Flagellated bacteria propel themselves through an aqueous medium by rotating their helical flagellar filaments. The torque required for this motion is generated in the flagellar motor, which anchors the filament to the cell wall. The motor measures only about 25 nm in diameter, and electron micrographs of the isolated organelle show several rings arranged on a rod, which is connected to the filament by a curved hook. Flagellar rotation is driven by the protonmotive force across the cytoplasmic membrane. In recent years, a motile strain of Streptococcus has emerged as the organism of choice in studies of the flagellar motor, chiefly because its protonmotive force is easily manipulated. Since this bacterium lacks endogenous energy reserves, it can be starved until no metabolic protonmotive force remains. The grampositive cell wall leaves the cytoplasmic membrane accessible to ionophores, which facilitates the generation of artificial proton gradients. Flagellar rotation can be visualized by tethering a bacterium to a glass surface by one of its flagellar filaments, which causes the cell body to spin about the point of attachment. Recently it has also become possible to measure the motor's rotation rate in swimming cells. This thesis contains a variety of functional studies as well as some theoretical considerations of torque generation in the flagellar motor.
\r\n\r\nThe rotational angular symmetry of the motor was probed by a study of the angular positions at which it can stop. Only 5 or 6 discrete stopping angles were found. This constraint may result from static interactions between the rod of the motor and components of the cell wall.
\r\n\r\nA technique was developed to measure the external torque required to stop flagellar rotation. This stall torque did not depend noticeably on the motor's angular position. It was equal to the running torque measured in a rotating tethered cell. In particular, a decrease in the running torque at very low and very high pH, as well as its saturation at large protonmotive force, were also observed for the stall torque. These results show that the running torque is not limited by the rates of torque-generating processes associated with motor rotation. The apparent saturation of the torque as a function of protonmotive force seems to result from difficulties in generating large potassium diffusion potentials. Similarly, the artificially generated protonmotive force might be affected at extreme values of the pH.
\r\n\r\nThis thesis also reports the first sucessful measurements of the proton flux associated with flagellar rotation. These studies required an investigation of the total proton flux through the cytoplasmic membrane. An unusually large proton conductance was found, ca 10 times higher than the values reported for the membrane of Streptococcus lactis. After energization with an artificial, inwardly directed protonmotive force the rate of proton uptake by the cells decreased by a factor of 10 in 30 to 60 s with roughly exponential time course. Under certain conditions this influx was followed by slow extrusion of protons, part of which could be mediated by passive antiport of protons against other cations. Exchange of H+ for Na+ and Li+ was observed directly. The time course of proton movements accelerated by a factor of 6 as the temperature varied from 16\u00b0C to 32\u00b0C. The rate of proton uptake was reduced by about 25% through the action of DCCD, an inhibitor of the proton-translocating ATPase. In D2O the flux of hydrogen ions was ca. 20% lower than in H2O.
\r\n\r\nOnly a small fraction of the initial proton influx was associated with flagellar rotation, as determined from measurements on cells whose motors could not turn because their filaments were cross-linked with an antibody. The rotation-dependent component of the proton flux varied proportionally to the speed of the motor, with ca. 1100 protons transferred in one revolution of the filament. These observations support the hypothesis that proton flux and flagellar rotation are tightly coupled by the flagellar motor in a constant stoichiometric ratio. Measurements of the torque acting on the filament suggest that the conversion from electrochemical energy to mechanical work occurs with an efficiency of the order of 5% in swimming cells. At the low speeds of tethered cells the motor generates a larger torque, and the efficiency might be close to unity.
\r\n\r\nA hypothetical mechanism for torque generation, originally proposed in 1982, is analyzed in detail and compared to other models found in the literature. Its predictions are at odds with the experimental evidence presented in this thesis. However, the model can be altered by the assumption that the conduction of protons through the motor limits its rotation rate at very low torque. In particular, it is suggested that protons may be transferred across the membrane along chains of discrete binding sites. This could account for the strong dependence of the motor's maximal speed on the temperature and the hydrogen isotope.
\r\n", "doi": "10.7907/rv9w-sm36", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:17519", "collection": "thesis", "collection_id": "17519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07112025-215055238", "primary_object_url": { "basename": "Lowe_G_1987.pdf", "content": "final", "filesize": 51427723, "license": "other", "mime_type": "application/pdf", "url": "/17519/1/Lowe_G_1987.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Rotation of Bacterial Flagella at High Frequency", "author": [ { "family_name": "Lowe", "given_name": "Graeme", "orcid": "0000-0002-6998-2503", "clpid": "Lowe-Graeme" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "orcid": "0000-0002-6958-9605", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "orcid": "0000-0002-6958-9605", "clpid": "Berg-Howard-C" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Goodstein", "given_name": "David L.", "clpid": "Goodstein-D-L" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Flagellated bacteria propel themselves through their fluid\r\nenvironment by rotating one or more helical filaments which are driven\r\nat their base by a reversible motor powered by a protonmotive force. In\r\nsome species many filaments join. together to form a single flagellar\r\nbundle during swimming. Past work has characterized the functional\r\nproperties of the motor at low speed and high torque from studies of\r\ncells tethered by a single filament. This thesis describes work in which\r\nproperties of the motor at high speed and low torque were investigated\r\nby studying free swimming cells.
\r\n\r\nA method was developed for measuring the rotation rates of filaments\r\nin bundles of swimming cells. Images of cell bodies were projected onto\r\nthe photocathode of a photomultiplier tube whose sensitivity is\r\nspatially inhomogeneous, and the power spectral density of the output\r\nwas computed using the Fast Fourier Transform. Averages of many spectra\r\nrevealed a peak at high frequency due to the vibration of the cell body\r\nby the rotating filaments. This method was analysed in detail by\r\nmechanical simulations and computer models. Techniques were also\r\ndeveloped for following the rotation of single motors by attaching\r\nmarkers to sheared flagella. Most experiments were done with a motile\r\nstrain of Streptococcus. At 22\u00b0C, filaments of this organism rotate at\r\nca. 100 Hz relative to the cell body. Higher frequencies were seen in\r\nEscherichia coli at the same temperature, ca. 180 Hz.
\r\n\r\nThe relation between torque and speed of the flagellar motor at\r\nfixed protonmotive force was determined by varying the viscosity of the\r\nmedium. Torque was found to drop linearly with increasing speed over the\r\nupper half of the speed range. A comparison with the torque generated by\r\ntethered cells suggests that linearity may hold over the entire speed\r\nrange. This behavior is consistent with a scheme whereby the free energy\r\navailable per proton is dissipated in a series of small steps.
\r\n\r\nThe bundle frequency of glycolysing Streptococcus was found to\r\nincrease linearly with temperature, by a factor of 7 from 10\u00b0C to 42\u00b0C,\r\ncorresponding to an increase in torque by a factor of 3. The\r\nprotonmotive force did not vary by more than 10% from 16\u00b0C to 32\u00b0C.\r\nConditions were found under which cells swam when artificially energized\r\nby a combination of transmembrane pH gradient and potassium diffusion\r\npotential. These cells exhibited a large deuterium isotope effect, their\r\nspeed dropping by 30 - 50% in D20. Thus, proton transfer reactions\r\nappear to be limiting the rate of motor rotation in swimming cells.
", "doi": "10.7907/yh7f-q174", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" } ]