Oligomerization and activation of the FliI ATPase central to bacterial flagellum assembly

Abstract

FliI is the peripheral membrane ATPase pivotal to the type III protein export mechanism underlying the assembly of the bacterial flagellum. Gel filtration and multiangle light scattering showed that purified soluble native FliI protein was in a monomeric state but, in the presence of ATP, FliI showed a propensity to oligomerize. Electron microscopy revealed that FliI assembles to a ring structure, the yield of which was increased by the presence of a non-hydrolysable ATP analogue. Single particle analysis of the resulting electron micrograph images, to which no symmetry was applied, showed that the FliI ring structure has sixfold symmetry and an external diameter of approximately 10 nm. The oligomeric ring has a central cavity of 2.5-3.0 nm, which is comparable to the known diameter of the flagellar export channel into which export substrates feed. Enzymatic activity of the FliI ATPase showed positive co-operativity, establishing that oligomerization and enzyme activity are coupled. Escherichia coli phospholipids increased enzyme co-operativity, and in vitro cross-linking demonstrated that they promoted FliI multimerization. The data reveal central facets of the structure and action of the flagellar assembly ATPase and, by extension, the homologous ATPases of virulence-related type III export systems.

Fig. 1

Multiangle light scattering of purified FliI in buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM ATP and 5 mM MgCl₂). The continuous line represents refractive index on an arbitrary scale, with the height of peaks directly proportional to protein concentration. The dotted line (arrowed) indicates molecular mass, and corresponding mass determinations are shown above each peak.

Fig. 2

Electron microscopy of FliI. Purified FliI, preincubated with AMP-PNP, was stained with 2% uranyl acetate and visualized by the transmission electron microscope. A. Typical field of view at 46 000× magnification. The white scale bar indicates 50 nm. Arrows highlight representative particles. B. Selected images of individual ring-like particles. C. Averaged images lowpass band filtered to their final resolution. The size of each frame is 25 × 25 nm.

Fig. 3

Dependence of FliI ATPase activity on protein and substrate concentration. Specific activity of FliI was measured at (A) different enzyme concentrations in the presence of 5 mM ATP, and (B) different ATP concentrations with 0.5 μM FliI. The plot of the logarithmic form of the Hill equation is shown inset and the Hill coefficient and K_0.5 for ATP are derived from the formula. Activity is expressed as μmol of Pi released min⁻¹ mg⁻¹ FliI.

Fig. 4

In vitro cross-linking of FliI. Purified FliI protein was incubated in cross-linking buffer either alone or with E. coli liposomes, before DSG was added to 0.1 mM. Aliquots were precipitated and subjected to electrophoresis through 4–10% acrylamide gradient gels containing 0.1% SDS.

Fig. 5

Substrate dependence of FliI ATPase activity in the presence of E. coli phospholipids. ATP hydrolysis was measured as a function of ATP concentration in the presence of 0.5 μM FliI plus 300 μg ml⁻¹ phospholipids. The plot of the logarithmic form of the Hill equation is shown inset and the Hill coefficient and K_0.5 for ATP are derived from the formula.