SUMMARY We describe a mechanism of flagellar motor control by the bacterial signaling molecule c-di-GMP, which regulates several cellular behaviors. E. coli and Salmonella have multiple c-di-GMP cyclases and phosphodiesterases, yet absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di-GMP-binding protein, for motility inhibition. We demonstrate that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, most strongly in the presence of c-di-GMP. This interaction reduces the efficiency of torque generation and induces CCW motor bias. We present a “backstop brake” model showing how both effects can result from disrupting the organization of the FliG C-terminal domain, which interacts with the stator protein MotA to generate torque. Inhibition of motility and chemotaxis may represent a strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.

Bacterial f lagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the f lagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.Many species of bacteria are propelled by flagella, each consisting of a thin helical propeller attached, via a flexible coupling, to a rotary motor in the cell membrane (for recent reviews, see refs. 1 and 2). The energy for rotation of the flagella comes from the transmembrane gradient of protons (3-5) or, in certain marine or alkaliphilic species, sodium ions (6). The molecular mechanism of torque generation is not understood. The proteins most closely involved are MotA, MotB, and FliG. MotA and MotB are integral membrane proteins (7-11) that function in proton conduction (12)(13)(14) and that are believed to form the stator, or nonrotating part, of the motor (9,(15)(16)(17). FliG is located on the cytoplasmic side of the membrane, and is thought to be a component of the rotor (18)(19)(20)(21)(22).In hypotheses for the mechanism of the flagellar motor, charged residues have often been suggested to play key roles, serving, for example, to regulate movements of the rotor or to control proton flow (5,(23)(24)(25). We recently identified charged residues essential for motor function in both the rotor protein FliG and the stator protein MotA in mutational analyses of these proteins (26,27). In FliG, the residues Arg-281, Asp-288, and Asp-289 are of primary importance for function, whereas residues Lys-264 and Arg-297 might have secondary roles [FliG residue numbers here are higher by two than those given in the previous study, owing to recent corrections to the Escherichia coli fliG sequence (28)]. In MotA, the charged residues most important for function are Arg-90 and Glu-98, whereas Glu-150 may have a secondary role. Mutant phenotypes suggest that charge is the most important feature of these residues and that the charged residues in each protein function redundantly. Single charge-neutralizing replacements had only mild effects on function, whereas double rep...

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane 1 . This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion 2,3 . The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export 1,4 . Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the nonflagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a protondriven protein exporter and that ATP hydrolysis is not essential for type III secretion.Flagellar assembly begins with structures in the cytoplasmic membrane and proceeds through steps that add the exterior structures in a proximal-to-distal sequence (Fig. 1) 1 . Assembly of the rod, hook and filament requires the action of the secretion apparatus, which transports the needed subunits into a central channel through the structure that conducts them to their site of incorporation at the tip ( Fig. 1). Flagellar export is notably fast: in the early stages of filament growth flagellin is delivered at a rate of several 55 kDa subunits per second 5 .ATP hydrolysis by FliI was thought to provide the energy for export because mutations that delete or reduce the activity of FliI block flagellar synthesis at the stage of rod assembly 1,4,6 (Fig. 1). Homologues of FliI also occur in the type III secretion apparatus of injectisomes and are usually assumed to energize export in those systems as well. Some evidence for a different view has also been reported: it was observed that type III secretion in Yersinia enterocolitica was prevented by the protonophore CCCP 7 , and it was shown that the secretion ATPase InvC of Salmonella functions to dissociate export substrate from the chaperone 8 , a role distinct from transport itself. The energy source for type III secretion thus remains uncertain.To address the energy requirements for type III secretion, we first measured the effect of the uncoupler CCCP on flagellar export in S. enterica, assayed by accumulation of the export substrate FlgM in the medium. FlgM export was prevented by 10 mM or more CCCP (Fig. 2a). Overall cellular energy levels seemed unaffected, because cells grew normally in 10 mM CCCP (growth data not shown) and ATP levels were unchanged ( Supplementary Fig. 1). The effect was reversible: FlgM export was largely re...

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.

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.

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We investigate clogging of microchannels at the single-pore level using microfluidic devices as model porous media. The process of clogging is studied at low volume fractions and high flow rates, a technologically important regime. We show that clogging is independent of particle flow rate and volume fraction, indicating that collective effects do not play an important role. Instead, the average number of particles that can pass through a pore before it clogs scales with the ratio of pore to particle size. We present a simple model that accounts for the data.

The development of the gradient force optical particle trap ('optical tweezers') has made it possible to manipulate biological materials using a single beam of laser light. Optical traps can produce forces in the microdyne range on intact cells without causing overt damage: such forces are sufficient to arrest actively swimming bacteria and can overcome torque generated by the flagellar motor of a bacterium tethered to a glass surface by a flagellar filament. By calibrating the trapping force against Stokes' drag and measuring the twist that is sustained by this force, we determined the torsional compliance of flagella in tethered Escherichia coli and a motile Streptococcus. Flagella behaved as linear torsion springs for roughly half a revolution, but became much more rigid when turned beyond this point in either direction.