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Escherichia coli and other bacteria are said to be the lowest forms of life in the biological world. Nevertheless, bacteria are magnificent organisms that can recognize the ambient environment, propagate ambient information into its body, and (in the case of most bacteria, at least…) possess the capacity to swim toward a more favorable environment; these are systems that no man-made machine could even hope to imitate. This complex machinery is constructed in an extremely small unit of space about a femtoliter. Because this organism is so small, its proteins function in far smaller numbers than seen in other organisms (on the order of about 100 protein molecules involving methylation and demethylation, and 1000 phosphorylated protein molecules work in a cell). Let us gain an understanding of the chemotaxis and motility of bacteria through the following conversation between a mother and her daughter.

In response to an attractant or repellant, an Escherichia coli cell controls the rotational direction of its flagellar motor by a chemotaxis system. When an E. coli cell senses an attractant, a reduction in the intracellular concentration of a chemotaxis protein, phosphorylated CheY (CheY-P), induces counterclockwise (CCW) rotation of the flagellar motor, and this cellular response is thought to occur in several hundred milliseconds. Here, to measure the signaling process occurring inside a single E. coli cell, including the recognition of an attractant by a receptor cluster, the inactivation of histidine kinase CheA, and the diffusion of CheY and CheY-P molecules, we applied a serine stimulus by instantaneous photorelease from a caged compound and examined the cellular response at a temporal resolution of several hundred microseconds. We quantified the clockwise (CW) and CCW durations immediately after the photorelease of serine as the response time and the duration of the response, respectively. The results showed that the response time depended on the distance between the receptor and motor, indicating that the decreased CheY-P concentration induced by serine propagates through the cytoplasm from the receptor-kinase cluster toward the motor with a timing that is explained by the diffusion of CheY and CheY-P molecules. The response time included 240 ms for enzymatic reactions in addition to the time required for diffusion of the signaling molecule. The measured response time and duration of the response also revealed that the E. coli cell senses a similar serine concentration regardless of whether the serine concentration is increasing or decreasing. These detailed quantitative findings increase our understanding of the signal transduction process that occurs inside cells during bacterial chemotaxis.

Escherichia coli cells swim toward a favorable environment by chemotaxis. The chemotaxis system regulates the swimming behavior of the bacteria by controlling the rotational direction of their flagellar motors. Extracellular stimuli sensed by chemoreceptors are transduced to an intracellular signal molecule, phosphorylated CheY (CheY-P), that switches the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) or from CW to CCW. Many studies have focused on identifying the proteins involved in the chemotaxis system, and findings on the structures and intracellular localizations of these proteins have largely elucidated the molecular pathway. On the other hand, quantitative evaluations of the chemotaxis system, including the process of intracellular signaling by the propagation of CheY-P and the rotational switching of flagellar motor by binding of CheY-P molecules, are still uncertain. For instance, scientific consensus has held that the flagellar motors of an E. coli cell switch rotational direction asynchronously. However, recent work shows that the rotational switching of any two different motors on a single E. coli cell is highly coordinated; a sub-second switching delay between motors is clearly correlated with the relative distance of each motor from the chemoreceptor patch located at one pole of the cell. In this review of previous studies and our recent findings, we discuss the regulatory mechanism of the multiple flagellar motors on an individual E. coli cell and the intracellular signaling process that can be inferred from this coordinated switching.

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).

The bacterial flagellar motor is driven by the electrochemical potential of specific ions, H + or Na + . The motor consists of a rotor and stator, and their interaction generates rotation. The stator, which is composed of PomA and PomB in the Na + motor of Vibrio alginolyticus, is thought to be a torque generator converting the energy of ion flux into mechanical power. We found that specific mutations in PomB, including D24N, F33C and S248F, which caused motility defects, affected the assembly of stator complexes into the polar flagellar motor using green fluorescent protein-fused stator proteins. D24 of PomB is the predicted Na + -binding site. Furthermore, we demonstrated that the coupling ion, Na + , is required for stator assembly and that phenamil (an inhibitor of the Na + -driven motor) inhibited the assembly. Carbonyl cyanide m-chlorophenylhydrazone, which is a proton ionophore that collapses the sodium motive force in this organism at neutral pH, also inhibited the assembly. Thus we conclude that the process of Na + influx through the channel, including Na + binding, is essential for the assembly of the stator complex to the flagellar motor as well as for torque generation.

The bacterial chemotaxis system regulates the rotational direction of flagellar motors through an intracellular signaling molecule, the phosphorylated form of CheY (CheY-P). The binding of CheY-P to a motor is believed to switch the motor's rotational direction from counterclockwise to clockwise. We demonstrated that the rotational switch of a motor was directly regulated by the binding and dissociation of CheY-P by simultaneously visualizing CheY tagged with green fluorescent protein and the rotational switching of a motor in live cells. The binding of 13 ± 7 CheY-P molecules was sufficient to induce clockwise rotation, and CheY-P molecules bound to and dissociated from a motor within ~100 ms during switching. Thus, we have directly measured the regulation of the output from a signal transduction pathway by intracellular signaling proteins.

In pancreatic islets, insulin secretion occurs via synchronous elevation of Ca(2+) levels throughout the islets during high glucose conditions. This Ca(2+) elevation has two phases: a quick increase, observed after the glucose stimulus, followed by prolonged oscillations. In these processes, the elevation of intracellular ATP levels generated from glucose is assumed to inhibit ATP-sensitive K(+) channels, leading to the depolarization of membranes, which in turn induces Ca(2+) elevation in the islets. However, little is known about the dynamics of intracellular ATP levels and their correlation with Ca(2+) levels in the islets in response to changing glucose levels. In this study, a genetically encoded fluorescent biosensor for ATP and a fluorescent Ca(2+) dye were employed to simultaneously monitor the dynamics of intracellular ATP and Ca(2+) levels, respectively, inside single isolated islets. We observed rapid increases in cytosolic and mitochondrial ATP levels after stimulation with glucose, as well as with methyl pyruvate or leucine/glutamine. High ATP levels were sustained as long as high glucose levels persisted. Inhibition of ATP production suppressed the initial Ca(2+) increase, suggesting that enhanced energy metabolism triggers the initial phase of Ca(2+) influx. On the other hand, cytosolic ATP levels did not fluctuate significantly with the Ca(2+) level in the subsequent oscillation phases. Importantly, Ca(2+) oscillations stopped immediately before ATP levels decreased significantly. These results might explain how food or glucose intake evokes insulin secretion and how the resulting decrease in plasma glucose levels leads to cessation of secretion., インスリン分泌における重要因子が変動する様子を可視化 -蛍光タンパク質センサーを用いたライブイメージング法で-. 京都大学プレスリリース. 2014-01-30.

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.

In Escherichia coli , rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na + -driven polar flagellum of Vibrio alginolyticus , but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli . The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42°C and was restored to the original level approximately 10 min after the temperature was returned to 20°C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.

Escherichia coli can form linear trails and move in a flagellum-independent manner on semisolid agar containing carbon sources. Trail formation seemed to correlate with the growth speed and/or carbon metabolism. Cell morphology in linear trails changed into larger cell sizes. We speculate that the flagellum-independent trail formation is a new mechanism for migration of E. coli cells.