Recombinant Flagellar protein fliO (fliO)

Advanced Research Questions

• How do suppressor mutations in FliP bypass the requirement for FliO?

  The mechanism by which FliP suppressor mutations bypass FliO deficiency provides significant insight into the functional relationship between these proteins. When a ΔfliO mutant is incubated in motility agar, pseudorevertants emerge with enhanced motility after approximately 42 hours . Genetic analysis identified two key suppressor mutations in FliP: R143H and F190L .

  These suppressor mutations appear to stabilize FliP in the absence of FliO. Experimental evidence shows that engineered strains encoding ΔfliO fliP(F190L) display similar motility to spontaneous pseudorevertants, confirming that this mutation is responsible for suppression . The ΔfliO fliP(R143H) strain showed less motility than the corresponding pseudorevertant strain, suggesting additional unknown suppressors .

  The functional significance of these suppressors lies in understanding how FliO regulates FliP. Current models suggest that FliO normally stabilizes or assists in the correct folding of FliP, and the suppressor mutations render FliP intrinsically more stable or better folded in the absence of this assistance .

• What are the current methodologies for studying FliO-FliP interactions and complex formation?

  Several complementary approaches have been developed to study the interactions between FliO and FliP:

  1. 2D Blue Native PAGE (BN-PAGE): This technique has been crucial for revealing FliP-FliR complex formation. By using anti-FLAG Western blot of 2D BN-PAGE from crude membrane extracts of strains encoding chromosomal FliO-3×FLAG and FliP-3×FLAG, researchers can visualize protein complexes under near-native conditions .

  2. Co-expression studies: By co-expressing FliO with FliP and measuring FliP expression levels, researchers have demonstrated that FliO increases FliP stability .

  3. Genetic suppressor analysis: The identification of suppressor mutations in fliP that bypass fliO deletions has been instrumental in understanding their functional relationship .

  4. Protein truncation analysis: By expressing truncated versions of FliO and testing their ability to complement ΔfliO mutants, researchers have identified functional domains involved in FliP interaction .

  5. Membrane topology analysis: PhoA and GFP fusion studies have helped determine the topology of FliO, which is essential for understanding how it interacts with other membrane components .

• How does FliO function within the larger context of the flagellar type III secretion system?

  FliO is part of a complex flagellar type III secretion system that includes several membrane proteins: FliO, FliP, FliQ, FliR, FlhA, and FlhB . The interactions between these proteins create the export gate for flagellar proteins.

  Within this system, FliO appears to have a specialized role in regulating FliP . FliP interacts with FlhA and is a core component of the export gate . Recent research has shown that FliO, FliP, and FliQ interact with FlhA, while FliR interacts with both FlhA and FlhB .

  The flagellar protein export system operates with remarkable efficiency, coordinating protein export with assembly in a well-controlled manner . The export apparatus contains an export gate complex made of six membrane proteins, and FliO appears to be peripheral to this core complex but essential for its proper assembly and function .

  Experimental evidence for interactions within this complex has been obtained through photo-cross-linking experiments. For example, researchers have shown that when tryptophan residues in FliH (another component of the export apparatus) are replaced with p-benzoyl-phenylalanine (a photo-cross-linkable amino acid), the modified protein cross-links with FlhA but not with other gate proteins, demonstrating specific interaction points .

• What experimental approaches can resolve contradictions in FliO function across different bacterial species?

  Researchers face several conflicting data points about FliO function across bacterial species. For example, in Pseudomonas aeruginosa, FliO appears to be involved in adhesion to mucin, a function not reported in Salmonella . To resolve these contradictions, several experimental approaches are recommended:

  1. Comparative genomics and phylogenetic analysis: Analyzing FliO conservation and variation across species can identify functional domains under different selective pressures.

  2. Cross-species complementation experiments: Testing whether FliO from one species can complement deletions in another provides functional insights into conserved mechanisms.

  3. Domain swapping experiments: Creating chimeric FliO proteins with domains from different species can identify which regions are responsible for species-specific functions.

  4. High-resolution structural studies: Comparing the structures of FliO from different species can reveal conformational differences that explain functional divergence.

  5. Standardized phenotypic assays: Developing consistent assays for flagellar assembly, motility, and protein export across species allows direct comparison of FliO function.

  In Pseudomonas aeruginosa, for instance, FliO has been implicated in both flagellar assembly and adhesion to mucin . A ΔfliO mutant in P. aeruginosa was found to be both nonmotile and nonadhesive to mucin, suggesting a dual function . Similar comprehensive phenotypic characterization in other species would help clarify whether these functions are conserved or species-specific.

• How can researchers effectively study the dynamic assembly of the flagellar export apparatus including FliO?

  Studying the dynamic assembly of membrane protein complexes like the flagellar export apparatus presents significant technical challenges. Several cutting-edge methodologies have proven effective:

  1. Single-molecule fluorescence microscopy: This technique has been used to study the stoichiometry and assembly dynamics of flagellar proteins with single-molecule precision. For example, FliI-YFP fusion proteins have been tracked to determine their association with the flagellar basal body and exchange rates .

  2. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS): This approach can define solvent-accessible regions of proteins and evaluate their dynamic behavior, as demonstrated with the flagellar capping protein FliD . Similar approaches could be applied to study FliO dynamics.

  3. Time-course cryo-electron tomography (cryo-ET): By capturing flagellar structures at different assembly stages, researchers can visualize intermediate complexes containing FliO and other components.

  4. Genetic approaches using assembly checkpoints: Using mutants blocked at specific stages of flagellar assembly, such as ΔrpoN mutants that lack rod and other periplasmic structures, researchers can isolate and characterize intermediate complexes .

  5. In vitro reconstitution systems: Purified components can be combined in controlled environments to study assembly steps and requirements.

  These approaches have revealed that flagellar assembly is a highly dynamic process, with proteins like FliI undergoing rapid exchange between flagellar basal body-localized and free-diffusing forms . Similar dynamics likely apply to FliO and other export apparatus components.

• What is the mechanism of interaction between FliO and the flagellar export gate complex?

  The precise mechanism of FliO interaction with the flagellar export gate complex remains an active area of research, but several key findings provide insights:

  1. FliO appears to function primarily by stabilizing FliP, which is a core component of the export gate .

  2. Recent studies of the flagellar type III secretion system have shown that FliO, FliP, and FliQ interact with FlhA, one of the central components of the export gate .

  3. The cytoplasmic domain of FliO (residues 43-125) is sufficient to restore motility when overexpressed in a ΔfliO mutant, indicating that this domain contains key functional elements for interaction with the export gate .

  4. Residue L91 in the cytoplasmic domain of FliO is critical for function, suggesting its involvement in protein-protein interactions .

  5. Unlike other export gate components that form stable complexes detectable by blue native PAGE, FliO appears to have a more transient or regulatory association with the core complex .

  Current models suggest that FliO acts as a chaperone or assembly factor for the export gate, rather than being a structural component of the mature complex. This would explain why FliO deletion can be partially bypassed by mutations that stabilize FliP . Further structural studies of the complete flagellar export apparatus will be needed to fully elucidate the molecular details of these interactions.