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Flagellin polymerisation control by a cytosolic export chaperone1

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Abstract

Assembly of the long helical filament of the bacterial flagellum requires polymerisation of ca 20,000 flagellin (FliC) monomeric subunits into the growing structure extending from the cell surface. Here, we show that export of Salmonella flagellin is facilitated specifically by a cytosolic protein, FliS, and that FliS binds to the FliC C-terminal helical domain, which contributes to stabilisation of flagellin subunit interactions during polymerisation. Stable complexes of FliS with flagellin were assembled efficiently in vitro, apparently by FliS homodimers binding to FliC monomers. The data suggest that FliS acts as a substrate-specific chaperone, preventing premature interaction of newly synthesised flagellin subunits in the cytosol. Compatible with this view, FliS was able to prevent in vitro polymerisation of FliC into filaments.

Introduction

The principal component of the bacterial flagellum is the long helical filament that rotates as a propeller when torque is transmitted from a motor housed in the basal body.1 Each filament comprises ∼20,000 subunits of monomeric flagellin (FliC) under the filament cap FliD.2 FliC and the other axial proteins making up the contiguous flagellar substructures are exported sequentially by a specialised type III export apparatus in the cytoplasmic membrane, and polymerise at the distal end of the growing cell-surface structure.3, 4, 5 They are assumed to move through the flagellum central channel of 25–30 Å as partially unfolded monomers.6, 7, 8

It seems probable, therefore, that premature folding and interaction of these monomers in the cytosol would have to be actively prevented. This is particularly so for flagellin, which is synthesised in such large amounts and has strong oligomerisation potential. Indeed, FliC polymerises readily into filaments in vitro.9, 10 It is likely that such a cytosolic control mechanism would be targeted especially at the N and C-terminal regions of monomeric FliC, as they are solvent-exposed and unfolded, and their removal hinders filament assembly.11, 12, 13 Upon polymerisation at the distal end of the filament, they become organized into a compact structure and are thought to play a role in quaternary interactions between subunits, maintaining the overall stability and conformation of the axial structure.13, 14, 15, 16

A possible effector of cytosolic polymerisation control would be a dedicated export chaperone, especially as such substrate-specific chaperones bind to the hook-filament junction proteins FlgK and FlgL and the FliD cap, also called the hook-associated proteins or HAPs.17, 18 A candidate chaperone for the flagellin subunit is FliS, which is encoded by a flagellar operon but does not have a known structural or regulatory function in flagellar biogenesis. FliS is small (15 kDa) and predicted to be predominantly α-helical with an acidic pI (4.9), characteristics common to the HAP chaperones and the chaperones of the type III virulence secretion systems thought to have evolved from the “archetypal” flagellar export machinery.19, 20 Compatible with this possibility, a Salmonella typhimurium fliS mutant has been reported to produce short flagella.21 Here, we provide evidence that FliS is an export chaperone specific for FliC, and that it binds to flagellin in vitro and prevents its polymerisation.

Section snippets

FliS is specifically required for the export of flagellin subunits

The fliS gene of motile S. typhimurium SJW1103 was mutated by inserting a chloramphenicol-resistance cassette. Because of polarity exerted on fliT expression (not shown) in the fliDST operon,22 this mutant was named fliST. Loss of FliT specifically impairs export of its substrate, the filament cap FliD, causing the release of unpolymerised, low molecular mass FliC into the extracellular medium.23, 24 Coomassie blue staining of FliC precipitated from culture supernatant shows that the fliT

Discussion

Flagellin translocates through the hollow core of the growing flagellum and polymerises at the distal end of the filament under the FliD cap.28 The N and C-terminal regions of flagellin have been suggested to be responsible for the regulation of FliC polymerisation.8, 16, 29 They are solvent-exposed and have no ordered tertiary structure in the monomeric form, but become folded and ordered upon incorporation in the filament.11, 12, 13 It has been reasonably assumed that the presence of unfolded

Bacterial strains and recombinant DNA manipulations

Wild-type S. typhimurium SJW1103 is motile,36 its derivatives are mutated in flagellar genes flhDC (SJW1368) or fliC (SJW2536).26 Generation of the fliS null mutant was achieved as described for flgN and fliT mutants.24 Briefly, the fliDST operon was amplified by PCR using primers D1 and T2 (Table 2) and cloned into plasmid pBlueScript II SK. The fliS gene was then inactivated by insertion of a chloramphenicol omega cassette after codon 37, and the disrupted locus subcloned into suicide vector

Acknowledgements

We thank R.D. Hayward for assistance with electron microscopy, L. Claret for assistance with FPLC, and E. McGhie and A. Ozin for critical reading of the manuscript. This work was supported by a Wellcome Trust Programme grant (C.H.), an EMBO fellowship (F.A.), and BBSRC (J.T.) and MRC (G.F.) studentships.

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    1

    Edited by I. B. Holland

    2

    Present address: G. M. Fraser, Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.

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