquisitions used an anisotropic 3D segmented k-space phase contrast gradient echo sequence with a short EPI readout, with prospective ECG and diaphragm navigator gating. The 2D acquisitions used segmented k-space phase contrast with prospective ECG and diaphragm navigator gating. Quantitative flow analyses were performed retrospectively with dedicated software for both the in vivo and in vitro acquisitions.</p> <p>Results</p> <p>Analysis of in vitro data found the 3D technique to have overestimated the continuous flow rate by approximately 5% across the entire applied flow range. In vivo, the 2D and the 3D techniques yielded similar volumetric flow curves and measurements. Aortic flow: (mean ± SD), 2D = 89.5 ± 13.5 ml & 3D = 92.7 ± 17.5 ml. Pulmonary flow: 2D = 98.8 ± 18.4 ml & 3D = 94.9 ± 19.0 ml). Each in vivo 3D acquisition took about 8 minutes or more.</p> <p>Conclusion</p> <p>Flow measurements derived from the 3D and 2D acquisitions were comparable. Although time consuming, comprehensive 3D velocity acquisition could be relatively operator independent, and could potentially yield information on flow through several retrospectively chosen planes, for example in patients with congenital or valvular heart disease.</p

Repurposing a chemosensory macromolecular machine

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost

The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates

Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates. Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation. Results: Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins.Apoptosis and ER stress were inducible in the cells and caspase 3/7 activity was elevated in response to cytokines, but not affected by the saturated fatty acid palmitate.By screening of various proteins and peptides, we found Bombesin (BB) receptor agonists and Pituitary Adenylate Cyclase-Activating Polypeptides (PACAP) to significantly induce insulin secretion and the proteins SerpinA6, STC1, and APOH to significantly stimulate proliferation.ER stress was readily induced by Tunicamycin and resulted in a reduction of insulin mRNA. Somatostatin (SST) was found to be expressed by 1% of the cells and manipulation of the SST receptors was found to significantly affect insulin secretion. Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates. Keywords: EndoC-βH1, Pseudoislets, Glucose stimulated insulin secretion, Somatostatin signaling, Proliferatio

Filament Depolymerization Can Explain Chromosome Pulling during Bacterial Mitosis

Chromosome segregation is fundamental to all cells, but the force-generating mechanisms underlying chromosome translocation in bacteria remain mysterious. Caulobacter crescentus utilizes a depolymerization-driven process in which a ParA protein structure elongates from the new cell pole, binds to a ParB-decorated chromosome, and then retracts via disassembly, pulling the chromosome across the cell. This poses the question of how a depolymerizing structure can robustly pull the chromosome that disassembles it. We perform Brownian dynamics simulations with a simple, physically consistent model of the ParABS system. The simulations suggest that the mechanism of translocation is “self-diffusiophoretic”: by disassembling ParA, ParB generates a ParA concentration gradient so that the ParA concentration is higher in front of the chromosome than behind it. Since the chromosome is attracted to ParA via ParB, it moves up the ParA gradient and across the cell. We find that translocation is most robust when ParB binds side-on to ParA filaments. In this case, robust translocation occurs over a wide parameter range and is controlled by a single dimensionless quantity: the product of the rate of ParA disassembly and a characteristic relaxation time of the chromosome. This time scale measures the time it takes for the chromosome to recover its average shape after it is has been pulled. Our results suggest explanations for observed phenomena such as segregation failure, filament-length-dependent translocation velocity, and chromosomal compaction