Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine.

The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation, Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out alanine-scanning mutagenesis of the methylation sites. Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability. Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found. Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism are also discussed.

Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus.

Chemotactic responses in Vibrio alginolyticus, which has lateral and polar flagellar systems in one cell, were investigated. A lateral-flagella-defective (Pof+ Laf-) mutant, which has only a polar flagellum, usually swam forward by the pushing action of its flagellum and occasionally changed direction by backward swimming. When the repellent phenol was added, Pof+ Laf- cells moved frequently forward and backward (tumbling state). The tumbling was derived from the frequent changing between counter-clockwise and clockwise (CW) rotation of the flagellar motor, as was confirmed by the tethered-cell method. Furthermore, we found that the tumbling cells did not adapt to the phenol stimulus. When the attractant serine was added, the phenol-treated cells ceased tumbling and swam smoothly, adapting to the attractant stimulus after several minutes. We isolated chemotaxis-defective (Che-) mutants from the Pof+ Laf- mutant; the tumbling mutants were not isolated. One interesting mutant swam backwards continuously, with its flagellum leading the cell and its flagellar motor rotating CW continuously. A polar-flagella-defective mutant (Pof- Laf+) stopped swimming after phenol addition and then recovered swimming ability within 10 min, indicating that lateral flagella can adapt to the repellent stimulus. This may represent a functional difference between the two flagellar systems in Vibrio cells, and between the chemotaxis systems affecting the two types of flagella.

Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus.

By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof+ Laf- cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof- Laf+ cells swam at 20 microns/s. The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further. These results show the functional difference between polar and lateral flagella in viscous environments.

Modulation of the thermosensing profile of the Escherichia coli aspartate receptor tar by covalent modification of its methyl-accepting sites.

The Escherichia coli aspartate receptor Tar is involved in the thermotactic response. We have studied how its thermosensing function is affected by the modification of the four methyl-accepting residues (Gln295, Glu302, Gln309, and Glu491), which play essential roles in adaptation. We found that the primary translational product of tar mediates a chemoresponse, but not a thermoresponse, and that Tar comes to function as a thermoreceptor, once Gln295 or Gln309 is deamidated. This is the first identification of a thermosensing-specific mutant form, suggesting that the methylation sites of Tar constitute at least a part of the region required for thermoreception, signaling, or both. We have also investigated the inverted thermoresponse mediated by Tar in the presence of aspartate. We found that, whereas the deamidated-and-unmethylated form functions as a warm receptor, eliciting a smooth-swimming signal upon increase of temperature, the heavily methylated form functions as a cold receptor, eliciting a smooth-swimming signal upon decrease of temperature. Thus, it is suggested that Tar exists in at least three distinct states, each of which allows it to function as a warm, cold, or null thermoreceptor, depending on the modification patterns of its methylation sites.

Rotational fluctuation of the sodium-driven flagellar motor of Vibrio alginolyticus induced by binding of inhibitors.

Rotation of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated under the influence of inhibitors specific to the motor, amiloride and phenamil. The rotation rate of a single flagellum on a cell stuck to a glass slide was examined using laser dark-field microscopy. In the presence of 50 mM NaCl, the average rotation rate (omega) was about 600 r.p.s. with a standard deviation (sigma omega) of 9% of omega. When omega was decreased to about 200 r.p.s. by the presence of 1.5 mM amiloride, sigma omega increased to 15% of omega. On the other hand, when omega was decreased to about 200 r.p.s. by the addition of 0.6 microM phenamil, a large increase in sigma omega up to 50% of omega, was observed. Similarly large fluctuations were observed at other concentrations of phenamil. These observations suggest that dissociation of phenamil from the motor was much slower than that of amiloride. A very low concentration of phenamil caused a transient but substantial reduction in rotation rate. This might suggest that binding of only a single molecule of phenamil strongly inhibits the torque generation in the flagellar motor.

The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression.

Certain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella. The polar and the lateral flagellar motors are powered by sodium- and proton-motive forces, respectively. The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum. The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum. To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium-driven motor, on lateral flagellar gene (laf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced laf transcription in media devoid of viscous agents in a dose-dependent manner. The relationship between the average swimming speed and laf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed. These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for laf induction. Alternative mechanisms for laf induction are also discussed.

Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.

Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus.

High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus.

The Na(+)-driven flagellar motor in Vibrio alginolyticus rotates very fast. Rotation of a single flagellum on a stuck cell was measured by laser darkfield microscopy with submillisecond temporal resolution. The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s. at 300 mM NaCl. The Na+ influx through the motor should determine the rotation period (tau) and affect the speed stability. Fluctuation of the rotation period was analyzed at various rotation rates (from approximately 50 r.p.s. to approximately 1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor. At high rotation rates (over 400 r.p.s.), the observed rotation was stable, and the standard deviation of tau (sigma tau) ranged from 7% to 16% of the average rotation period (< tau >). At low rotation rates (under 100 r.p.s), the rotation period tended to fluctuate, and the distributions of tau were non-Gaussian. The value of sigma tau ranged from 10 to 30% of < tau >. However, the observed minimum value of sigma tau at various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament. These results suggest that the torque was stably generated at various Na+ influxes through the motor. We observed large fluctuations that cannot be explained by rotational diffusion. We discuss the factors that induce the large fluctuation.

In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.

The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein.

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions.

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TM2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed 'smooth', biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.

Biological activities of 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 and its 24-epimer.

Biological activities of two epimeric 24-fluorinated vitamin D-2 analogs, 24-fluoro-1 alpha,25-dihydroxyvitamin D-2 [24-F-1,25-(OH)2D2] and its 24-epimer [24-epi-24-F-1,25-(OH)2D2], were studied and compared with 1 alpha,25-dihydroxyvitamin D-3 [1,25-(OH)2D3] and 1 alpha,25-dihydroxyvitamin D-2 [1,25-(OH)2D2]. 24-F-1,25-(OH)2D2 was nearly as active as 1,25-(OH)2D3 and 1,25-(OH)2D2 both in regulating calcium metabolism in vivo including bone mineral mobilization and intestinal calcium transport and in inducing differentiation of HL-60 cells. While 24-epi-24-F-1,25-(OH)2D2 showed distinct properties in these two types of the actions. Though the 24-epimer was nearly as potent as 1,25-(OH)2D3 in inducing differentiation of HL-60 cells, it showed little activity in regulating calcium metabolism in vivo. The fluorine atom introduced at the 24-position of either 1,25-(OH)2D2 or its 24-epimer had no potentiating effect. This is in sharp contrast with the cases of 24- and 26,27-multifluorinated analogs of active vitamin D-3.

Successive inactivation of the force-generating units of sodium-driven bacterial flagellar motors by a photoreactive amiloride analog.

Like amiloride, 6-iodoamiloride (6-IA) competitively and reversibly inhibits rotation of the Na(+)-driven flagellar motors of alkalophilic Bacillus cells. However, when 6-IA-treated cells are irradiated with UV light, motility is irreversibly inhibited. This treatment does not alter the membrane potential or affect Na(+)-coupled alpha-aminoisobutyrate transport. An increase in the Na+ concentration during UV irradiation substantially protects the motors from irreversible inhibition. Thus, photoactivated 6-IA seems to bind specifically and covalently at or around the Na(+)-interaction site of the force-generating units of the motors to inhibit motor rotation irreversibly. Rotation of each motor, which is monitored using tethered alkalophilic Bacillus cells, is also inhibited by photoactivated 6-IA. In this case, however, the rotation rate during UV irradiation decreases stepwise, suggesting the presence of several independently functioning force-generating units in a motor. From the data of 14 tethered cells, the number of units/motor is estimated to be 5-9.

Study of the torque of the bacterial flagellar motor using a rotating electric field.

Bacterial flagella are driven by a rotary motor that is energized by an electrochemical ion gradient across the cell membrane. In this study the torque generated by the flagellar motor was measured in tethered cells of a smooth-swimming Escherichia coli strain by using rotating electric fields to determine the relationship between the torque and speed over a wide range. By measuring the electric current applied to the sample cell and combining the data obtained at different viscosities, the torque of the flagellar motor was estimated up to 55 Hz, and also at negative rotation rates. By this method we have found that the torque of the flagellar motor linearly decreases with rotation rate from negative through positive rate of rotation. In addition, the dependence of torque upon temperature was also investigated. We showed that torque at the high speeds encountered in swimming cells had a much steeper dependence on temperature that at the low speeds encountered in tethered cells. From these results, the activation energy of the proton transfer reaction in the torque-generating unit was calculated to be about 7.0 x 10(-20) J.

Inhibition of aspartate chemotaxis of Escherichia coli by site-directed sulfhydryl modification of the receptor.

Thr-154 of the chemoreceptor Tar in Escherichia coli is important for aspartate sensing. Taking advantage of the fact that Tar has no Cys residues, we have further investigated the role of Thr-154 by replacing it with Cys in order to subject it to SH modification. Tar-T154C retained the abilities of aspartate sensing and repellent sensing. However, when cells with Tar-T154C were treated with an SH-modifying reagent, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), they specifically lost the ability to sense aspartate; the ability was restored by the reducing reagent, 1,4-dithiothreitol. DTNB showed no detectable effect on the function of wild-type Tar or serine-replaced Tar, Tar-T154S. Thus, DTNB modifies Cys-154 of Tar-T154C in intact cells and causes a specific defect in the aspartate-sensing ability of Tar. The addition of 1 mM or higher concentrations of aspartate resulted in protection of Cys-154 from the modification; serine had no effect in this regard. These results that not only is Thr-154 important for aspartate sensing but also, it may be located at the actual aspartate-binding site.

Cloning and characterization of the Salmonella typhimurium-specific chemoreceptor Tcp for taxis to citrate and from phenol.

Salmonella typhimurium shows an attractant response to citrate and a repellent response to phenol, and a chemoreceptor mediating these responses has been identified and named Tcp (taxis to citrate and away from phenol). Tcp is one of the methyl-accepting chemotaxis proteins that have a molecular mass of approximately 60 kDa estimated by SDS/PAGE, and its methylation level is increased by citrate and decreased by phenol. Tcp also mediates an attractant response to metal-citrate complexes. The complete nucleotide sequence of the tcp coding region has been determined. The deduced amino acid sequence of Tcp, consisting of 547-amino acid residues, is homologous with that of the aspartate chemoreceptor of S. typhimurium. Thus, Tcp is another member of the bacterial transmembrane chemoreceptor family. Because citrate is a good carbon source for S. typhimurium but is not a carbon source for the closely related species Escherichia coli and because citrate utilization is used as a key diagnostic character to distinguish these species, it is reasonable to assume that Tcp is specific to S. typhimurium.

Amiloride at pH 7.0 inhibits the Na(+)-driven flagellar motors of Vibrio alginolyticus but allows cell growth.

Amiloride, a specific inhibitor for the Na(+)-driven flagellar motors of alkalophilic Bacillus, is known to inhibit secondarily the growth of alkalophiles. The motility of a marine Vibrio, V. alginolyticus, was almost completely inhibited by 2 mM amiloride either at pH 7.0 or 8.5. We found that this concentration of amiloride inhibited the cell growth completely at pH 8.5 but only slightly at pH 7.0. Kinetic analysis of the inhibition of motility by amiloride at pH 7.0 showed that the inhibition was competitive with Na+ in the medium. Thus, amiloride at pH 7.0 is really a specific and useful tool for the analysis of the Na(+)-driven flagellar motors of Vibrio.

Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces.

Various species of marine Vibrio produce two distinct types of flagella, each adapted for a different type of motility. A single, sheathed polar flagellum is suited for swimming in liquid medium, and numerous unsheathed lateral flagella, which are produced only under viscous conditions, are suited for swarming over viscous surfaces. Both types of flagella are driven by reversible motors embedded in the cytoplasmic membrane. Here we report that the energy source for the polar flagellar motor of Vibrio parahaemolyticus is the sodium-motive force, whereas the lateral flagellar motors are driven by the proton-motive force. This is evidence that two distinct types of flagella powered by different energy sources are functionally active in one cell.

Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli.

The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap). Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli. However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C. These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.