Rotation of the polar flagellum of Vibrio alginolyticus is driven by a Na؉ -type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of the Vibrio FliF protein produced at high levels in Escherichia coli was found in the soluble fraction. Soluble FliF purifies as an oligomer of ϳ700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data from in vitro binding assays using the soluble FliF multimer correlate with data from in vivo functional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation in Vibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na ؉ -driven flagellar motor, both in vivo and in vitro. Many motile bacteria can swim in liquid environments by means of a motility organelle, the flagellum. Bacteria propel themselves by rotating a helical flagellar filament to move forward, and flagellar rotation is driven by a reversible rotary motor at its base. The flagellum is divided into three parts: the filament (screw), the hook (universal joint), and the basal body (motor). About 50 gene products are required for flagellar assembly and function (1, 2). The energy source for the flagellar motor is the electrochemical gradient of protons or, in some species, sodium ions. The ion flux through stator units that are incorporated around the rotary part of the motor (rotor) is coupled with the generation of torque. The flagellar motor can rotate up to 1,700 revolutions per second (rps) (in the case of the Na ϩ -driven Vibrio motor) and can switch its rotational direction within a millisecond, properties which identify it as an elaborate biological nanomachine (3). However, the key question, how is the rotor-stator interaction coupled to ion flux to generate motor torque, has remained a mystery.Genetic, biochemical, and structural analyses identified key proteins that are most closely involved in torque generation: the stator complex and FliG in the rotor (Fig. 1A) (4). The stator is composed of two membrane proteins (MotA and MotB or their orthologs) that form an ion-conducting force-generating unit (5, 6). In the H ϩ -driven Escherichia coli or Salmonella motor, MotA, with 4 transmembrane (TM) segments, and M...

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The precipitation of human fibrinogen by ZnCl2 was studied by measuring the turbidity and the protein concentration in the supernatant (referred as solubility). Different kinetic patterns, either fast or slow, were observed in both solubility and turbidity depending on whether ZnCl2 concentration (CM) was larger or smaller than the critical concentration. In the slow pattern, the increase of the turbidity and the decrease of the solubility both occurred slowly and almost linearly with time. In the fast pattern, these changes occurred rapidly and stationary values were reached within 30 min for the turbidity and 2 h for the solubility. These two stationary values were found to be linearly related to each other. The stationary solubility decreased as CM increased, while it increased with the total amount of the protein. The dissolution of the precipitates was very slow resulting in a practically irreversible precipitation. A gelation model was proposed to account for these characteristic features, according to which fibrinogen molecules were linked to each other through the ’bond’ mediated by a zinc atom.

Kinetics of the precipitation of human fibrinogen by the addition of ZnCl2 was followed by the turbidity at 350 or 500 nm. Fast or slow precipitation occurred depending on whether the metal concentration (CM) was higher or lower than its critical value. In fast precipitations, the turbidity reached the stationary value within about 30 min. From the stationary values of the turbidity the critical concentration could be determined. A sigmoidal increase of the turbidity was often observed at low protein concentrations (CP); the lag time decreased with both CM and CP. At the final stage of the precipitation, the turbidity reached the stationary value exponentially; the characteristic time constant was almost independent of both CM and CP. These kinetic behaviors were consistent with the gelation model proposed to the present precipitation. When CuCl2 was used in place of ZnCl2, similar kinetic behaviors were obtained in many respects, suggesting the similar mechanism of precipitation for both ZnCl2 and CuCl2. Changes from slow to fast precipitation were observed when ionic strength was lowered.

SflA from p]asmid repTessed motitity and biosynthesis of tlagellin. In addition, the A flhFG AsfiA triple deletion mutant represented the same phenotypeastheAflhFG-supstrain.Thus,weconc]udedthatmutationinsflAis responsible for suppressor phenotype of the AtlhFG-sup strain. We propose that sflA was a novel gene involved in the flagellation of V. algino]yticus.