Bacterial motility is commonly achieved by rotation of cell surface flagella. The well-characterized flagellum of Escherichia coli and Salmonella is a long helical filament, the propeller, attached to a flexible hook anchored to the cell envelope by the flagellar basal body (Macnab, 1996; Namba and Vonderviszt, 1997). The flagellum is assembled by polymerization of subunits that form the rod, hook, hook–filament junction, filament cap and filament at the distal end of the growing flagellar structure. These subunits diffuse through the channel at the centre of the flagellum, but they must first be exported from the cytosol by a dedicated type III export mechanism (Macnab, 1999; Aizawa, 2001).
The flagellar export apparatus comprises six integral membrane proteins, and it has been speculated that these could sit in the patch of membrane within the MS ring of the flagellar basal body (Fan and Macnab, 1996; Minamino and Macnab, 1999; Kihara et al., 2001). Delivery of structural subunits to the membrane apparatus is aided by several subunit-specific chaperones (Fraser et al., 1999; Auvray et al., 2002), but essential to the export mechanism is the flagellar ATPase FliI that provides energy to the export process (Vogler et al., 1991; Dreyfus et al., 1993), and is also assumed to be centrally involved in the series of protein–protein interactions underlying the translocation of substrates to the membrane apparatus. It is known that FliI interacts with another flagellar export component, FliH, in the cytosol and/or at the membrane and that this negatively regulates FliI activity (Minamino and Macnab, 2000a; Minamino et al., 2001), and that FliJ, which is proposed to be a general flagellar chaperone, interacts with FliH (Minamino et al., 2000; Minamino and Macnab, 2000b;Gonzalez-Pedrajo et al., 2002) and FliI (L. Claret, unpublished). Furthermore, interactions have been reported between both FliI and FliH and the cytosolic domains of the integral membrane proteins FlhA and FlhB (Minamino and Macnab, 2000b;Zhu et al., 2002). Despite the emerging view of these and other interactions, little is known about the behaviour of FliI, in particular how the flagellar ATPase activity is coupled to the export process. It has been reported that FliI is a cytosolic monomeric protein (Minamino and Macnab, 2000a), and it has been suggested that active FliI monomers ‘scavenge’ for substrates and target them for export at the flagellar basal body (Macnab, 1996) or, alternatively, FliI monomer could be held inactive by FliH in the cytosol until it docks as a complex with delivered substrate at the membrane machinery within the MS ring where ATP hydrolysis is triggered (Macnab, 1999; Minamino and Macnab, 2000a). We have subsequently shown that, although FliI lacks transmembrane domains, it behaves as a peripheral membrane protein when expressed physiologically in Salmonella (Auvray et al., 2002), and that purified FliI has an intrinsic affinity for E. coli phospholipids, i.e. even without the integral membrane machinery of the export pathway (Auvray et al., 2002). Our experiments also showed that these phospholipids stimulate FliI ATPase activity in vitro (Auvray et al., 2002). These experimental findings provided direct support for the view that FliI interacts with the membrane, while leaving open the many questions regarding which interactions with cytosolic and membrane proteins of the flagellar pathway precede or follow such interaction. By extension, the results would indicate a comparable membrane interaction by the homologous ATPases of virulence-related type III export systems (Venkatesan et al., 1992; Eichelberg et al., 1994; Woestyn et al., 1994).
This view of membrane interaction appears to be strengthened by the similar peripheral membrane localization of ATPases energizing bacterial type IV transport across the cytoplasmic membrane despite lacking predicted transmembrane domains (Bhattacharjee et al., 2001). Provocatively, these ATPases have been shown to exist in an oligomeric state, specifically forming hexameric ring structures (Krause et al., 2000a,b; Yeo et al., 2000). These findings in another bacterial export pathway suggested to us the possibility that FliI activity might be coupled to the assembly of an active, multimeric form at the membrane. Therefore, we have assessed in this study whether the flagellar ATPase FliI can assemble to a multimeric state, if there might be a connection between such multimerization and enzymatic activation and whether this might be promoted by contact with phospholipid.