Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Oct;194(19):5353-60.
doi: 10.1128/JB.01028-12. Epub 2012 Jul 27.

Interaction of the extreme N-terminal region of FliH with FlhA is required for efficient bacterial flagellar protein export

Noritaka Hara  1 , Yusuke V Morimoto, Akihiro Kawamoto, Keiichi Namba, Tohru Minamino
Affiliations

Interaction of the extreme N-terminal region of FliH with FlhA is required for efficient bacterial flagellar protein export

Noritaka Hara et al. J Bacteriol. 2012 Oct.

Abstract

The flagellar type III protein export apparatus plays an essential role in the formation of the bacterial flagellum. FliH forms a complex along with FliI ATPase and is postulated to provide a link between FliI ring formation and flagellar protein export. Two tryptophan residues of FliH, Trp7 and Trp10, are required for the effective docking of the FliH-FliI complex to the export gate made of six membrane proteins. However, it remains unknown which export gate component interacts with these two tryptophan residues. Here, we performed targeted photo-cross-linking of the extreme N-terminal region of FliH (FliH(EN)) with its binding partners. We replaced Trp7 and Trp10 of FliH with p-benzoyl-phenylalanine (pBPA), a photo-cross-linkable unnatural amino acid, to produce FliH(W7pBPA) and FliH(W10pBPA). They were both functional and were photo-cross-linked with one of the export gate proteins, FlhA, but not with the other gate proteins, indicating that these two tryptophan residues are in close proximity to FlhA. Mutant FlhA proteins that are functional in the presence of FliH and FliI but not in their absence showed a significantly reduced function also by N-terminal FliH mutations even in the presence of FliI. We suggest that the interaction of FliH(EN) with FlhA is required for anchoring the FliI hexamer ring to the export gate for efficient flagellar protein export.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Characterization of weakly motile revertants isolated from a fliI null mutant. (A) Motility of the ΔfliI and ΔfliI fliH(T11P) mutants in soft agar. (B) Motility of the ΔfliI and ΔfliI fliH(T11P) mutants transformed with pET19b-based plasmid encoding FliI-YFP. (C) Effect of the fliH(T11P) mutation on the subcellular localization of FliI-YFP in the ΔfliI and ΔfliI fliH(T11P) mutants. Fluorescent (Epi) and bright-field (BF) images of these cells were observed by epifluorescence microscopy after the cells were exponentially grown in LB at 30°C.
Fig 2
Fig 2
Cysteine modification of the FliH-Cys variants by mPEG-maleimide. FliH containing single cysteines at the indicated positions was expressed. After modification by mPEG-maleimide, the reaction was terminated by adding β-mercaptoethanol, and results were analyzed by immunoblotting with polyclonal anti-FliH antibody. The positions of free and modified FliH are indicated.
Fig 3
Fig 3
Complementation test of FliH amber mutants in the presence of pBPA. (A) Motility of ΔfliH null strain expressing FliH amber mutants. The plates were incubated at 30°C for 6 h with (+) or without (−) 1 mM pBPA. V, pUC19; WT, wild type; W7amber, FliH(W7amber); W10amber, FliH(W10amber). (B) Secretion assay of FliC. Proteins in the culture supernatants (Sup) prepared from the strains listed above were precipitated by TCA, subjected to SDS-PAGE, and analyzed by CBB staining.
Fig 4
Fig 4
Photo-cross-linking between FliHEN and the export gate proteins. Cells coproducing FliH(WT) or FliH(W7pBPA) with FlhA (A), FlhB (B), FliO-FLAG (C), FliP-FLAG (D), FliQ-FLAG (E), FliR-FLAG (F), FlhA(1-351)-YFP (G), or FlhAC (H) were treated (+) or not (−) with UV irradiation for 5 min, followed by SDS-PAGE and finally by immunoblotting with their respective antibodies.
Fig 5
Fig 5
Effect of FliH on the export function of point mutant FlhA proteins. (A) Motility of the ΔfliH ΔflhA strain transformed with pUC19-based plasmids encoding wild-type or mutant FlhA. (B, C) Motility of the ΔfliH ΔflhA strain harboring pUC19-based plasmids encoding wild-type or mutant FlhA and pKG116-based plasmids encoding wild-type FliH (B) or FliH(W7A) (C). (D) Motility of the ΔfliH ΔflhA strain harboring pKG116-based plasmids encoding wild-type or point mutant FlhA and pTrc99A-based plasmid encoding FliI. Fresh transformants were inoculated onto soft agar plates and incubated at 30°C. V, pUC19; WT, wild-type FlhA; R85A, FlhA(R85A); R94K, FlhA(R94K); D208E, FlhA(D208E).
See this image and copyright information in PMC

References

    1. Bange G, et al. 2010. FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system. Proc. Natl. Acad. Sci. U. S. A. 107:11295–11300 - PMC - PubMed
    1. Barker CS, Meshcheryakova IV, Kostyukova AS, Samatey FA. 2010. FliO regulation of FliP in the formation of the Salmonella enterica flagellum. PLoS Genet. 6:e1001143 doi:10.1371/journal.pgen.1001143 - DOI - PMC - PubMed
    1. Chin JW, Martin AB, King DS, Wang L, Schultz PG. 2002. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 99:11020–11024 - PMC - PubMed
    1. Claret L, Susannah CR, Higgins M, Hughes C. 2003. Oligomerisation and activation of the FliI ATPase central to bacterial flagellum assembly. Mol. Microbiol. 48:1349–1355 - PMC - PubMed
    1. Cornelis GR. 2006. The type III secretion injectisome. Nat. Rev. Microbiol. 4:811–825 - PubMed

Publication types

MeSH terms