Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins

M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins

BACKGROUND: Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values. CONCLUSIONS/SIGNIFICANCE: Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins

An energy transduction mechanism used in bacterial flagellar type III protein export

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton–protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH–FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus

Development of a low-background HPGe detector at Kamioka Observatory

A new ultra-low background high-purity germanium (HPGe) detector has been installed at the Kamioka underground experimental site. The background count rate in the energy range from 40 keV to 2700 keV is about 25% lower than that of the first HPGe detector installed in 2016, which has the same detector specification and similar shielding geometry. This paper describes the shielding configuration, including the cleaning of the material surface, the comparison of calibration data and simulation, the time variation of the background spectra, the sample measurement procedure, and some results of the radioactivity in the selected samples

Structural Insight into the Rotational Switching Mechanism of the Bacterial Flagellar Motor

Structural analysis of a clockwise-biased rotation mutant of the bacterial flagellar rotor protein FliG provides a new model for the arrangement of FliG subunits in the motor, and novel insights into rotation switching

Distillation of Liquid Xenon to Remove Krypton

A high performance distillation system to remove krypton from xenon was constructed, and a purity level of Kr/Xe = $\sim 3 \times 10^{-12}$ was achieved. This development is crucial in facilitating high sensitivity low background experiments such as the search for dark matter in the universe.Comment: 15 pages, 11 figure

Self-shielding effect of a single phase liquid xenon detector for direct dark matter search

Liquid xenon is a suitable material for a dark matter search. For future large scale experiments, single phase detectors are attractive due to their simple configuration and scalability. However, in order to reduce backgrounds, they need to fully rely on liquid xenon's self-shielding property. A prototype detector was developed at Kamioka Observatory to establish vertex and energy reconstruction methods and to demonstrate the self-shielding power against gamma rays from outside of the detector. Sufficient self-shielding power for future experiments was obtained.Comment: 8 pages, 8 figure

Baby MIND: A magnetised spectrometer for the WAGASCI experiment

The WAGASCI experiment being built at the J-PARC neutrino beam line will measure the difference in cross sections from neutrinos interacting with a water and scintillator targets, in order to constrain neutrino cross sections, essential for the T2K neutrino oscillation measurements. A prototype Magnetised Iron Neutrino Detector (MIND), called Baby MIND, is being constructed at CERN to act as a magnetic spectrometer behind the main WAGASCI target to be able to measure the charge and momentum of the outgoing muon from neutrino charged current interactions.Comment: Poster presented at NuPhys2016 (London, 12-14 December 2016). Title + 4 pages, LaTeX, 6 figure

Synchronization of the Distributed Readout Frontend Electronics of the Baby MIND Detector

Baby MIND is a new downstream muon range detector for the WGASCI experiment. This article discusses the distributed readout system and its timing requirements. The paper presents the design of the synchronization subsystem and the results of its test

Baby MIND Experiment Construction Status

Baby MIND is a magnetized iron neutrino detector, with novel design features, and is planned to serve as a downstream magnetized muon spectrometer for the WAGASCI experiment on the T2K neutrino beam line in Japan. One of the main goals of this experiment is to reduce systematic uncertainties relevant to CP-violation searches, by measuring the neutrino contamination in the anti-neutrino beam mode of T2K. Baby MIND is currently being constructed at CERN, and is planned to be operational in Japan in October 2017.Comment: Poster presented at NuPhys2016 (London, 12-14 December 2016). 4 pages, LaTeX, 7 figure