121 research outputs found
Bifurcations in the wake of a thick circular disk
Using DNS, we investigate the dynamics in the wake of a circular disk of aspect ratio χ = d/w = 3(where d is the diameter and w the thickness) embedded in a uniform flow of magnitude U0 perpendicular to its symmetry axis. As the Reynolds number Re = U0d/ν is increased, the flow is shown to experience an original series of bifurcations leading to chaos. The range Re ∈ [150, 218] is analysed in detail. In this range, five different non-axisymmetric regimes are successively encountered, including states similar to those previously identified in the flow past a sphere or an infinitely thin disk, as well as a new regime characterised by the presence of two distinct frequencies. A theoretical model based on the theory of mode interaction with symmetries, previously introduced to explain the bifurcations in the flow past a sphere or an infinitely thin disk (Fabre et al. in Phys Fluids 20:051702, 2008), is shown to explain correctly all these results. Higher values of the Reynolds number, up to 270, are also considered. Results indicate that the flow encounters at least four additional bifurcations before reaching a chaotic state
Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation
Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome
Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-di-AMP
Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg2+, Mn2+ or Co2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis
Cellular Levels and Binding of c-di-GMP Control Subcellular Localization and Activity of the Vibrio cholerae Transcriptional Regulator VpsT
The second messenger, cyclic diguanylate (c-di-GMP), regulates diverse cellular processes in bacteria. C-di-GMP is produced by diguanylate cyclases (DGCs), degraded by phosphodiesterases (PDEs), and receptors couple c-di-GMP production to cellular responses. In many bacteria, including Vibrio cholerae, multiple DGCs and PDEs contribute to c-di-GMP signaling, and it is currently unclear whether the compartmentalization of c-di-GMP signaling components is required to mediate c-di-GMP signal transduction. In this study we show that the transcriptional regulator, VpsT, requires c-di-GMP binding for subcellular localization and activity. Only the additive deletion of five DGCs markedly decreases the localization of VpsT, while single deletions of each DGC do not impact VpsT localization. Moreover, mutations in residues required for c-di-GMP binding, c-di-GMP-stabilized dimerization and DNA binding of VpsT abrogate wild type localization and activity. VpsT does not co-localize or interact with DGCs suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model in which c-di-GMP acts at a distance. Furthermore, VpsT localization in a heterologous host, Escherichia coli, requires a catalytically active DGC and is enhanced by the presence of VpsT-target sequences. Our data show that c-di-GMP signaling can be executed through an additive cellular c-di-GMP level from multiple DGCs affecting the localization and activity of a c-di-GMP receptor and furthers our understanding of the mechanisms of second messenger signaling
O circuito social da fotografia em Porto Alegre (1922 e 1935)
Photography has been increasingly investigated by Brazilian historians, thus bringing
about diverse methodological approaches for the analysis of such visual images. A fundamental
component in the studies of this nature is research on photographys social circuit. Within this
scope, there is a body of information that is related to the production, circulation and use of
photography understood as a visual, creative and disseminating image for certain social
imaginaries, and as an artifact that had a place in peoples lives in different contexts. In this
article, the author investigates photographys social circuit in the city of Porto Alegre in the
1920s and 1930s, in an attempt to find clues of the presence of this modern invention in the
life of Porto Alegre citizens and of the urban vistas that it disseminated
Cyclic di-GMP is Essential for the Survival of the Lyme Disease Spirochete in Ticks
Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host
A Global Metabolic Shift Is Linked to Salmonella Multicellular Development
Bacteria can elaborate complex patterns of development that are dictated by temporally ordered patterns of gene expression, typically under the control of a master regulatory pathway. For some processes, such as biofilm development, regulators that initiate the process have been identified but subsequent phenotypic changes such as stress tolerance do not seem to be under the control of these same regulators. A hallmark feature of biofilms is growth within a self-produced extracellular matrix. In this study we used metabolomics to compare Salmonella cells in rdar colony biofilms to isogenic csgD deletion mutants that do not produce an extracellular matrix. The two populations show distinct metabolite profiles. Even though CsgD controls only extracellular matrix production, metabolite signatures associated with cellular adaptations associated with stress tolerances were present in the wild type but not the mutant cells. To further explore these differences we examine the temporal gene expression of genes implicated in biofilm development and stress adaptations. In wild type cells, genes involved in a metabolic shift to gluconeogenesis and various stress-resistance pathways exhibited an ordered expression profile timed with multicellular development even though they are not CsgD regulated. In csgD mutant cells, the ordered expression was lost. We conclude that the induction of these pathways results from production of, and growth within, a self produced matrix rather than elaboration of a defined genetic program. These results predict that common physiological properties of biofilms are induced independently of regulatory pathways that initiate biofilm formation
Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer
The reversible Y–O•/Y–OH redox properties of the α[subscript 3]Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α[subscript 3]Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0 ± 0.1) of the unnatural residue. Square-wave voltammetry on α[subscript 3](3,5)F[subscript 2]Y provides an E°′(Y–O•/Y–OH) of 1026 ± 4 mV versus the normal hydrogen electrode (pH 5.70 ± 0.02) and shows that the fluoro substitutions lower the E°′ by −30 ± 3 mV. These results illustrate the utility of combining the optimized α[subscript 3]Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°′ values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.National Institutes of Health (U.S.) (Grant GM29595
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