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. 2013 Feb;195(3):466-73.
doi: 10.1128/JB.01711-12. Epub 2012 Nov 16.

Interaction between FliJ and FlhA, components of the bacterial flagellar type III export apparatus

Tatsuya Ibuki  1 , Yumiko Uchida, Yusuke Hironaka, Keiichi Namba, Katsumi Imada, Tohru Minamino
Affiliations

Interaction between FliJ and FlhA, components of the bacterial flagellar type III export apparatus

Tatsuya Ibuki et al. J Bacteriol. 2013 Feb.

Abstract

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH(X)-FliI(6) ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues-Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83-of FliJ. A structural model of the FliI(6)-FliJ ring complex suggests that they extend out of the FliI(6) ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.

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Figures

Fig 1
Fig 1
Interaction of GST-FliJ with FlhA. (A) Motility assays of MKM40 (ΔfliJ) transformed with pTrc99AFF4 (V), pGEX6p-1 (GST), pMM404 (wild-type FliJ, indicated as FliJWT), or pMMJ1001 (GST-FliJ) in soft agar plates. The plates were incubated at 30°C for 6 h. (B) Dominant-negative effect of GST-FliJ on motility of wild-type cells (upper panel) and the ΔfliH-fliI flhB(P28T) bypass mutant (lower panel). The motility of SJW1103 (wild type) and MMHI0017 (ΔfliH-fliI, flhB*) transformed with pGEX-6p-1 (GST) or pMMJ1001 (GST-FliJ) in soft agar plates was evaluated. (C) Effect of external pH on FlgD secretion by the ΔfliH-fliI flhB(P28T) bypass mutant. Immunoblotting was performed with the polyclonal anti-FlgD antibody of whole-cell and culture supernatant fractions prepared from the ΔfliH-fliI flhB(P28T) bypass mutant overexpressing GST or GST-FliJ grown at external pH values of 6.0 and 7.0. (D) Pulldown assays by GST affinity chromatography. The soluble fractions (indicated as L) from the ΔfliH-fliI flhB(P28T) bypass mutant overproducing GST or GST-FliJ were loaded onto a GST column. After being washed with 10 ml of PBS, the proteins were eluted with a buffer containing 10 mM reduced glutathione. The eluted factions containing GST or GST-FliJ were analyzed by CBB staining (upper panels), while the eluted FlhA protein was done by immunoblotting with polyclonal anti-FlhAC antibody (lower panels).
Fig 2
Fig 2
Molecular model of the FliI6-FliJ ring complex. (A) Cα backbone trace of the FliI6-FliJ complex. The FliI subunits are colored blue, violet, green, yellow, lilac, and red, and FliJ is colored cyan. (B) Model viewed from the top of panel A. (C) The eight conserved residues are labeled and highlighted in red. The FliI subunits are colored gray. (D) Binding region of FliJ for FliI. The subunit shown in violet in panel A is removed for better visualization of FliJ. Residues 13 to 24 of FliJ are shown in magenta. The region including these residues interacts with FliI.
Fig 3
Fig 3
Effect of FliJ mutations on the FliJ-FlhA interaction. (A) Expression levels of various point mutant variants of GST-FliJ. Whole-cell lysates were prepared from the ΔfliH-fliI flhB(P28T) bypass mutant (ΔfliH-fliI flhB*) transformed with pGEX-6p-1-based plasmids encoding various forms of GST-FliJ and subjected to SDS-PAGE, followed by CBB staining. Lane 1, GST; lane 2, GST-FliJ (indicated as wild type [WT]); lane 3, GST-FliJ(Q38A) (indicated as Q38A); lane 4, GST-FliJ(L42A) (indicated as L42A); lane 5, GST-FliJ(Y45A) (indicated as Y45A); lane 6, GST-FliJ(Y49A) (indicated as Y49A); lane 7, GST-FliJ(F72A) (indicated as F72A); lane 8, GST-FliJ(L76A) (indicated as L76A); lane 9, GST-FliJ(A79S) (indicated as A79S); lane 10, GST-FliJ(H83A) (indicated as H83A). (B) Dominant-negative effect of GST-FliJ point mutant variants on motility of the ΔfliH-fliI flhB(P28T) bypass mutant (ΔfliH-fliI flhB*). The motility of the same transformants in soft agar was evaluated. The plates were incubated at 30°C for 7 h. (C) Pulldown assays by GST affinity chromatography. The soluble fractions (L) prepared from the above strains were loaded onto a GST column. After extensive washing with 10 ml of PBS, the proteins were eluted with a buffer containing 10 mM reduced glutathione. The eluted fractions were analyzed by CBB staining (upper panel) and immunoblotting with polyclonal anti-FlhA antibody (lower panel).
Fig 4
Fig 4
Effect of the F72A and L76A mutations on the export function of FliJ. (A) Motility (left panel) and secretion assays (right panel) of a ΔfliJ mutant harboring pTrc99AFF4 (V), pMM404 (pTrc99AFF4/wild-type FliJ, indicated as FliJWT), pTIJ105 [pTrc99AFF4/FliJ(F72A), indicated as F72A], or pTIJ106 [pTrc99AFF4/FliJ(F76A), indicated as F76A]. The plates were incubated at 30°C for 4.5 h. Immunoblotting with polyclonal anti-FlgD or anti-FliJ antibody of whole-cell (Cell) and culture supernatant (Sup) fractions prepared from the above transformants. (B) Motility (left panel) of and FlgD secretion (right panel) by a ΔfliH-fliI-fliJ flhB(P28T) mutant transformed with the above plasmids. The plates were incubated at 30°C for 8 h. The level of FlgD secretion was analyzed by immunoblotting with the polyclonal anti-FlgD antibody. The expression level of FliJ was detected by immunoblotting with polyclonal anti-FliJ antibody.
Fig 5
Fig 5
Effect of the F72A and L76A mutations on the interaction with FliI ATPase. (A) Pulldown assays by GST affinity chromatography. A mixture of the soluble fractions (L) prepared from a ΔflhDC-cheW mutant expressing GST (first row), GST-FliJ (second row), GST-FliJ(F72A) (third row), or GST-FliJ(L76A) (fourth row) with those from the ΔflhDC-cheW mutant producing His-FliI were loaded onto a GST column. The eluted fractions were analyzed by both CBB staining (upper panels) and immunoblotting with polyclonal anti-FliI antibody (lower panels). (B) Effect of FliJ substitutions on the ATPase activity of FliI. The ATPase activity of FliI alone and the mixture of FliI with wild-type FliJ (FliJWT), FliJ(F72A) (F72A), or FliJ(L76A) (L76A) at the molar ratio of 6 FliI to 1 FliJ was measured at 37°C with an enzyme-coupled ATP-regenerating system. At least three independent experiments were carried out for each mixture. Vertical bars indicate the standard deviations.
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