Abstract
Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane1. 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' secretion2, 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 export1, 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 non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven 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 second5.
Figure 1: Steps in flagellar assembly.
Dashed boxes indicate the proteins that function in flagellar secretion, either in the membrane-bound part of the apparatus or in delivery of substrate. Flagellar components that depend on export are indicated in light- (early substrates) or dark- (late substrates) grey; these include the structural proteins that form the rod, hook and filament, the transcriptional regulator FlgM, and the hook-length regulator FliK. OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane.
High resolution image and legend (143K)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 assembly1, 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 CCCP7, and it was shown that the secretion ATPase InvC of Salmonella functions to dissociate export substrate from the chaperone8, 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 
M or more CCCP (Fig. 2a). Overall cellular energy levels seemed unaffected, because cells grew normally in 10 M CCCP (growth data not shown) and ATP levels were unchanged (Supplementary Fig. 1). The effect was reversible: FlgM export was largely restored following a 30-min washout of the 10 M CCCP (Fig. 2b). FlgM inhibits transcription of its own gene, and so reduced FlgM export might partially reflect decreased cellular levels of the protein9. To circumvent this autoinhibitory effect the experiment was repeated with a strain in which flgM was placed under control of a non-native (ParaB) promoter. In this strain, the cytoplasmic level of FlgM remained nearly constant, whereas FlgM secretion was again prevented by 10 M or more CCCP (Fig. 2c).
Figure 2: Inhibition of FlgM secretion by CCCP.
a, Secretion in Salmonella strain TH3730 (Tet-inducible flhDC). b, Partial restoration of export following a 30-min wash into CCCP-free buffer. c, Secretion in strain TH10874 (arabinose-inducible flgM). d, Inhibition of FlgM secretion by CCCP in an ATP-synthase defective (
atpA) strain (TH11802). e, ATP levels in the atpA mutant at various times following treatment with CCCP. Open circles, no treatment; open square, 10 M CCCP; and filled circles, 30 M CCCP. f, Inhibition of secretion of other flagellar substrates (FlgK, FlgL, FliC and FliK) by CCCP.
The maintenance of normal ATP levels in the presence of CCCP was noted previously in experiments with Y. enterocolitica7 and is thought to be due to a regulatory mechanism that inhibits the hydrolytic activity of the ATP synthase when the membrane is de-energized10. Because this protective mechanism may not act instantaneously, cellular ATP levels might undergo a transient drop following CCCP treatment that would escape detection in our measurements. To rule out such an effect, we measured FlgM export in a atpA strain that lacks a major subunit of the ATP synthase. FlgM secretion was again prevented by 10 M or higher CCCP, and ATP levels were unaffected (Fig. 2d, e).
The flagellum exports more than a dozen substrates, which are classified as early or late according to whether they are secreted during assembly of the hook/basal-body or the filament1 (Fig. 1). To test the generality of the PMF requirement we examined the effect of CCCP on secretion of additional substrates, assayed by their accumulation in the culture medium11. We observed the early substrate FliK and the late-export substrates FlgK, FlgL and FliC in culture supernatants of LB-grown cells. Accumulation of all four substrates was prevented by treatment with 20 M CCCP (Fig. 2f). The band between FliC and FlgK does not correspond to the size of a known flagellar protein and is likely to be a stable protein secreted in (relatively) low amounts.
CCCP functions as a proton carrier to discharge both the electric potential (
) and concentration (pH) components of the PMF. To examine the contribution of the component separately, we measured FlgM secretion in cells treated with the K+-ionophore valinomycin. In medium containing 150 mM KCl, FlgM secretion was inhibited by 10 M valinomycin (Fig. 3a). Thus, the electrical potential component seems essential for export under the conditions of this experiment (extracellular pH = 7.3). Next, the pH component of the gradient was discharged by the weak acid acetate (34 mM), which crosses the membrane in neutral (protonated) form and releases protons inside the cell. At an external pH of 7, FlgM secretion was not affected by treatment with acetate (Fig. 3b), indicating that flagellar export can be supported by alone. At an external pH of 5, acetate prevented secretion (Fig. 3b), presumably owing to acidification of the cytoplasm and the resulting protonation of one or more functionally important acidic groups. A similar effect was reported with the flagellar motor, which ceased rotating when the cytosolic pH was lowered to 5 (ref. 12).
Figure 3: Effect of 
and pH on FlgM export.
![Figure 3 : Effect of |[Dgr]||[psgr]| and |[Dgr]|pH on FlgM export. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com](/nature/journal/v451/n7177/images/nature06497-f3.0.jpg)
a, Inhibition of FlgM secretion by valinomycin and K+. Where indicated, cells were pretreated with Tris (120 mM) to permeabilize the outer membrane to valinomycin. b, Inhibition of FlgM secretion by acetate (34 mM) at pH 5.
High resolution image and legend (73K)We characterized export requirements further using an assay based on secretion of a FlgE–Bla (hook/
-lactamase) fusion protein. Cells were deleted for the rod proteins FlgB and FlgC (Fig. 1) to direct the fusion protein into the periplasm, allowing export to be quantified by the MIC (minimum inhibitory concentration) for ampicillin13. The MIC value was reduced by uncoupler, from a value of 25 g ml-1 in the untreated control to about 4 g ml-1 in 30 M CCCP (Table 1, and Supplementary Information).
If energy for flagellar transport comes from the proton gradient, then ATP hydrolysis by FliI may be less important than has been supposed. To examine the FliI requirement more closely we measured FlgE–Bla export in a strain deleted for fliI and the flanking genes fliH and fliJ. FliH is a regulator of FliI14 and FliJ interacts with the FliHI complex and with other export components15, 16. The MIC measured for the fliHIJ strain was 12 g ml-1, reproducibly larger than that of a negative-control strain lacking the MS-ring gene fliF (<3 g ml-1) or a strain with all the flagellar genes repressed by downregulation of the master regulators flhDC (<3 g ml-1) (Table 1). Furthermore, the MIC value of the fliHIJ strain was greatly increased (to 800 g ml-1) by overexpression of FliR, a membrane-associated part of the export apparatus (Table 1, and Supplementary Data). Prompted by this evidence of export in the absence of FliI, we examined motility of fliHIJ cells. The fliHIJ cells migrated in soft agar at about one-tenth the wild-type rate (Fig. 4a), and a fraction of the cells were observed to swim in liquid media. Cells isolated from the centre, edges or intermediate positions in the swarm showed the same phenotype when purified and re-tested (not shown), and so the slow motility is a property of the population and is not due to suppressing mutations. Staining showed flagella on a small fraction (<1%) of the cells (Fig. 4b, left panel). A fliI mutant swarmed more slowly than the fliHIJ strain (Fig. 4a) and also showed reduced export in the MIC assay (Table 1), consistent with the more severe motility defect reported previously for a fliI mutant4, 17.
Figure 4: FliI is non-essential for flagellar assembly and function.
a, Swarming of the fliHIJ deletion strain and a fliHIJ invC ssaN strain with all type-III secretion ATPases deleted. The fliF strain, blocked in the earliest step of flagellar assembly (Fig. 1), is included as a negative control. Plates were incubated at 32 °C. b, Flagella on cells of the fliHIJ mutant (left panel) and the fliHIJ invC ssaN triple-deletion (right panel). Flagella were visualized with FITC-conjugated anti-FliC antibody (green)26. DNA was stained with DAPI (blue), and membranes by FM64 (red).
In addition to the flagella apparatus, members of Salmonella spp. contain two non-flagellar (injectisome) type III secretion systems, with associated ATPases InvC and SsaN8, 18. To rule out any involvement of InvC or SsaN in the secretion observed in fliHIJ cells, we repeated the experiments in a invC ssaN fliHIJ strain. The triple-deletion mutant swarmed equally as well as the fliHIJ strain in soft agar (Fig. 4a), and flagella were again seen on a few cells (Fig. 4b, right). Thus, none of the secretion ATPases is required for flagellar export, assembly or function.
Our conclusions are consistent with previous observations of a PMF requirement for flagellar growth from more than 25 years ago19, and extend the earlier findings in showing that export can be energized by PMF alone in the absence of any type III secretion ATPase7. Use of the proton gradient is perhaps not surprising given the speed of type III secretion and the likely advantage of tapping a proximal energy source. Rapid subunit export presumably requires a rapid supply of energy, which might be more easily delivered by a proton current than by ATP hydrolysis. Given that type III secretion is energized by PMF, future studies should focus on the molecular mechanism of proton movement through the apparatus and its coupling to movement of substrate.
Methods Summary
Media, growth conditions, methods for phage-mediated transduction and motility assays were performed as described previously9, 20, 21. Carbonylcyanide m-chlorophenylhydrozone (CCCP) and valinomycin were from Sigma (analytical grade). Potassium acetate was from J. T. Baker, growth media from Difco, and buffers from Sigma.
FlgM secretion was assayed by accumulation of the protein in the culture medium, using procedures described previously22 with minor modifications described in Methods. Cells at mid-log growth stage were treated with the PMF-discharging agents (CCCP or valinomycin) at the concentrations indicated in the figures, introduced from freshly prepared stocks. Following centrifugation to remove cells, supernatants were filtered and FlgM was detected by immunoblotting, essentially as described23.
ATP was measured using the firefly luciferase assay and sample-processing procedures of ref. 24.
Ampicillin resistance in FlgE–Bla-exporting cells was assayed as described13, with minor modifications described in Methods. Briefly, cells were diluted to D600 = 0.01, cultured for 4.5 h in LB media containing ampicillin in a series of dilutions ranging from 800 g ml-1 to 1.56 g ml-1, and the minimum inhibitory concentration (MIC) was taken as the lowest ampicillin concentration giving D600 < 0.05. The periplasmic fractionation and anti-Bla immunoblot detection of FlgE/Bla in the cellular and periplasmic fractions was performed as described by ref. 25.
Swarming motility was assayed in plates containing tryptone broth and 0.28% bacto-agar. Plates were incubated at 32 °C. Flagellar immunostaining used the methods of ref. 26.
Full methods accompany this paper.
subunit in response to proton motive force and ADP/ATP balance
28-dependent transcription in Salmonella enterica is independent of flagellar shearing
SDS sample buffer
-lactamase activity
