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 ΔfliH-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 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

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

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

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

Angular dependence of columnar recombination in high pressure xenon gas using time profile of scintillation emission

The angular dependence of the columnar recombination in xenon gas, if observed for low energy nuclear tracks, can be used for a direction-sensitive dark matter search. We measured both scintillation and ionization to study columnar recombination for 5.4 MeV alpha particles in a high pressure gas detector filled with 8 atm xenon. Since the recombination photons are emitted several~$\mu$s after de-excitation emission, scintillation photons are separated to the fast and slow components. The fast component does not show dependence on the track angle relative to the drift electric field, on the other hand, the slow component increases when the track is aligned with the electric field. The result indicates that the track angle relative to the electric field can be reconstructed from the scintillation time profile.Comment: 11 pages, 11figure

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

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

A chain mechanism for flagellum growth.

Bacteria swim by means of long flagella extending from the cell surface. These are assembled from thousands of protein subunits translocated across the cell membrane by an export machinery at the base of each flagellum. Unfolded subunits then transit through a narrow channel at the core of the growing flagellum to the tip, where they crystallize into the nascent structure. As the flagellum lengthens outside the cell, the rate of flagellum growth does not change. The mystery is how subunit transit is maintained at a constant rate without a discernible energy source in the channel of the external flagellum. We present evidence for a simple physical mechanism for flagellum growth that harnesses the entropic force of the unfolded subunits themselves. We show that a subunit docked at the export machinery can be captured by a free subunit through head-to-tail linkage of juxtaposed amino (N)- and carboxy (C)-terminal helices. We propose that sequential rounds of linkage would generate a multisubunit chain that pulls successive subunits into and through the channel to the flagellum tip, and by isolating filaments growing on bacterial cells we reveal the predicted chain of head-to-tail linked subunits in the transit channel of flagella. Thermodynamic analysis confirms that links in the subunit chain can withstand the pulling force generated by rounds of subunit crystallization at the flagellum tip, and polymer theory predicts that as the N terminus of each unfolded subunit crystallizes, the entropic force at the subunit C terminus would increase, rapidly overcoming the threshold required to pull the next subunit from the export machinery. This pulling force would adjust automatically over the increasing length of the growing flagellum, maintaining a constant rate of subunit delivery to the tip

Drift field generation with Cockcroft-Walton voltage multiplier in xenon gas for AXEL 0vββ search detector

16th International Conference on Topics in Astroparticle and Underground Physics (TAUP 2019) 9-13 September 2019, Toyama, JapanFor noble gas Time Projection Chambers (TPCs) in the field of rare event searches, operation of high voltage to generate an electric field is a key point. We designed a new structure of electrodes to shape a strong and uniform drift field without electric discharge, in which electrodes of two different radius are used. We also developed Cockcroft-Walton voltage multiplier as a high voltage generator inside a pressure vessel. We achieved −30.0 kV output and examined such kind of voltage generator is feasible as a high voltage supplier in a TPC