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H C Berg

Publications and source records attributed to H C Berg.

At least 73 records · Page 4Linked to original sources
PubMed1989

Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli.

If cells of Escherichia coli deleted for genes that specify transducers and all known cytoplasmic chemotaxis proteins are reconstituted with CheA, CheW, and CheY, they spin their flagella alternately clockwise and counterclockwise. If the aspartate receptor also is present, clockwise rotation is suppressed upon addition of aspartate. If either CheA or CheW is absent, the fraction of time that the flagella spin clockwise is reduced and responses to aspartate do not occur.

Bacterial Proteins↗
PubMed1988

Restoration of torque in defective flagellar motors.

Paralyzed motors of motA and motB point and deletion mutants of Escherichia coli were repaired by synthesis of wild-type protein. As found earlier with a point mutant of motB, torque was restored in a series of equally spaced steps. The size of the steps was the same for both MotA and MotB. Motors with one torque generator spent more time spinning counterclockwise than did motors with two or more generators. In deletion mutants, stepwise decreases in torque, rare in point mutants, were common. Several cells stopped accelerating after eight steps, suggesting that the maximum complement of torque generators is eight. Each generator appears to contain both MotA and MotB.

Bacterial Proteins↗
PubMed1988

Acetyladenylate plays a role in controlling the direction of flagellar rotation.

Cells of Escherichia coli deleted for genes that code for the transducers and all the known cytoplasmic Che proteins except CheY responded reversibly to the addition of acetate by spinning their flagellar motors clockwise. By varying growth conditions and using metabolic inhibitors and mutants deficient in acetate metabolism, this effect was shown to require acetate-CoA synthetase [acetate:CoA ligase (AMP-forming); EC 6.2.1.1], an enzyme that catalyzes the formation of acetyl-CoA from acetate by an acetyladenylate intermediate. A mutant deficient in this enzyme but retaining the chemotaxis genes was deficient for chemotaxis. Thus, acetyladenylate appears to play a role in generating clockwise rotation at the level of CheY or the motor.

Acetate-CoA Ligase↗
PubMed1988

A physicist looks at bacterial chemotaxis.

What is distinctive about bacterial chemotaxis, as compared to, for example, taste in the elephant, is the time over which decisions must be made. The lower limit is set by diffusion of chemicals to and from the cell surface, which demands long times for statistically significant counts. The upper limit is set by diffusion of the cell itself, which demands short times for well-defined swimming paths. For an organism the size of E. coli, temporal comparisons of the concentrations of chemicals in the environment must be made within a few seconds. Although such short time spans might be difficult for the biochemist, they are not so difficult for E. coli, because diffusion can carry a small molecule across the cell in about 1 msec. E. coli has the opposite problem: How does it integrate inputs from many receptors over periods 1000 times as long? The mechanisms for this signal processing are beginning to be understood. We know how most chemical attractants are identified, how temporal comparisons might be made, and how the behavioral output is effected. We know less about how sensory information crosses the cytoplasmic membrane, how the reactions that link the receptors to the flagella generate such high gain, and what actually controls the direction of flagellar rotation. One thing is quite clear: E. coli demands our admiration and respect.

Chemotaxis↗
PubMed1987

The proton flux through the bacterial flagellar motor.

Bacterial flagella are driven by a rotary motor that utilizes the free energy stored in the electrochemical proton gradient across the cytoplasmic membrane to do mechanical work. The flux of protons coupled to motor rotation was measured in Streptococcus and found to be directly proportional to motor speed. This supports the hypothesis that the movement of protons through the motor is tightly coupled to the rotation of its flagellar filament. Under this assumption the efficiency of energy conversion is close to unity at the low speeds encountered in tethered cells but only a few percent at the high speeds encountered in swimming cells. This difference appears to be due to dissipation by processes internal to the motor. The efficiency at high speeds exhibits a steep temperature dependence and a sizable deuterium solvent isotope effect.

Flagella↗
PubMed1987

Response of the flagellar rotary motor to abrupt changes in extracellular pH.

Cells of a motile Streptococcus were starved, tethered to a quartz coverslip, energized with a potassium diffusion potential, and exposed to sudden decrements in external pH generated by flash photolysis of 2-hydroxyphenyl-1-(2-nitro)phenyl phosphate. The rotation rate of the cells increased following the flash but only after a brief time lag. Lags of the order of 0.1 second were observed in a dilute buffer (0.05 mM), confirming results obtained earlier. These lags were longer when the buffer was prepared in D2O. However, lags as short as 0.01 second were found in more concentrated buffers (1 and 3 mM). In this case, there was no deuterium solvent isotope effect. These differences arise from the extra time required for diffusion of protons from a dilute medium into the cell wall, which has a large buffering capacity. The short lags observed in concentrated media could be inherent to the flagellar motor, but the possibility that they are due to buffered diffusion through the cell wall or to elastic filtering by the tether has not been ruled out.

Cell Movement↗
PubMed1987

The stall torque of the bacterial flagellar motor.

The bacterial flagellar motor couples the flow of protons across the cytoplasmic membrane to the rotation of a helical flagellar filament. Using tethered cells, we have measured the stall torque required to block this rotation and compared it with the torque of the running motor over a wide range of values of proton-motive force and pH. The stall torque and the running torque vary identically: both appear to saturate at large values of the proton-motive force and both decrease at low or high pH. This suggests that up to speeds of approximately 5 Hz the operation of the motor is not limited by the mobility of its internal components or the rates of proton transfer reactions coupled to flagellar rotation.

Cell Membrane↗
PubMed1987

Avoidance of Phycomyces in a controlled environment.

The sporangiophore of the fungus Phycomyces bends away from nearby objects without ever touching them. It has been thought that these objects act as aerodynamic obstacles that damp random winds, thereby generating asymmetric distributions of a growth-promoting gas emitted by the growth zone. In the interest of testing this hypothesis, we studied avoidance in an environmental chamber in which convection was suppressed by a shallow thermal gradient. We also controlled pressure, temperature, and relative humidity of the air, electrostatic charge, and ambient light. A protocol was established that yielded avoidance rates constant from sporangiophore to sporangiophore to within +/- 10%. We found that avoidance occurred at normal rates in the complete absence of random winds. The rates were smaller at 100% than at lower values of relative humidity, but not by much. Remarkably, at a distance as great as 0.5 mm, avoidance from a 30-micron diam glass fiber (aligned parallel to the sporangiophore) was about the same as that from a planar glass sheet. However, the rate for the fiber fell more rapidly with distance. The rate for the sheet remained nearly constant out to approximately 4 mm. We conclude that avoidance depends either on adsorption by the barrier of a growth-inhibiting substance or emission by the barrier of a growth-promoting substance; it cannot occur by passive reflection. Models that can explain these effects are analyzed in the Appendix.

Mathematics↗
PubMed1987

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.

Bacterial Proteins↗
PubMed1986

Temporal comparisons in bacterial chemotaxis.

Responses of tethered cells of Escherichia coli to impulse, step, exponential-ramp or exponentiated sine-wave stimuli are internally consistent, provided that allowance is made for the nonlinear effect of thresholds. This result confirms that wild-type cells exposed to stimuli in the physiological range make short-term temporal comparisons extending 4 sec into the past: the past second is given a positive weighting, the previous 3 sec are given a negative weighting, and the cells respond to the difference. cheRcheB mutants (defective in methylation and demethylation) weight the past second in a manner similar to the wild type, but they do not make short-term temporal comparisons. When exposed to small steps delivered iontophoretically, they fail to adapt over periods of up to 12 sec; when exposed to longer steps in a flow cell, they partially adapt, but with a decay time of greater than 30 sec. cheZ mutants use a weighting that extends at least 40 sec into the past. The gain of the chemotactic system is large: the change in occupancy of one receptor molecule produces a significant response.

Bacterial Proteins↗
PubMed1985

Constraints on flagellar rotation.

The motion of tethered cells of Streptococcus was analyzed at low values of protonmotive force (delta p). Cells repeatedly energized and de-energized stopped at discrete angular positions, indicating a rotational symmetry of barriers to rotation of order 5 or 6. At values of delta p smaller than -30 mV, constraints imposed by these barriers were evident when cells were starved and gradually energized, but not when they were energized first and then gradually de-energized. At values of delta p larger than about -30 mV, the cells behaved as if there were no barriers. Cells spinning in this regime also executed rotational Brownian movement. At energy levels above threshold, the motor determines torque; it does not fix the position of the rotor relative to the stator.

Diffusion↗
PubMed1985

Chimeric chemosensory transducers of Escherichia coli.

The tar and tsr genes of Escherichia coli encode homologous transducer proteins that mediate distinct chemotactic responses. We report here the construction of two tasr chimeric genes in which the 5' coding region of the tar gene is fused to the 3' coding region of the tsr gene at either of two conserved restriction sites. Both chimeric genes code for chemotactically functional proteins. Results of analyses of behavior and methylation in cells carrying the chimeric genes support existing models for the disposition of transducer domains across the cell membrane and reveal that the receptors for internal pH map in a specific region of the COOH-terminal (cytoplasmic) domain.

Bacterial Proteins↗
PubMed1985

Chemotactic signaling in filamentous cells of Escherichia coli.

Video techniques were used to record chemotactic responses of filamentous cells of Escherichia coli stimulated iontophoretically with aspartate. Long, nonseptate cells were produced from polyhook strains either by introducing a cell division mutation or by growth in the presence of cephalexin. Markers indicating rotation of flagellar motors were attached with anti-hook antibodies. Aspartate was applied by iontophoretic ejection from a micropipette, and the effects on the direction of rotation of the markers were measured. Motors near the pipette responded, whereas those sufficiently far away did not, even when the pipette was near the cell surface. The response of a given motor decreased as the pipette was moved away, but it did so less steeply when the pipette remained near the cell surface than when it was moved out into the external medium. This shows that there is an internal signal, but its range is short, only a few micrometers. These experiments rule out signaling by changes in membrane potential, by simple release or binding of a small molecule, or by diffusion of the receptor-attractant complex. A likely candidate for the signal is a protein or ligand that is activated by the receptor and inactivated as it diffuses through the cytoplasm. The range of the signal was found to be substantially longer in a cheZ mutant, suggesting that the product of the cheZ gene contributes to this inactivation.

Aspartic Acid↗
PubMed1984

Chemical modification of Streptococcus flagellar motors.

Video techniques were used to record changes in motility of cells of Streptococcus sp. strain V4051 exposed to a variety of protein modification reagents. Starved cells were tethered to glass by a single flagellum, energized metabolically with glucose, or treated with valinomycin and energized artificially via shifts to media containing low concentrations of potassium ion. Experiments were devised that distinguished reagents that lowered the proton motive force from those that blocked the generation of torque (damaged the flagellar motors). Imidazole reagents blocked the generation of torque. Amino, sulfhydryl, dithiol, and disulfide reagents did not. Some of the imidazole, amino, and sulfhydryl reagents had long-term effects on the direction of flagellar rotation.

Cell Movement↗
PubMed1983

Isotope and thermal effects in chemiosmotic coupling to the membrane ATPase of Streptococcus.

We measured rates of ATP synthesis by the proton-translocating ATPase of the motile Streptococcus strain V4051. Starved cells were energized artificially by exposing their membranes to a variable electrical potential difference (internal medium negative) and a fixed pH difference (internal medium alkaline). The initial rates of ATP synthesis increased exponentially with protonmotive force. The results were the same in D2O and H2O; there was no solvent isotope effect. At a fixed protonmotive force, the rates were strongly dependent on temperature, as expected for a reaction with a large enthalpy of activation. At a different protonmotive force, the rates varied with temperature in an identical fashion; there was no change in the enthalpy of activation. We conclude that protonation-deprotonation steps are not rate limiting and that the protons that cross the membrane drive ATP synthesis by mass action. The transmembrane electric field acts by changing the concentrations of the reactants, not by changing the configuration of the enzyme-substrate complex.

Adenosine Triphosphatases↗
PubMed1983

Isotope and thermal effects in chemiosmotic coupling to the flagellar motor of Streptococcus.

The torque generated by the flagellar motor of Streptococcus strain V4051 has been determined from rates of rotation of cells tethered by a single flagellum in media of different isotopic composition and temperature. Starved cells were energized artificially with either a potassium diffusion potential or a pH gradient. The torque increased linearly with protonmotive force. Identical results were obtained in media made with D2O or H2O; there was no solvent isotope effect. At a fixed protonmotive force, the torque was approximately constant over a temperature range of 4 degrees -38 degrees C. In cells chemotactically inert to changes in cytoplasmic pH, the motor turned counterclockwise when protons moved inward and clockwise when they moved outward. We conclude that the motor is a reversible engine driven by simple acid-base dissociation. A detailed model is discussed.

Chemotaxis↗