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 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...

The direction of rotation of the Escherichia coli flagellum is controlled by an assembly called the switch complex formed from multiple subunits of the proteins FliG, FliM, and FliN. Structurally, the switch complex corresponds to a drum-shaped feature at the bottom of the basal body, termed the C-ring. Stimulus-regulated reversals in flagellar motor rotation are the basis for directed movement such as chemotaxis. In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. In response to the chemotaxis signaling molecule phospho-CheY (CheY P ), the motors can switch to clockwise (CW) rotation, causing dissociation of the filament bundle and reorientation of the cell. CheY P has previously been shown to bind to a conserved segment near the N terminus of FliM. Here, we show that this interaction serves to capture CheY P and that the switch to CW rotation involves the subsequent interaction of CheY P with FliN. FliN is located at the bottom of the C-ring, in close association with the C-terminal domain of FliM (FliM C ), and the switch to CW rotation has been shown to involve relative movement of FliN and FliM C . Using a recently developed structural model for the FliN/FliM C array, and the CheY P -binding site here identified on FliN, we propose a mechanism by which CheY P binding could induce the conformational switch to CW rotation.switching | cell motility | signal transduction | molecular motors M any motile bacteria control the direction of their swimming by regulating the sense of flagellar rotation in response to sensory cues. In the well-studied enteric species Escherichia coli and Salmonella (Salmonella enterica serovar typhimurium), counterclockwise (CCW) rotation allows the several flagellar filaments on a cell to join in a bundle to propel the cell smoothly, whereas clockwise (CW) rotation of one or more flagella disrupts the bundle and causes the cell to tumble. In the absence of external stimuli, a cell executes smooth runs of about a second punctuated by brief tumbles that send it in a new, essentially random, direction; by delaying the switch to CW rotation in response to attractant stimuli, cells prolong runs that happen to be in a favorable direction and so bias their movement toward nutrients, temperatures, or other factors conducive to survival (1, 2).

Rotation and switching of the bacterial flagellum depends on a large rotor-mounted protein assembly composed of the proteins FliG, FliM and FliN, with FliG most directly involved in rotation. The crystal structure of a complex between the central domains of FliG and FliM, in conjunction with several biochemical and molecular-genetic experiments, reveals the arrangement of the FliG and FliM proteins in the rotor. A stoichiometric mismatch between FliG (26 subunits) and FliM (34 subunits) is explained in terms of two distinct positions for FliM: one where it binds the FliG central domain and another where it binds the FliG C-terminal domain. This architecture provides a structural framework for addressing the mechanisms of motor rotation and direction switching and for unifying the large body of data on motor performance. Recently proposed alternative models of rotor assembly, based on a subunit contact observed in crystals, are not supported by experiment.

The switch complex at the base of the bacterial flagellum is essential for flagellar assembly, rotation, and switching. In Escherichia coli and Salmonella, the complex contains about 26 copies of FliG, 34 copies of FliM, and more then 100 copies of FliN, together forming the basal body C ring. FliG is involved most directly in motor rotation and is located in the upper (membrane-proximal) part of the C ring. A crystal structure of the middle and C-terminal parts of FliG shows two globular domains connected by an ␣-helix and a short extended segment. The middle domain of FliG has a conserved surface patch formed by the residues EHPQ 125-128 and R 160 (the EHPQR motif), and the C-terminal domain has a conserved surface hydrophobic patch. To examine the functional importance of these and other surface features of FliG, we made mutations in residues distributed over the protein surface and measured the effects on flagellar assembly and function. Mutations preventing flagellar assembly occurred mainly in the vicinity of the EHPQR motif and the hydrophobic patch. Mutations causing aberrant clockwise or counterclockwise motor bias occurred in these same regions and in the waist between the upper and lower parts of the C-terminal domain. Pull-down assays with glutathione S-transferase-FliM showed that FliG interacts with FliM through both the EHPQR motif and the hydrophobic patch. We propose a model for the organization of FliG and FliM subunits that accounts for the FliG-FliM interactions identified here and for the different copy numbers of FliG and FliM in the flagellum.