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Projects -Fluorescent chemotaxis proteins -Chemotactic signaling studied by FRET -Models of the chemotactic system
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Motor force generationDuty ratio. Will Ryu utilized the technique of attaching beads to flagellar stubs to study the resurrection of motors at low torque. Here, cells are nonmotile because of a mutation in a gene encoding a protein that is part of a force-generating element. A copy of the wild-type (good) gene is supplied on a plasmid under the control of a promoter that can be turned on by the addition of a chemical. As good proteins are made, the bad ones are replaced, and the motor starts spinning. In the low-speed, high-torque domain studied earlier with tethered cells, the motor speeds up in a series of equally-spaced steps, indicating the presence of at least 8 independent force-generating units. In the high-speed, low-torque domain, the steps get progressively smaller. This is consistent with a model in which the force-generating units are attached to the rotor most of the time (have a high duty ratio) and generate force by a powerstroke mechanism. Pmf dependence. Chris Gabel attached small beads to tethered cells and compared the rotation rates of the beads to those of the cells, as the cells were de-energized by addition of a respiratory poison or an uncoupler. We know from earlier work that when a cell is spinning slowly (as does a tethered cell), speed is proportional to membrane potential. Chris found that the speeds of the beads were proportional to the speeds of the cells, extending the proportionality of speed to pmf over the full accessible dynamic range (0-270 Hz). This adds a further constraint to motor models.Behavior near zero load. Junhua Yuan attached 60 nm gold beads to flagellar hooks (of cells lacking flagellar filaments) and followed their rotation by imaging scattered laser light on a pinhole in front of a photomultiplier. At room temperature, the beads spun ~ 300 Hz. Now, in a resurrection experiment (when bad force-generating units were replaced by good ones) full speed was restored in a single step. This method was also utilized to look at the temperature & solvent-isotope effects on the flagellar motor near zero load. We monitored changes in speed when cells were subjected to changes in temperature or shifted from a medium made with H2O to one made with D2O. In H2O, the speed increased with temperature in a near-exponential manner, with an activation enthalpy of 52 ± 4 kJ/mol (12.0 ± 1.0 kcal/mol). In D2O, the speed increased in a similar manner, with an activation enthalpy of 50 ± 4 kJ/mol. The speed in H2O was higher than that in D2O by a factor 1.53 ± 0.14. ReferencesBerg, H.C. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19-54 (2003).Fung, D.C. and Berg, H.C. Powering the flagellar motor of Escherichia coli with an external voltage source. Nature 375, 809-812 (1995). Gabel, C.V. and Berg, H.C. The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force. Proc. Natl. Acad. Sci. USA 100, 8748-8751 (2003). Ryu, W.S., Berry, R.M. and Berg, H.C. Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio. Nature 403, 444-447 (2000). Yuan, J. and Berg, H.C. Resurrection of the flagellar rotary motor near zero load. Proc Natl. Acad. Sci. USA 105, 1182-1185 (2008). Yuan, J., and Berg, H.C. Following the behavior of the flagellar rotary motor near zero load. Exp. Mech. (published online 09 September 2009). Yuan, J. and Berg, H.C. Thermal and solvent-isotope effects on the flagellar rotary motor near zero load. Biophys. J. (in press).
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Overview
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Last modified Tuesday, July 23, 2008.
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