Recombinant Flagellar protein FliL (fliL)

What is the molecular structure of FliL and how does it organize around the flagellar motor?

FliL forms a circular structure approximately 10 nm in diameter that surrounds the stator complex in its extended conformation. Crystallographic studies have revealed that FliL oligomerizes into a ring-like structure through lateral subunit interactions. In Borrelia burgdorferi, FliL forms a decameric ring with an interior diameter of approximately 36 Å that encircles the portion of the stator unit MotB extending above MotA in the inner membrane . The ring structure's interior is primarily composed of polar and charged residues, with 10 pairs of inward-pointing positively charged residues (R89 and K103) forming a constriction . This ring formation appears essential for FliL's function, as mutations of hydrophobic residues on the subunit interface to hydrophilic ones disrupted FliL localization at the motor .

How is FliL conserved across bacterial species?

FliL is highly conserved across diverse bacterial phyla but shows functional variations between species. It has been identified and studied in Gammaproteobacteria (Escherichia coli, Salmonella enterica, Proteus mirabilis, Pseudomonas aeruginosa, and Vibrio alginolyticus), Alphaproteobacteria (Bradyrhizobium diazoefficiens, Caulobacter crescentus, Rhodobacter sphaeroides, Silicibacter sp., and Sinorhizobium meliloti), Betaproteobacteria (Herminiimonas arsenicoxydans), Campylobacterota (Helicobacter pylori), Firmicutes (Bacillus subtilis), and Spirochetes (Borrelia burgdorferi) . Despite this conservation, FliL's essentiality varies; it is absolutely required for motility in some bacteria like Rhodobacter sphaeroides , while in others it plays a conditional role, primarily enhancing motility under high-load conditions .

What evidence supports the interaction between FliL and the stator complex?

Multiple lines of evidence demonstrate FliL's interaction with the stator complex:

• Photo-cross-linking experiments using p-benzoyl-l-phenylalanine (pBpa) have shown that FliL residues V74 and V165 are in close proximity to PomB (a stator component) in vivo .

• Cryo-electron tomography has directly visualized the FliL ring surrounding the stator complex in B. burgdorferi .

• Genetic studies show that mutations in MotB can suppress fliL deletion phenotypes. In R. sphaeroides, eight independent pseudorevertants of a fliL mutant contained single nucleotide changes in motB affecting only three residues on the periplasmic side of MotB .

• The length of the flexible linker in PomB (a MotB homolog) is critical for proper FliL localization around the motor in V. alginolyticus .

What are the optimal methods for expressing and purifying recombinant FliL for structural studies?

Recombinant FliL can be successfully expressed and purified using the following approach, as demonstrated with H. pylori FliL:

• Expression system: The C-terminal domain of FliL (amino acid residues 81-183) can be cloned and expressed in E. coli .

• Purification method: Affinity chromatography followed by size exclusion chromatography achieves >98% homogeneity .

• Solution behavior: Purified recombinant FliL behaves as a monomer in solution, though it forms oligomers at high concentrations .

• Crystallization: Crystals can be obtained using the hanging-drop vapor-diffusion method with ammonium phosphate monobasic as a precipitant. For H. pylori FliL, crystals belonged to space group P1 with unit-cell parameters a = 62.5, b = 82.6, c = 97.8 Å, α = 67.7, β = 83.4, γ = 72.8° .

• Data collection: High-resolution structural data (2.8 Å resolution for H. pylori FliL) can be collected using synchrotron radiation .

For V. alginolyticus FliL, researchers successfully used X-ray diffraction data from synchrotron beamline BL41XU at the SPring-8 facility to determine the structure .

How can researchers effectively visualize FliL localization in living bacterial cells?

Green Fluorescent Protein (GFP) fusions provide an effective means to visualize FliL localization in living bacteria:

• Construct design: A GFP-FliL fusion can be created by amplifying fliL without the initiation codon, in frame with the coding region of gfp. The fusion should be cloned into an appropriate expression vector with a strong promoter and ribosome binding site .

• Microscopy approach: Fluorescence microscopy can be used to observe the intracellular localization of GFP-FliL. In R. sphaeroides, GFP-FliL forms polar and lateral fluorescent foci that show different spatial dynamics .

• Controls: The expression of flagellar genes controlled by the master regulator FleQ is required for proper localization of GFP-FliL, suggesting that additional components of the flagellar regulon are necessary for correct FliL positioning .

• Quantification: The number and intensity of fluorescent foci can be quantified and compared between wild-type and mutant strains to assess the impact of various genetic backgrounds on FliL localization .

What genetic approaches are most effective for studying FliL function?

Several genetic approaches have proven effective for investigating FliL function:

• Gene deletion: Constructing a fliL deletion strain through homologous recombination. This approach involves:

  • Amplifying upstream and downstream homologous fragments (~800 bp) of the fliL gene

  • Creating a fusion fragment through overlapping PCR

  • Cloning into a suicide vector like pK18mobsacB

  • Using two-step selection (first for integration, then for excision) to obtain clean deletions

• Complementation studies: Cross-species complementation experiments are particularly informative. For example, fliL from Bacillus (FliL Bc) and Proteus (FliL Pr) can complement an E. coli fliL null mutant for both swimming and swarming, while Proteus FliL can also complement Salmonella fliL .

• Suppressor mutant analysis: Isolating pseudorevertants from fliL mutant strains has identified compensatory mutations in motB. In R. sphaeroides, eight independent pseudorevertants revealed single nucleotide changes affecting only three residues on the periplasmic side of MotB .

• Site-directed mutagenesis: Targeted mutagenesis of specific residues, particularly at the FliL oligomerization interface, can disrupt FliL localization and function .

How does FliL enhance flagellar motor function under high-load conditions?

FliL enhances flagellar motor function under high-load conditions through several mechanisms:

• Stator stabilization: FliL forms a ring structure that wraps around the stator complex in its extended, active conformation, stabilizing it for optimal motor function . The interaction between the FliL ring and MotB plays a crucial role in maintaining this extended state .

• Stator recruitment: FliL helps recruit stator complexes to the motor, as evidenced by the observation that only a partial FliL ring forms in a ΔmotB mutant . This suggests that as the stator complex is recruited to the motor, the FliL partial ring engages it before oligomerizing into a full ring that wraps around the stator in its active conformation .

• Enhanced torque generation: FliL is particularly important for swarming motility, which requires higher torque. In P. plecoglossicida, deletion of fliL significantly decreases swarming ability on 1.25% agar plates but has a lesser effect on swimming in 0.25% agar .

• Ion channel modulation: The structural similarity between FliL and SPFH domain proteins implies that FliL may be involved in regulating the channel activity of the stator, possibly enhancing ion flow through the channel under high-load conditions .

What is the role of FliL in bacterial surface sensing and virulence?

FliL plays important roles in bacterial surface sensing and virulence:

• Surface sensing: In Proteus mirabilis and other bacteria, FliL is involved in mechanosensing pathways that detect contact with surfaces . This sensing mechanism allows bacteria to adapt their motility patterns when transitioning from swimming to swarming.

• Virulence attenuation: Deletion of the fliL gene in P. plecoglossicida results in twelve-fold lower virulence compared to the wild-type strain, as measured by LD50 values in infection models :

  Strain	LD50 (CFU/fish)	95% Confidence Interval
  Wild-type	5.0 × 10³	2.1 × 10³–1.2 × 10⁴
  ΔfliL	6.3 × 10⁴	1.9 × 10⁴–2.1 × 10⁵
  C-ΔfliL (complemented)	1.3 × 10³	4.8 × 10²–3.7 × 10³

• Biofilm formation: The deletion of fliL in P. plecoglossicida significantly reduces biofilm formation, with the OD590 value of the ΔfliL strain (1.22 ± 0.08) being significantly lower than that of the wild strain (1.57 ± 0.19) .

• Adhesion ability: FliL affects bacterial adhesion to host surfaces. The number of ΔfliL strain bacteria attached to mucus-coated slides is significantly less than that of wild-type bacteria under the same visual field .

How does the flexible linker of PomB contribute to FliL localization and function?

The flexible linker region of PomB (a MotB homolog in V. alginolyticus) plays a critical role in FliL localization and function:

• Essential for localization: While the flexible linker region (residues 60 to 121) is not essential for basic motor function, it is crucial for FliL localization. In-frame deletion mutations in the linker region completely disrupt the localization of polar FliL at the flagellar motor .

• Length dependence: It appears that the length of the linker, rather than specific amino acid sequences within it, is critical for FliL localization. A series of in-frame deletion mutants lacking 20 amino acid residues in different parts of the linker region all showed disrupted FliL localization .

• Motor efficiency: The flexible linker region contributes to motor efficiency. While deletion mutants could still form swimming rings in soft-agar plates, the proper interaction with FliL likely enhances motor performance under more challenging conditions .

• Structural implications: The requirement for an intact linker suggests that proper spacing between the membrane-embedded portion of PomB and its periplasmic domain is necessary for FliL interaction and localization .

What is the relationship between FliL's structure and its homology to stomatin-like proteins?

FliL's structure reveals interesting relationships to stomatin-like proteins that provide insights into its function:

How do FliL paralogs contribute to flagellar function in different bacterial species?

Some bacteria possess FliL paralogs that show distinct but complementary roles in flagellar function:

• MotF in Sinorhizobium meliloti: MotF is a highly divergent FliL paralog that shares structural similarity to the core of V. alginolyticus FliL despite low sequence conservation . Both FliL and MotF are essential for normal motor function and torque generation in S. meliloti .

• Functional specialization: In bacteria with FliL paralogs, each protein may have specialized for different aspects of motor function. For example, in S. meliloti, FliL is indispensable for flagellation, while both FliL and MotF contribute to torque generation .

• Evolutionary implications: The presence of FliL paralogs in some bacterial lineages suggests evolutionary adaptation to specific ecological niches or motility requirements. MotF has been identified in related Rhizobiaceae species, including Agrobacterium tumefaciens (51% identity) and Rhizobiaceae bacterium LC148 (49% identity) .

• Comparative analysis: Structural alignment reveals that despite low sequence similarity, the globular domains of FliL paralogs maintain similar three-dimensional structures, highlighting the importance of structural conservation for function .

What are the molecular dynamics of FliL assembly around the stator complex?

The assembly of FliL around the stator complex involves a coordinated sequence of molecular events:

• Sequential assembly: Evidence from B. burgdorferi suggests that FliL initially forms a partial ring that engages the stator complex before it can fully oligomerize into a complete ring. In ΔmotB mutants, only four FliL periplasmic units could be docked into the partial FliL ring, compared to the decameric ring in wild-type bacteria .

• Stator-dependent oligomerization: The full oligomerization of FliL appears dependent on interaction with the stator complex. This suggests a cooperative assembly process where initial FliL-stator interactions promote further FliL recruitment and complete ring formation .

• Collar protein enclosure: In B. burgdorferi, the FliL-stator complex is further enclosed by flagellar collar proteins, indicating an ordered, cooperative assembly necessary for proper localization and stability of individual stator complexes .

• Motor-independent assembly: FliL can assemble around the stator complex without the flagellar basal body present. This was demonstrated by cross-linking experiments in E. coli BL21(DE3) cells, which have no flagellum but still showed FliL-PomB interaction when both were expressed .

What are common challenges in cross-species complementation studies with FliL?

Cross-species complementation studies with FliL present several challenges:

• Variable complementation efficiency: FliL from different species shows variable ability to complement defects in other species. For example, Proteus FliL (FliL Pr) more effectively complements E. coli fliL defects than E. coli's own FliL expressed from a plasmid .

• Expression timing issues: Proper incorporation of FliL at the basal body may depend on the order of gene expression from the native operon. Ectopic expression from plasmids may be untimely, resulting in suboptimal complementation .

• Stoichiometry considerations: The amount of FliL expressed may affect complementation efficiency. Too little or too much FliL might disrupt proper assembly of the flagellar structures .

• Specificity of interactions: Despite FliL's conservation across species, specific interactions with other flagellar components may vary. For example, while FliL Pr effectively complemented both E. coli and Salmonella fliL defects, FliL Bc only complemented E. coli fliL .

• Negative results interpretation: Negative results in cross-species complementation should be interpreted cautiously, as they may reflect the technical difficulty of FliL complementation rather than true functional incompatibility .

How can researchers effectively study FliL-stator interactions?

Several effective approaches for studying FliL-stator interactions include:

• Photo-cross-linking with unnatural amino acids:

  • Incorporate photoreactive unnatural amino acids like p-benzoyl-l-phenylalanine (pBpa) at specific positions in FliL

  • Co-express with stator proteins (PomA/PomB or MotA/MotB)

  • Use UV irradiation to create covalent bonds between closely positioned proteins

  • Analyze cross-linked products by Western blotting

• Two-hybrid assays:

  • Yeast two-hybrid assays can detect interactions between the periplasmic domains of FliL and stator components

  • These assays have shown that the periplasmic domain of FliL can interact with itself but not directly with the periplasmic domain of MotB in R. sphaeroides

• Pulldown assays:

  • Combine with two-hybrid approaches to validate protein-protein interactions

  • These have confirmed FliL self-interaction but no direct interaction with MotB in some species

• Cryo-electron tomography:

  • Provides direct visualization of FliL-stator interactions in situ

  • Has revealed the FliL ring structure surrounding the stator complex in B. burgdorferi

• Genetic suppressor analysis:

  • Isolate suppressors of fliL mutant phenotypes

  • Sequence to identify compensatory mutations, often found in stator components like MotB

What are the key considerations when designing mutations in FliL for functional studies?

When designing mutations in FliL for functional studies, researchers should consider:

• Domain-specific effects: Target mutations to specific functional domains based on structural information:

  • The periplasmic region for stator interactions

  • The oligomerization interface for ring formation

  • The transmembrane region for membrane anchoring

• Conservation analysis: Focus on highly conserved residues, which are more likely to be functionally important. Comparative sequence analysis across bacterial species can identify these residues .

• Hydrophobicity changes: Mutations that change hydrophobic residues to hydrophilic ones at the subunit interface have been shown to disrupt FliL localization at the motor .

• Oligomerization interface: Target residues involved in lateral subunit interactions that are crucial for FliL oligomerization and ring formation .

• Proximity to stator components: Residues like V74 and V165 in V. alginolyticus FliL have been identified as being in close proximity to PomB through cross-linking studies .

• Functional readouts: Design appropriate assays to evaluate the effects of mutations:

  • Swimming assays in soft agar (0.25%) for basic motility

  • Swarming assays on firmer surfaces (1.25% agar) for high-load conditions

  • Fluorescence microscopy with GFP-FliL to assess localization

  • Bacterial attachment assays to evaluate surface interaction phenotypes

What are the most significant unanswered questions about FliL function?

Despite significant advances in understanding FliL, several important questions remain:

• Regulatory mechanisms: How is FliL expression and activity regulated in response to environmental conditions? The mechanisms controlling FliL's involvement in the transition between swimming and swarming are not fully understood.

• Ion flow modulation: The precise mechanism by which FliL regulates ion flow through the stator channels remains to be established. Does it act as a direct modulator or indirectly by affecting stator conformation?

• Signal transduction: How does FliL transduce mechanical signals to biochemical responses during surface sensing? The complete signaling pathway connecting FliL to changes in gene expression remains to be elucidated.

• Pathogenic relevance: While FliL contributes to virulence in some pathogens, the molecular mechanisms linking FliL function to pathogenicity need further investigation to identify potential therapeutic targets.

• Species-specific functions: The basis for the varying essentiality of FliL across bacterial species requires further comparative studies to understand evolutionary adaptations.