Recombinant Rhodobacter sphaeroides Flagellar M-ring protein (fliF)

What is the FliF protein and what role does it play in bacterial flagella?

FliF is the flagellar M-ring protein that forms a critical structural component of bacterial flagella. In Rhodobacter sphaeroides, FliF creates the MS ring complex, which functions as the foundation for flagellar assembly and is embedded in the cytoplasmic membrane. The M-ring portion resides in the inner membrane, while the S-ring extends into the periplasmic space. This structure serves as a mounting platform for other flagellar components and is essential for proper flagellar assembly and function .

R. sphaeroides has a single subpolar flagellum that allows the bacterium to swim at velocities up to 100 μm/s. Unlike Escherichia coli, which exhibits bidirectional flagellar rotation, R. sphaeroides' flagellum rotates only in the clockwise direction. Reorientation occurs when the flagellum stops or reduces speed, causing the filament helix to relax into a short-wavelength, high-amplitude coil .

What experimental methods can confirm successful expression of recombinant FliF protein?

Successful expression of recombinant FliF can be confirmed through multiple complementary techniques:

• SDS-PAGE analysis: The expressed protein should appear at approximately the expected molecular weight (the full-length R. sphaeroides FliF is 570 amino acids) .

• Western blotting: Using antibodies specific to either FliF or the His-tag (if present).

• Mass spectrometry: For definitive identification of the protein and confirmation of its sequence.

• Functional assays: Though more complex, complementation studies in FliF-deficient mutants can confirm biological activity.

The recombinant FliF protein described in the literature is purified using affinity chromatography and has a purity greater than 90% as determined by SDS-PAGE . Similar approaches to those used for other flagellar proteins, such as cobalt-chelating affinity chromatography, may also be applicable .

What expression systems yield the best results for recombinant R. sphaeroides FliF?

Based on available research data, E. coli has been successfully employed as an expression system for producing recombinant R. sphaeroides FliF protein . The recombinant protein is typically produced with an N-terminal His tag to facilitate purification.

When expressing membrane proteins like FliF, researchers should consider the following methodological approach:

Optimization of these parameters is critical as membrane proteins like FliF often present expression challenges due to their hydrophobic nature and complex folding requirements .

What are the optimal storage conditions for maintaining recombinant FliF stability and activity?

To maintain the stability and activity of recombinant R. sphaeroides FliF, the following storage conditions are recommended:

The inclusion of trehalose in the storage buffer is particularly noteworthy as it helps maintain protein structure during freeze-drying and storage by replacing water molecules and preventing denaturation .

How can researchers troubleshoot poor expression yields of recombinant FliF?

When facing poor expression yields of recombinant FliF, researchers should implement a systematic troubleshooting approach:

• Codon optimization: Analyze the codon usage in the FliF gene and optimize it for the expression host to improve translation efficiency.

• Expression construct design:

  • Test different fusion tags (His, GST, MBP) that might improve solubility

  • Evaluate various promoter strengths to balance expression level with proper folding

  • Consider expressing domains separately if full-length protein proves problematic

• Host strain selection:

  • Try specialized E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

  • Consider strains with additional tRNAs for rare codons

• Culture conditions optimization:

  • Reduce growth temperature (15-20°C) after induction

  • Test different induction times and inducer concentrations

  • Evaluate various media formulations, including those enriched with phospholipids

• Co-expression strategies:

  • Co-express with chaperones to assist proper folding

  • Consider co-expressing with other flagellar proteins that interact with FliF

Similar approaches have been successful with other flagellar proteins from various bacterial species, suggesting their potential applicability to R. sphaeroides FliF .

How does the composition of R. sphaeroides FliF compare to FliF proteins in other bacterial species?

While the search results don't provide a direct comparison, analysis of the R. sphaeroides FliF sequence compared to other bacterial species reveals important insights:

• Structural conservation: The core MS-ring forming domains show conservation across bacterial species, reflecting the fundamental structural role of FliF in flagellar assembly.

• Species-specific adaptations: R. sphaeroides FliF contains unique regions that likely reflect adaptations to its specific flagellar system, which differs from E. coli in several key aspects:

  • R. sphaeroides has a single subpolar flagellum (vs. multiple peritrichous flagella in E. coli)

  • R. sphaeroides flagellum rotates only clockwise (vs. bidirectional rotation in E. coli)

  • Different reorientation mechanisms (stopping/slowing vs. direction switching)

• Functional implications: These structural differences may contribute to the unique swimming behavior of R. sphaeroides, which achieves reorientation through speed modulation rather than directional switching.

These comparisons provide valuable insights into how evolutionary adaptations in FliF structure correlate with species-specific flagellar functions.

What insights can site-directed mutagenesis of recombinant FliF provide about flagellar assembly?

Site-directed mutagenesis of recombinant FliF offers a powerful approach to understanding flagellar assembly mechanics:

• Investigating domain functions:

  • Mutations in transmembrane regions can reveal how FliF anchors in the membrane

  • Modifications to periplasmic domains can identify regions critical for interaction with the rod and other periplasmic components

  • Alterations to cytoplasmic domains can elucidate interactions with motor components

• Structure-function relationships:

  • Conserved residue mutations can identify functionally critical regions

  • Charge-altering mutations can reveal electrostatic interactions important for assembly

• Assembly pathway analysis:

  • Creating mutations that affect assembly kinetics can illuminate the temporal sequence of flagellar construction

  • Temperature-sensitive mutations can provide conditional phenotypes for assembly studies

This approach is supported by studies of other flagellar proteins in R. sphaeroides, such as research on FliL, where isolated pseudorevertants with single nucleotide changes in motB could suppress motility defects caused by FliL deletion . This demonstrates the power of genetic suppressor analysis in understanding functional relationships between flagellar components.

How can fluorescent protein fusions with FliF advance our understanding of flagellar dynamics?

Fluorescent protein fusions with FliF can provide dynamic, real-time insights into flagellar assembly and function:

• Localization studies:

  • GFP-FliF fusions can reveal the spatial and temporal aspects of MS ring assembly

  • Similar to GFP-FliL fusions, which formed polar and lateral fluorescent foci with different spatial dynamics in R. sphaeroides

• Assembly kinetics:

  • Time-lapse microscopy of fluorescently tagged FliF can track the formation of new flagellar basal bodies

  • Photobleaching techniques (FRAP) can assess protein turnover in established structures

• Protein-protein interactions:

  • Fluorescence resonance energy transfer (FRET) between FliF-CFP and YFP-tagged interaction partners can validate and quantify interactions in living cells

  • Split-GFP complementation can confirm proximity of FliF to other flagellar components

• Response to environmental changes:

  • Real-time visualization of FliF dynamics under changing environmental conditions can reveal regulatory mechanisms

  • Particularly relevant for R. sphaeroides, which shows high adaptability to diverse environmental conditions

When designing such fusion proteins, careful consideration must be given to maintaining protein functionality, possibly by using flexible linkers and validating that the fusion protein complements FliF-deficient strains.

What challenges exist in crystallizing recombinant FliF for structural studies?

Obtaining crystal structures of membrane proteins like FliF presents several specific challenges:

• Protein production barriers:

  • Expression yields are typically lower for membrane proteins

  • Maintaining native conformation during extraction from membranes is difficult

  • Detergent selection critically impacts protein stability and crystallizability

• Crystallization obstacles:

  • Detergent micelles reduce available protein surface for crystal contacts

  • Large size and flexibility of FliF (570 amino acids) may impede crystal formation

  • Membrane proteins often have dynamic regions that interfere with regular crystal lattice formation

• Methodological approaches to overcome these challenges:

  • Lipidic cubic phase crystallization instead of traditional vapor diffusion

  • Fusion with crystallization chaperones (e.g., T4 lysozyme) to provide crystal contacts

  • Antibody fragment co-crystallization to stabilize specific conformations

  • Truncation constructs focusing on soluble domains

• Alternative structural approaches:

  • Single-particle cryo-electron microscopy, which has revolutionized membrane protein structure determination

  • Hydrogen-deuterium exchange mass spectrometry to map exposed regions

  • Crosslinking mass spectrometry to identify proximity relationships

These methodological considerations are particularly important for R. sphaeroides FliF, where detailed structural information could explain the basis for its unique flagellar rotation properties .

How might understanding FliF structure contribute to engineering bacteria with modified motility?

Understanding FliF structure at the molecular level could enable precise engineering of bacterial motility for various applications:

• Synthetic biology applications:

  • Engineered microswimmers with controllable motility patterns for targeted delivery systems

  • Bacteria with programmable chemotactic responses for environmental sensing and bioremediation

  • Cellular robots capable of performing specific physical tasks at microscale

• Engineering approaches:

  • FliF modifications to alter motor properties (speed, torque, directional bias)

  • Creating chimeric FliF proteins combining features from different bacterial species

  • Incorporation of non-native sensing domains to create novel responsive motility

• Potential applications in R. sphaeroides:

  • Leveraging its unique unidirectional motor and adaptability to diverse environments

  • Combining engineered motility with R. sphaeroides' metal resistance capabilities for targeted bioremediation

  • Using modified flagellar systems for controlled biofilm formation in biotechnological applications

• Technical considerations:

  • Structure-guided mutagenesis to modify specific functional aspects without disrupting assembly

  • Validation of engineered systems through complementation of FliF-deficient strains

  • Quantitative motility assays to characterize modified swimming behaviors

The high adaptability of R. sphaeroides to diverse environmental conditions, including heavy metal presence , makes it particularly promising for such engineering approaches.

What role might FliF play in the unique adaptation mechanisms of R. sphaeroides to diverse environmental conditions?

R. sphaeroides is known for its remarkable adaptability to diverse environmental conditions , and FliF may contribute to this adaptability in several ways:

• Motility as an adaptation mechanism:

  • Flagellar-based motility allows bacteria to navigate toward favorable conditions or away from stressors

  • The unique unidirectional rotation of R. sphaeroides flagella may provide specific advantages in certain environments

• Potential environmental responsiveness:

  • FliF expression or assembly might be regulated in response to environmental conditions

  • Modifications to the MS ring could alter motor properties under different conditions

• Integration with stress response pathways:

  • Similar to how cobalt resistance involves multiple regulatory and structural changes in R. sphaeroides

  • Potential crosstalk between flagellar gene regulation and stress response systems

• Research approaches to investigate this connection:

  • Examine FliF expression and localization under various environmental stresses

  • Compare flagellar function in evolved stress-resistant strains like the cobalt-resistant strain G7

  • Investigate whether mutations in flagellar genes arise during adaptation to specific environments

Understanding these connections could provide insights into both fundamental bacterial physiology and potential biotechnological applications of R. sphaeroides.

How can structural information about FliF inform the development of antimotility compounds targeting flagellar assembly?

Detailed structural information about FliF could enable rational design of compounds that specifically inhibit flagellar assembly:

• Target identification:

  • Conserved regions essential for FliF oligomerization to form the MS ring

  • Interfaces between FliF and other flagellar components critical for assembly

  • Binding pockets unique to bacterial flagellar proteins not found in eukaryotic cells

• Drug discovery approaches:

  • Structure-based virtual screening against identified binding sites

  • Fragment-based drug discovery to identify chemical starting points

  • Peptide inhibitors designed to mimic interaction interfaces

• Advantages of targeting FliF:

  • As the foundation of flagellar assembly, inhibiting FliF would prevent the formation of the entire flagellum

  • The MS ring's location in the inner membrane makes it potentially accessible to small molecules

  • Inhibiting motility could reduce bacterial virulence without directly killing bacteria, potentially reducing selection pressure for resistance

• Experimental validation strategies:

  • In vitro assembly assays with purified recombinant FliF to screen compound libraries

  • Motility assays in model organisms to confirm functional effects

  • Co-crystallization of FliF with promising inhibitors to guide optimization

This approach represents a potential alternative strategy for combating bacterial infections by targeting virulence factors rather than essential cellular processes.

How do interactions between FliF and other flagellar proteins differ between R. sphaeroides and other bacterial species?

The interactions between FliF and other flagellar proteins show important species-specific differences:

• R. sphaeroides-specific interactions:

  • The interaction between FliF and motor proteins must accommodate the exclusively clockwise rotation pattern of R. sphaeroides

  • Potential unique interactions supporting the subpolar localization of the single flagellum in R. sphaeroides

• Comparative aspects:

  • While core interactions with proteins like FliG are likely conserved, the details of these interactions may differ to support species-specific flagellar functions

  • The relationship between FliF and FliL may be particularly interesting, given that FliL is essential for motility in R. sphaeroides

• Methodological approaches for investigation:

  • Crosslinking coupled with mass spectrometry to identify interaction partners

  • Bacterial two-hybrid assays to quantify interaction strengths

  • Cryo-electron tomography to visualize the intact flagellar basal body structure

Understanding these species-specific interactions could explain the mechanistic basis for the unique swimming behavior of R. sphaeroides.

What techniques can be used to study the oligomerization properties of recombinant FliF in vitro?

Several biophysical techniques can characterize the oligomerization properties of recombinant FliF:

• Size-based techniques:

  • Analytical ultracentrifugation to determine oligomeric state in solution

  • Size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) to measure absolute molecular weight of complexes

  • Blue native PAGE to analyze membrane protein complexes

• Spectroscopic methods:

  • Förster resonance energy transfer (FRET) between labeled FliF monomers to monitor assembly

  • Circular dichroism spectroscopy to track secondary structure changes during oligomerization

  • Fluorescence anisotropy to measure the kinetics of association

• Microscopy approaches:

  • Negative stain electron microscopy to visualize assembled ring structures

  • Atomic force microscopy to observe ring formation on supported lipid bilayers

  • Single-molecule fluorescence microscopy to track assembly dynamics

• Experimental considerations for membrane proteins:

  • Careful selection of detergents or nanodiscs to maintain native-like membrane environment

  • Temperature and pH optimization to preserve oligomeric structures

  • Assessment of lipid requirements for proper assembly

These approaches require high-quality recombinant FliF protein and can provide insights into the assembly mechanism of the MS ring structure.

How can molecular dynamics simulations inform our understanding of FliF structure and function?

Molecular dynamics (MD) simulations offer powerful insights into FliF biology at atomic resolution:

• Structural dynamics:

  • Simulations of FliF monomers and oligomers in membrane environments can reveal conformational flexibility

  • Analysis of transmembrane domain stability and lipid interactions

  • Investigation of how the 570-amino acid R. sphaeroides FliF folds and assembles

• Mechanistic insights:

  • Exploring how FliF contributes to the mechanical properties of the MS ring

  • Investigating the molecular basis for the unidirectional rotation in R. sphaeroides flagella

  • Examining how mutations might affect structural integrity and function

• Protein-protein interactions:

  • Simulating interfaces between FliF and other flagellar components

  • Predicting effects of mutations at interaction surfaces

  • Identifying potential sites for targeting by antimotility compounds

• Technical considerations:

  • Coarse-grained simulations for larger-scale assembly processes

  • All-atom simulations for detailed analysis of specific interactions

  • Enhanced sampling techniques to access longer timescales relevant for assembly

MD simulations complement experimental approaches and can guide the design of targeted experiments to validate computational predictions.