Recombinant Salmonella typhimurium Flagellar M-ring protein (fliF)

What is the FliF protein and what is its structural significance in bacterial flagella?

FliF is a 65-kDa protein that constitutes the main component of the MS ring in the flagellar basal body of Salmonella typhimurium. The MS ring is embedded in the cytoplasmic membrane and is positioned at the critical site where transmembrane proton potential energy is converted into the mechanical work necessary for flagellar rotation . FliF forms a planar, inner membrane-embedded ring structure that connects to both the type III secretion system (T3SS) at the cytoplasmic base and the axial structure that extends through the periplasmic space .

Methodologically, researchers identified FliF through immunological approaches using monoclonal antibodies directed against flagellar hook-basal body complexes. Immunoelectron microscopy confirmed FliF localization to the cytoplasmic-facing surface of the M ring . The protein was initially assigned as the product of the flaAII.1 gene before current nomenclature established it as the fliF gene product.

How does the MS ring interact with other flagellar components?

The MS ring acts as a central architectural element that houses and interacts with multiple flagellar components:

• It encloses the flagellar type III secretion system (fT3SS) export apparatus

• It provides the foundation for assembly of the cytoplasmic C ring

• It connects to the rod structure via the FliE protein adapter

Research has demonstrated specific physical interactions between FliF (MS ring) and FlhA (an integral membrane component of the export apparatus). This was established through genetic studies where mutations in FliF could be suppressed by second-site mutations in FlhA, providing evidence against the "floating island" model and supporting direct structural interaction .

Membrane fractionation experiments have shown that even certain fliF mutations that prevent export are still mild enough to permit both MS ring assembly and the subsequent attachment of the cytoplasmic C ring to the MS ring .

What techniques are most effective for studying FliF-protein interactions?

Several complementary approaches have proven valuable for investigating FliF interactions:

Genetic suppressor analysis: This approach identifies compensatory mutations that restore function in FliF mutants. Researchers have successfully isolated intergenic suppressors in genes coding for interacting proteins like FlhA . When mutations in fliF are suppressed by mutations in another gene, it suggests a direct physical interaction between the proteins.

Immunoelectron microscopy: This technique allows precise localization of FliF within the flagellar structure by using specific antibodies conjugated to electron-dense markers. This approach confirmed FliF's position at the cytoplasmic-facing surface of the M ring .

Protein-protein binding assays: In vitro studies using purified recombinant proteins can determine direct binding between FliF and other flagellar components.

Fluorescent protein fusions: FliM-GFP fusions have been used as proxies for monitoring flagellar numbers per cell, which can indirectly indicate MS ring assembly competence .

What are the optimal methods for cloning and expressing recombinant FliF protein?

For successful cloning and expression of recombinant FliF protein, researchers typically employ the following methodology:

Cloning strategy:

• Amplify the fliF gene from Salmonella typhimurium genomic DNA using PCR

• Create appropriate restriction sites (e.g., NdeI and BamHI) for directional cloning

• Insert the PCR product into an expression vector such as pTrc99A1de4

Expression system considerations:

• E. coli BL21(DE3) is commonly used as the host strain for expressing membrane proteins

• Expression at reduced temperatures (16-20°C) often improves proper folding

• Induction with lower concentrations of IPTG (0.1-0.5 mM) can reduce inclusion body formation

Optimization parameters:

• Growth medium composition (LB, TB, or minimal media depending on experimental needs)

• Cell density at induction point (typically mid-log phase, OD600 of 0.6-0.8)

• Duration of expression (4-16 hours)

It's important to note that as a membrane protein, FliF presents particular challenges for recombinant expression and may require detergent solubilization for downstream applications.

How can researchers evaluate the structural integrity and functionality of purified FliF protein?

Multiple complementary approaches should be employed to assess both structural integrity and functional activity:

Structural integrity analysis:

• Circular dichroism (CD) spectroscopy to evaluate secondary structure composition

• Size-exclusion chromatography to confirm proper oligomerization state

• Limited proteolysis to identify correctly folded domains resistant to degradation

• Thermal shift assays to measure protein stability

Functionality assessment:

• In vitro binding assays with known interaction partners (e.g., FlhA, FliE)

• Complementation studies in fliF mutant strains to assess ability to restore motility

• Electron microscopy to visualize MS ring formation in reconstitution experiments

A particularly effective approach is to create fliF gene fusions with reporter constructs like β-galactosidase to quantitatively measure expression levels and compare wild-type and mutant protein activity, similar to approaches used for other flagellar genes .

What mutagenesis strategies are most informative for studying FliF structure-function relationships?

Targeted mutagenesis approaches have proven highly valuable for dissecting FliF's functional domains:

Site-directed mutagenesis:
Focused on conserved amino acids or predicted functional domains based on structural information. Key targets include:

• Residues at protein-protein interaction interfaces

• Membrane-spanning segments

• Highly conserved motifs across bacterial species

Domain swapping:
Exchanging domains between FliF proteins from different bacterial species can identify regions responsible for species-specific functions.

Deletion analysis:
Similar to the approach used for FliE , creating a series of small deletions throughout the fliF coding sequence can map functional regions. In the FliE study, researchers replaced coding segments with tetracycline resistance cassettes, which can be subsequently removed using fusaric acid selection .

Suppressor screening:
After generating fliF mutants with motility defects, researchers can isolate spontaneous or induced suppressors. These suppressor mutations, particularly those in genes encoding interacting partners like flhA, provide valuable insights into protein interaction networks .

What is currently known about the stoichiometry and symmetry of FliF in the MS ring assembly?

The MS ring, primarily composed of FliF protein, exhibits specific symmetry properties that are crucial for its function. Based on current research findings:

• The MS ring displays rotational symmetry, which is critical for its interaction with the N-terminal α-helix of FliE .

• This symmetry alignment is essential for proper flagellar assembly and rotation.

• The exact number of FliF subunits in the MS ring has been estimated from structural studies, but remains an active area of investigation.

Researchers studying the interactions between FliE and FliF have found that the N-terminal α-helix of FliE specifically interacts with the MS ring with a defined rotational symmetry . This rotational arrangement is likely critical for proper opening of the export channel gate of the flagellar type 3 secretion system.

Methodologically, determining precise stoichiometry requires advanced structural biology techniques such as cryo-electron microscopy combined with image analysis and 3D reconstruction. Mass photometry is an emerging technique that could provide additional insights into the exact number of FliF subunits per MS ring.

How do mutations in FliF affect flagellar assembly and motility?

Mutations in FliF can produce a range of phenotypic effects on flagellar assembly and bacterial motility:

Effects on flagellar export:
Some fliF mutations prevent proper protein export through the flagellar type III secretion system while still allowing MS ring assembly and C-ring attachment . This suggests that specific regions of FliF are critical for export apparatus function but not structural assembly.

Impact on flagellar numbers:
Wild-type Salmonella typically produces approximately 6-10 flagella arranged peritrichously around the cell . Quantitative assessment of flagellar numbers can be performed using fluorescent protein fusions like FliM-GFP, which form spots associated with nascent C-rings that correlate with flagella numbers .

Motility phenotypes:
Mutations in fliF can be assessed using soft-agar motility plates, where the size of the motility ring correlates with swimming capability. This provides a straightforward phenotypic readout for functional analysis of FliF variants .

Suppressor interactions:
Certain mutations in fliF can be suppressed by second-site mutations in other flagellar genes, including flhA and fliE . These genetic interactions provide valuable insights into the functional relationships between FliF and other flagellar components.

The molecular mechanism by which FliF mutations affect flagellar function can be complex, involving perturbations in protein-protein interactions, alterations in the export apparatus function, or disruptions in the mechanical properties of the MS ring.

What is the relationship between FliF and the flagellar type III secretion system (fT3SS)?

FliF plays a pivotal role in the assembly and function of the flagellar type III secretion system:

• The MS ring formed by FliF houses the export apparatus components of the fT3SS .

• FliF interacts directly with components of the export apparatus, particularly FlhA .

• Specific mutations in FliF can be suppressed by second-site mutations in FlhA, indicating a functional relationship .

• FliF works in conjunction with FliE, which completes the T3S structure - an fliE null strain exhibits an 8-fold reduction in secretion of Hook (FlgE) protein .

Research indicates that the association between FliF and the export apparatus is not a "floating island" arrangement but involves specific physical interactions. Immunoelectron microscopy positioned FliR, another component of the export apparatus, in the vicinity of the cytoplasmic face of the MS ring .

The exit gate of the protein export channel appears to require proper interactions between FliF, FliR, FlgB, and FlgC, which are facilitated by the adapter protein FliE . This highlights the critical role of FliF in creating a functional secretion pathway for flagellar proteins.

How can researchers distinguish between direct and indirect effects when interpreting FliF mutation phenotypes?

Distinguishing between direct and indirect effects of FliF mutations requires a multi-faceted experimental approach:

Complementation analysis:

• Express wild-type FliF from a plasmid in the mutant background

• Quantify the degree of phenotypic rescue (motility, flagellar number)

• Complete restoration suggests direct effects, while partial rescue may indicate indirect effects

Dominance testing:
Expressing mutant FliF in a wild-type background can reveal whether mutations have dominant effects. Studies have shown that certain mutant FliF proteins are partially dominant over wild-type FliF in both wild-type and second-site FlhA backgrounds .

Structural analysis:
Membrane fractionation experiments can determine if mutations affect MS ring assembly itself or downstream processes. Some fliF mutations permit MS ring assembly and C-ring attachment while still preventing export, indicating structure-function separability .

Protein-protein interaction studies:
Direct binding assays between mutant FliF and its known interaction partners can help determine if mutations directly affect specific protein interfaces.

Epistasis analysis:
Testing genetic interactions with mutations in other flagellar genes helps place the effect of fliF mutations in the context of the flagellar assembly pathway.

What bioinformatic approaches are valuable for analyzing FliF conservation and predicting functional domains?

Several bioinformatic approaches provide insights into FliF structure and function:

Sequence conservation analysis:

• Multiple sequence alignment of FliF proteins across bacterial species

• Identification of highly conserved residues that likely play critical functional roles

• Analysis of co-evolving residues that may indicate interaction surfaces

Domain prediction:

• Identification of transmembrane segments using programs like TMHMM or Phobius

• Recognition of conserved motifs or domains using PFAM or SMART databases

• Secondary structure prediction to identify alpha-helical and beta-sheet regions

Structural modeling:

• Homology modeling based on related proteins with known structures

• Integration of experimental constraints from crosslinking or mutagenesis data

• Molecular dynamics simulations to predict protein flexibility and conformational changes

Co-evolution analysis:
Modern approaches like Direct Coupling Analysis (DCA) or Statistical Coupling Analysis (SCA) can identify pairs of residues that have co-evolved, suggesting physical proximity or functional relationships.

Genomic context analysis:
Examination of gene neighborhoods across species can provide insights into functional relationships between FliF and other flagellar components.

How can contradictory data regarding FliF interactions be reconciled in the context of flagellar assembly models?

Researchers often encounter seemingly contradictory data regarding protein interactions in complex molecular machines like the flagellum. Several approaches can help reconcile such conflicts:

Consider context-dependent interactions:
FliF may interact differently with partner proteins depending on the assembly state of the flagellum. Different experimental approaches may capture different states.

Evaluate experimental conditions:
Contradictions may arise from differences in:

• In vivo versus in vitro studies

• Detergent types used for membrane protein solubilization

• Buffer conditions affecting protein conformation

• Temperature effects on protein stability

Integrate multiple data types:
Combining genetic, biochemical, and structural data provides a more complete picture:

• Genetic suppressor data suggests interaction between FliF and FlhA

• Biochemical binding studies quantify direct interactions

• Structural studies visualize physical proximity

Consider protein dynamics:
FliF likely undergoes conformational changes during flagellar assembly and function:

• Some interactions may be transient

• Different domains may interact with different partners at different times

• Post-translational modifications could alter interaction profiles

Address methodological limitations:
Each approach has inherent limitations:

• Genetic studies may identify indirect effects

• Biochemical studies may not recapitulate the native membrane environment

• Structural studies typically capture static states

What are the latest advances in understanding the dynamic properties of FliF during flagellar rotation?

Recent research has begun to elucidate the dynamic behavior of the MS ring during flagellar function:

• The MS ring, composed primarily of FliF, is not merely a static structural component but likely undergoes conformational changes during flagellar rotation

• These dynamic properties may be essential for translating proton gradient energy into mechanical torque

• The interaction between FliF and other components, particularly FliE, appears to play a role in modulating these dynamic properties

The rotational symmetry of the MS ring is particularly important for its interaction with the N-terminal α-helix of FliE . This specific geometric arrangement likely facilitates the coordinated movements necessary for flagellar function.

Research employing advanced biophysical techniques such as high-speed atomic force microscopy and single-molecule FRET could provide further insights into these dynamic aspects of FliF function in the near future.

How is structural biology advancing our understanding of FliF's role in the flagellar motor?

Structural biology approaches are providing increasingly detailed insights into FliF architecture and function:

• Cryo-electron microscopy has enabled visualization of the MS ring at higher resolution

• Integration of genetic data with structural models has identified critical interaction interfaces

• Computational approaches complement experimental studies to predict conformational changes

Recent studies combining genetic mutant suppressor analysis with structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) have provided insights into how FliE serves as an essential adaptor connecting these components .

Structural investigations also support the model where FliF (MS ring) physically interacts with FlhA and other components of the export apparatus, rather than simply enclosing them without specific contacts .

Future advances in structural biology, particularly in situ structural techniques that can visualize protein complexes in their native environment, promise to further refine our understanding of how FliF contributes to the remarkable mechanical properties of the flagellar motor.

What emerging techniques show promise for studying FliF interactions in their native membrane environment?

Several cutting-edge techniques are becoming increasingly valuable for studying membrane proteins like FliF in their native environment:

Nanodiscs and lipid bilayer systems:
These membrane mimetics provide a more native-like environment for studying FliF structure and interactions compared to traditional detergent solubilization approaches.

Single-molecule tracking in live cells:
By tagging FliF with photoactivatable fluorescent proteins, researchers can track individual molecules in living bacteria, providing insights into dynamics and stoichiometry.

In-cell NMR spectroscopy:
This emerging technique allows detection of structural features and interactions within living cells, potentially revealing FliF conformational states during flagellar assembly.

Cross-linking mass spectrometry (XL-MS):
By introducing chemical crosslinks between interacting proteins followed by mass spectrometric analysis, researchers can identify specific contact points between FliF and its binding partners.

Cryo-electron tomography:
This technique enables visualization of flagellar structures in their cellular context at macromolecular resolution, potentially revealing the arrangement of FliF within the native basal body.

These methodological advances hold promise for addressing current knowledge gaps regarding how FliF integrates into the complex flagellar machine and how its interactions with other components contribute to the remarkable mechanical properties of the bacterial flagellum.

How might research on FliF contribute to developing novel antimicrobial strategies?

The flagellar system represents a potential target for antimicrobial development, with FliF playing a central role:

• FliF is essential for flagellar assembly and bacterial motility, which contributes to virulence in many pathogens

• The MS ring represents a structurally distinct target compared to traditional antibiotic targets

• Inhibiting FliF function could disrupt both motility and the flagellar export apparatus

Research approaches focused on antimicrobial applications might include:

• High-throughput screening for small molecules that bind specifically to FliF

• Peptide-based inhibitors designed to mimic interaction interfaces between FliF and other flagellar components

• Structure-based drug design targeting conserved functional domains of FliF

Methodologically, researchers could employ motility assays as phenotypic screens for potential inhibitors, followed by biochemical validation of direct binding to FliF. Genetic approaches using suppressor analysis could help identify the specific mechanisms by which candidates inhibit FliF function.

What computational models best capture the relationship between FliF structure and flagellar function?

Computational modeling approaches are increasingly valuable for understanding FliF's role in flagellar mechanics:

Multi-scale models:
Integrating atomic-level details of FliF structure with mesoscale models of the entire flagellar motor can provide insights into how molecular interactions translate into mechanical functions.

Molecular dynamics simulations:
These can reveal conformational changes in FliF during interactions with other flagellar components and predict the effects of mutations on protein stability and function.

Protein-protein docking:
Computational prediction of interaction interfaces between FliF and other flagellar proteins can guide experimental design and interpretation.

The most effective computational approaches will likely integrate experimental data from multiple sources, including genetic suppressors, structural studies, and biophysical measurements of flagellar rotation.

How do phase variation mechanisms affect FliF expression and flagellar assembly?

Flagellar phase variation in Salmonella involves the alternate expression of two different flagellin genes, fliC and fljB . While this process primarily affects the filament composition rather than the MS ring directly, it has implications for understanding flagellar gene regulation as a whole:

• The regulatory mechanisms controlling flagellar phase variation involve complex transcriptional and post-transcriptional controls

• Understanding these mechanisms provides insights into how bacteria coordinate expression of all flagellar components

• FljA protein prevents FliC production through interaction with the 5'-untranslated region of the fliC mRNA transcript

Research methodologies to study these regulatory networks include:

• Operon and gene fusions to reporter systems like β-galactosidase

• Western blot analysis to measure protein levels

• T2RNase protection assays to quantify transcript levels

• Creation of strains with the fljBA promoter locked in either the on or off orientation

These approaches have revealed that regulatory mechanisms operate at both transcriptional and translational levels, with FljA inhibiting fliC transcription fivefold while having a 200-fold effect on both transcription and translation .