Yasuo Imae

Flagellar motors of alkalophilic bacillus are powered by an electrochemical potential gradient of Na

Flagellated bacteria such as Bacillus subtilis and Escherichia coli SW& by rotating their flagella at the basal structures which are embedded in cytoplasmic membrane [ 1,2]. There is considerable evidence that the flagellar basal structure is a reversible rotary motor powered by the transmembrane electrochemical potential gradient of H’, ApH+ [3-81. The flagellar motor of B. subtilis requires at threshold ApH’ of about -30 mV for its rotation, and the rotation rate is saturated at a A/*H’of about -100 mV [9,10]. Some alkalophilic Bacillus whose optimal growth pH is between 9 and 11 swim vigorously at high pH [11,12].However,in [13,14] the ApH’ofanalkalophilic BaciZZus was only about -15 mV at pH 11. This result suggests that the flagellar motors of alkalophilic Bacillus may be powered by some energy sources other than ApH+. This communication reports that Na* in the medium was required for the swimming of two independently isolated alkalophilic Bacillus. This suggests that the flagellar motors of these bacteria are powered by the transmembrane electrochemical potential gradient of Na+, ApNa+.

Motility in Bacillus subtilis driven by an artificial protonmotive force.

Peritrichously flagellated bacteria swim by rotating their flagella as a bundle [ 1,2] . A motor-like model for the flagellar rotation was proposed by Berg [3] , in which a flagellum is nearly a rigid propeller and is attached to a motor, namely the basal structure of flagella. When bacterial cells were tethered on micro- scope slides by means of antibodies specific for flagella, the cell bodies did rotate [4] . This finding gave a strong support to the above model. Then, what is the energy source for the rotation of the motor? Larsen et al. [5] reported that the intermediate form in oxydative phosphorylation is the energy source for the motility in

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 (Iaf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced Iaf transcription in media devoid of viscous agents in a dose‐dependent manner. The relationship between the average swimming speed and Iaf 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 Iaf induction. Alternative mechanisms for Iaf induction are also discussed.

Na+-driven bacterial flagellar motors

Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H+-driven type found in neutrophiles such asBacillus subtilis andEscherichia coli and the other is the Na+-driven type found in alkalophilicBacillus and marineVibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na+-driven flagellar motors, which introduces several new aspects in the analysis.

Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.

Removal of the periplasmic DNase before electroporation enhances efficiency of transformation in the marine bacterium Vibrio alginolyticus

We have established a reliable procedure for electroporation in the marine bacterium Vibrio alginolyticus. Plasmids carrying the P15A replicon were found to be stably maintained in the Vibrio cells, and chloramphenicol, kanamycin or tetracycline were used for selection. Since we found that the Vibrio cells excrete DNase into the culture medium, cells were subjected to osmotic shock before extensive washing in order to remove the DNase from the periplasmic space. This manipulation resulted in about a 10-fold increase in the efficiency of transformation. In addition, cells were washed in the presence of 5-10 mM Mg2+in order to stabilize the outer membrane. The efficiency of transformation was found to be optimal when cells were harvested at early stationary phase, and when electroporation was carried out at an electric field strength between 5-0 and 7.5 kV cm-1. Under optimal conditions, about 105transformants per μg of input DNA were reproducibly obtained, which is tolerable for cloning.

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.

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.

Requirement of Na+ in flagellar rotation and amino-acid transport in a facultatively alkalophilic Bacillus

Abstract A facultatively alkalophilic Bacillus strain YN-2000 grew well between pH 6.8 and 10, although the available size of the protonmotive force for the cells was high around neutral pH, but considerably low at alkaline pH. The energy source for the cellular functions of this bacterium grown at different pHs was analyzed to test if the cells switch the energy source between the protonmotive force and another source, depending upon the available size of the protonmotive force. The flagellar motors and the transport system of glutamine in this bacterium showed optimal activities at an alkaline pH and absolutely required the presence of Na + in the medium, irrespective of the growth pH. These results are consistent with the idea that, like obligate alkalophiles, the flagellar motors and an amino acid transport system of this bacterium are designed to utilize only the electrochemical potential gradient of Na + and have no switch of the energy source, irrespective of the available size of the protonmotive force during cell growth.

Relationship between Na+-dependent cytoplasmic pH homeostasis and Na+-dependent flagellar rotation and amino acid transport in alkalophilic Bacillus

The cytoplasmic pH homeostasis of alkalophilic Bacillus strains required the presence of Na+ in the medium, and Li+ was found to be equivalently substitutable for Na+. Flagellar rotation and amino acid transport of these bacteria also required Na+ but Li+ was not substitutable for Na+. Na+ concentration of about 1 mM was enough for the cytoplasmic pH homeostasis, while more than 10 mM Na+ was required for the full activities of flagellar rotation and amino acid transport. The addition of 150 mM ethanolamine to the cells at pH 9.6 disrupted the pH homeostasis and increased the cytoplasmic pH close to the external pH. Under this condition, however, flagellar rotation and amino acid transport were not so much affected. Thus, it is clear that flagellar rotation and amino acid transport themselves require the presence of Na+ in the medium, independent of the Na+‐dependent cytoplasmic pH homeostasis.