The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Ozin, Amanda J.; Claret, Laurent; Auvray, Frédéric; Hughes, Colin

doi:10.1016/s0378-1097(02)01208-9

Cited by 53 publications

(62 citation statements)

References 25 publications

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“…In pseudomonads the role of the FliS protein remains unknown. Its distant homologue in enterobacteria has been described as a substrate-specific cytosolic chaperone that facilitates FliC secretion and contributes to the stabilization of the flagellin subunits during polymerization (Auvray et al, 2001;Ozin et al, 2003). The F113 fliS mutant has a very short and thin flagellum (Fig.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

et al. 2004

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The ability of plant-associated micro-organisms to colonize and compete in the rhizosphere is specially relevant for the biotechnological application of micro-organisms as inoculants. Pseudomonads are one of the best root colonizers and they are widely used in plant-pathogen biocontrol and in soil bioremediation. This study analyses the motility mechanism of the well-known biocontrol strain Pseudomonas fluorescens F113. A 6?5 kb region involved in the flagellar filament synthesis, containing the fliC, flaG, fliD, fliS, fliT and fleQ genes and part of the fleS gene, was sequenced and mutants in this region were made. Several non-motile mutants affected in the fliC, fliS and fleQ genes, and a fliT mutant with reduced motility properties, were obtained. These mutants were completely displaced from the root tip when competing with the wild-type F113 strain, indicating that the wild-type motility properties are necessary for competitive root colonization. A mutant affected in the flaG gene had longer flagella, but the same motility and colonization properties as the wild-type. However, in rich medium or in the absence of iron limitation, it showed a higher motility, suggesting the possibility of improving competitive root colonization by manipulating the motility processes. INTRODUCTIONThe study of rhizosphere colonization by micro-organisms is crucial for the efficient application of bacteria as inoculants, both in agricultural and in environmental biotechnology processes. Pseudomonas spp. can colonize the roots of a wide range of plants (Simons et al., 1996;Naseby & Lynch, 1998;Villacieros et al., 2003), being one of the best root colonizers, and are used as a model in root-colonization studies (Bloemberg et al., 2000; Chin-a-Woeng et al., 2000). The rhizosphere is a complex environment that supports a large and metabolically active microbial population, several orders of magnitude higher than the non-rhizospheric soil. Many bacterial genes and traits have been shown to be involved in plant-root colonization (Lugtenberg & Dekkers, 1999;Rainey, 1999;Lugtenberg et al., 2001). However, not only colonization but also the pseudomonads' ability to compete with the indigenous microbial population are essential to improve their biotechnological applications in the rhizosphere environment.The soil-borne fluorescent pseudomonads are used as biocontrol inoculants because of their ability to produce some antifungal metabolites (Dowling & O'Gara, 1994;Walsh et al., 2001). Other applications of pseudomonads include soil biofertilization and rhizoremediation (Ramos et al., 1991; Brazil et al., 1995; Höflich et al., 1995;Yee et al., 1998).The strain Pseudomonas fluorescens F113 was isolated from the sugarbeet rhizosphere and it is used as a biocontrol agent against the fungal pathogen Pythium ultimum, which causes damping-off disease in sugarbeet seedlings. The biocontrol abilities of this strain are due mainly to the production of the antifungal metabolite DAPG (2,4-diacetylphloroglucinol) (Shanahan et al., 1992). P. fluorescens ...

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Section: Discussionmentioning

confidence: 99%

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

et al. 2004

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“…Export of these structural components is strongly facilitated by cytosolic substrate-specific chaperones, which bind respectively to the C-terminal amphipathic domains of their substrates FlgK and FlgL (chaperone FlgN), FliD (FliT), and FliC (FliS) (6)(7)(8). Although primary sequence identity is low or inapparent between the chaperones of the flagellar and virulence type III export systems, most are small, homodimeric proteins with a predicted C-terminal amphipathic helix (6,9,10), and it is suggested that they act as ''bodyguards'' to maintain monomers in an export-competent form and prevent their interaction before export (6,(10)(11)(12)(13). In the flagellar chaperones FlgN and FliT and the virulence chaperone CesT, the C-terminal region has been shown to mediate substrate binding, whereas the N terminus is responsible for homodimerization (7,14).…”

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confidence: 99%

Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export

Thomas

Stafford

Hughes

2004

Proc. Natl. Acad. Sci. U.S.A.

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129

155

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Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells. The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus. We have identified a stalled intermediate export complex in the flagellar type III export pathway of Salmonella typhimurium by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium. These chaperone variants bound their specific export substrates strongly and severely reduced their export. They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway. Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus. Membrane localization persisted in fliOPQR, flhB, flhA, fliJ, and fliH null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI. After expression of the variant chaperones in Salmonella, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated. Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate. Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.

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“…In the case of SseB, it appears that the chaperone impinges upon domains (Fig. 6) likely to participate in subunit assembly, as demonstrated for the flagellar chapereone, FliS (Auvray et al, 2001;Ozin et al, 2003). In addition, a second instance where the stabilization and secretion functions of a chaperone can be uncoupled has been reported (Cheng & Schneewind, 1999;Fig.…”

Section: Discussionmentioning

confidence: 90%

“…A function provided by some virulence chaperones (Neyt & Cornelis, 1999; reviewed by Parsot et al, 2003) and by C-terminal binding flagellar chaperones (Auvray et al, 2001;Ozin et al, 2003) is to control association of subunits before export. It was anticipated that this is the role provided by SseA binding to SseB (Zurawski & Stein, 2003), and the location of the binding region supports this hypothesis.…”

Section: A Model For Ssea Functionmentioning

confidence: 99%

“…This suggests that at least part of the domain occupied by SseA involves the putative coiled-coil. Occupancy of a self-association domain, as demonstrated for flagellar subunit chaperones (Auvray et al, 2001;Ozin et al, 2003), is predicted to interfere with SseB assembly into a translocon sheath. In addition, heterologous protein-protein interactions occurring with SseB are also likely, as suggested by the EPEC paradigm for Esc TTS phylogenetic class.…”

Section: A Model For Ssea Functionmentioning

confidence: 99%

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The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Zurawski

Stein

2004

Microbiology

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SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

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The FliS chaperone selectively binds the disordered flagellin C-terminal D0 domain central to polymerisation

Cited by 53 publications

References 25 publications

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

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