1,276 research outputs found
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
Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ΞfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate
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 protonβprotein antiporter that uses the two components of proton motive force, ΞΟ and ΞpH, 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 FliHβFliI complex, which turns the export gate into a highly efficient, ΞΟ-driven protein export apparatus
Application of Hamamatsu MPPC to T2K Neutrino Detectors
A special type of Hamamatsu MPPC, with a sensitive area of 1.3x1.3mm^2
containing 667 pixels with 50x50um^2 each, has been developed for the near
neutrino detector in the T2K long baseline neutrino experiment. About 60 000
MPPCs will be used in total to read out the plastic scintillator detectors with
wavelength shifting fibers. We report on the basic performance of MPPCs
produced for T2K.Comment: Contribution to the proceedings of NDIP 2008, Aix-les-Bains, France,
June 15-20, 200
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
Performance of Multi-Pixel Photon Counters for the T2K near detectors
We have developed a Multi-Pixel Photon Counter (MPPC) for the neutrino
detectors of T2K experiment. About 64,000 MPPCs have been produced and tested
in about a year. In order to characterize a large number of MPPCs, we have
developed a system that simultaneously measures 64 MPPCs with various bias
voltage and temperature. The performance of MPPCs are found to satisfy the
requirement of T2K experiment. In this paper, we present the performance of
17,686 MPPCs measured at Kyoto University.Comment: 15 pages, 14 figure
Mass production test of Hamamatsu MPPC for T2K neutrino oscillation experiment
In the T2K near neutrino detectors, about 60 000 Hamamatsu Multi-Pixel Photon
Counters (MPPCs) will be used. The mass production of MPPC has started in
February 2008.In order to perform quality assurance and to characterize each
device, we have developed an MPPC test system. For each MPPC, gain, breakdown
voltage, noise rate, photo detection efficiency, and cross-talk and after-pulse
rate are measured as functions of the bias voltage and temperature. The design
of the test system and the measurement procedure are described.Comment: Contribution to the proceedings of NDIP 2008, Aix-les-Bains, France,
June 15-20, 200
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
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
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 = 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
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