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

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

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

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

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

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

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

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

HpaC Controls Substrate Specificity of the Xanthomonas Type III Secretion System

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition