Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits

Abstract

Many flagellar proteins are exported by a flagellum-specific export pathway. In an initial attempt to characterize the apparatus responsible for the process, we designed a simple assay to screen for mutants with export defects. Temperature-sensitive flagellar mutants of Salmonella typhimurium were grown at the permissive temperature (30°C), shifted to the restrictive temperature (42°C), and inspected in a light microscope. With the exception of switch mutants, they were fully motile. Next, cells grown at the permissive temperature had their flagellar filaments removed by shearing before the cells were shifted to the restrictive temperature. Most mutants were able to regrow filaments. However, flhA,fliH, fliI, and fiN mutants showed no or greatly reduced regrowth, suggesting that the corresponding gene products are involved in the process of flagellum-specific export. We describe here the sequences offliH,fliI, and the adjacent gene,flij; they encode proteins with deduced molecular masses of 25,782, 49,208, and 17,302 Da, respectively. The deduced sequence of Fliu shows significant similarity to the catalytic 0i subunit of the bacterial FOF, ATPase and to the catalytic subunits of vacuolar and archaebacterial ATPases; except for limited similarity in the motifs that constitute the nucleotide-binding or catalytic site, it appears unrelated to the ElE2 class of ATPases, to other proteins that mediate protein export, or to a variety of other ATP-utilizing enzymes. We hypothesize that FliI is either the catalytic subunit of a protein translocase for flagellum-specific export or a proton translocase involved in local circuits at the flagellum.The bacterial flagellum is a complex structure comprising intracellular, envelope-spanning, and extracellular components. In Salmonella typhimurium, it is encoded by about 40 genes (39).Of those flagellar proteins located within the cell envelope or outside the cell, only the outer-ring (P and L) proteins appear to be exported by the primary signal peptide-dependent pathway (33,36,42). The rest (four rod proteins, a hook protein, three hook-associated proteins, and the filament protein, flagellin) are thought to be exported by a unique, flagellum-specific pathway (30,34,38,59), travelling through the hollow core of the nascent structure (70) and assembling at its distal end (19, 37) (Fig. 1). The process must be a highly organized one, in which protein subunits are incorporated into the growing flagellum in the correct order and stoichiometry (45). The export apparatus obviously must be selective in the proteins it recognizes for export. It may also have to supply energy for the process. Given that the physical path for export is through the nascent structure, it seems likely that the export apparatus is located at the flagellar base (Fig. 1).Essentially nothing is known about this apparatus; specifically, its genetic origin and biochemical composition are completely unknown. We describe here initial attempts to identify its components genetically by use of temperaturesensitive ...

Bacterial flagellation and cell division

“…There are several lines of evidence suggesting that the export apparatus is localized to the basal structure (Vogler et al 1991;Fan et al 1997;Ohnishi et al 1997). A small dome with a dip found in the C ring, C rod, is a good candidate for the export apparatus (Katayama et al 1996).…”

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body

“…The subunits of the rod, hook, hook cap, hook-filament junction, filament and filament cap are thought to travel through a hollow channel in the nascent structure (Mimori et al, 1995;Morgan et al, 1995) and to assemble at its distal end (Iino, 1969;Emerson et al, 1970). The export apparatus was proposed to be at the base of the flagellum and to be assembled itself at an early stage of flagellar biosynthesis, probably after the assembly of the MS ring and switch complex Vogler et al, 1991;Kubori et al, 1997). There is little in the way of physical evidence for the presence of such an export machinery in the flagellar basal body.…”

“…There is little in the way of physical evidence for the presence of such an export machinery in the flagellar basal body. Filament assembly studies with mutants and sequence similarity to proteins in other type III systems have implicated several flagellar proteins in the export process: FlhA, FlhB, FliH, FliI, FliP, FliQ and FliR (Vogler et al, 1991;. Among them, FliI, which shows sequence similarity to the catalytic ␤-subunit of the F 0 F 1 proton-translocating ATP synthase (Vogler et al, 1991), was demonstrated to be an ATPase, presumably providing energy for the export process or possibly acting as a chaperone (Dreyfus et al, 1993;Fan and Macnab, 1996).…”

Energy source of flagellar type III secretion

“…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).…”

“…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).…”