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Interactions of FliJ with the Salmonella Type III Flagellar Export Apparatus

Gillian M. Fraser,^1, Bertha González-Pedrajo,¹ Jeremy R. H. Tame,^2, and Robert M. Macnab^1*

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114,¹ Protonic Nanomachine Project, ERATO, JST, 1-7 Hikaridai, Seika, Kyoto, 619-0237 Japan²

Received 20 May 2003/ Accepted 2 July 2003

FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for FliJ self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association. We present a model summarizing these multiple interactions.

Present address: Department of Pathology, Cambridge University, Cambridge, CB2, 1QP, United Kingdom.

Present address: Yokohama City University, 1-7-29, Suehiro, Tsurumi, Yokohama Kanagawa 230-0045, Japan.

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