Recombinant Flagellar basal body rod protein FlgB (flgB)

What is the structural composition of the FlgB protein?

FlgB is one of four structurally related proteins (including FlgC, FlgF, and FlgG) that form the rod structure in the flagellar basal body. It is a relatively small protein of approximately 138 amino acids that lacks a consensus signal sequence for the primary export pathway . The protein is not processed through the Sec-dependent secretion pathway but instead is exported through the flagellum-specific type III secretion (T3S) system . FlgB has had its N-terminal methionine removed post-translation, while the other rod proteins (FlgC, FlgF, and FlgG) are not processed at all . The protein exhibits significant sequence similarity with other rod proteins, particularly toward the N and C termini, suggesting evolutionary conservation of functionally important domains .

Where is FlgB located within the flagellar structure?

FlgB is located in the proximal portion of the flagellar rod structure. The rod is a cell-proximal part of a segmented axial structure of the flagellum, with FlgB believed to be positioned in one of the successive segments of the proximal rod . The complete rod structure consists of FlgB, FlgC, and FlgF located in successive segments of the proximal rod, followed by FlgG located in the distal rod . This axial structure then continues with the hook, hook-associated proteins (HAPs), and filament components to form the complete flagellum. Despite being external to the cell membrane, FlgB is not exported via the primary cellular pathway but through the flagellum-specific T3S pathway .

How does FlgB contribute to flagellar assembly?

FlgB functions as one of the first true rod subunits to be secreted during flagellar assembly . It forms part of the drive-shaft within the hook-basal body (HBB) structure that transmits motor rotation to the filament . The assembly process follows a specific order, with FlgB being secreted early in the process. Any mutations affecting FlgB secretion or assembly can disrupt the entire flagellar structure, as evidenced by decreased expression of downstream flagellar genes like fljB . The critical nature of FlgB in flagellar assembly is demonstrated by the observation that FlgB-Bla fusions can compete with wild-type FlgB for secretion and incorporation into the growing basal body, sometimes inhibiting further rod assembly .

What specific amino acid residues are critical for FlgB secretion and function?

Research has identified several amino acid regions in FlgB that are critical for its secretion and function. In-frame deletions of amino acids 9 through 18 and amino acids 39 through 58 decreased FlgB secretion levels . Further mutagenesis studies revealed that amino acid F45 is particularly critical for FlgB secretion . All amino acid substitutions at position F45 impaired rod assembly due to defects in FlgB secretion .

When analyzing the secretion capability of different amino acid substitutions at position 45, only three of twenty possible substitutions showed significant secretion: F45 (wild-type), W45, and Y45, with W45 and Y45 showing reduced secretion efficiency compared to the wild-type F45 . This suggests that an aromatic residue at position 45 is crucial for FlgB secretion, with phenylalanine being optimal. The table below summarizes the secretion capabilities of different amino acid substitutions at position 45:

How does FlgB interact with other components of the flagellar T3S system?

FlgB interacts specifically with the FlhB component of the flagellar T3S system, particularly through a hydrophobic pocket in the cleaved C-terminal domain of FlhB . This interaction is critical for the recognition and secretion of FlgB. Research has shown that the F45 residue of FlgB is essential for this interaction, as mutations at this position severely impair FlgB secretion .

The study by Evans et al. revealed that the purified FlhB C-terminal cytoplasmic domain (CCD) binds to early secretion substrates . Further research identified specific residues within a hydrophobic pocket of the FlhB CCD that, when deleted, no longer bound to early secretion substrates . Genetic evidence supports a direct interaction between the F45 residue of FlgB and the hydrophobic pocket of FlhB CCD .

Interestingly, certain mutations in FlhB can partially suppress the secretion defects caused by mutations in FlgB. For example, the FlhB (A286A A341V L344E) mutant permitted increased secretion of the FlgB-Bla F45R variant compared to wild-type FlhB . This suggests a specific recognition mechanism between these two proteins that can be altered through compensatory mutations.

What are the evolutionary implications of the conserved domains in FlgB?

The four rod proteins (FlgB, FlgC, FlgF, and FlgG) show clear sequence relationships, especially toward the N and C termini . This conservation suggests important functional and/or structural roles for these regions. A particularly notable feature is a six-residue consensus motif located approximately 30 residues from the N terminus across these proteins .

This conserved motif may serve as a recognition signal for the flagellum-specific export pathway or may reflect higher-order structural similarities within the rod . The conservation of these domains across different rod proteins suggests evolutionary pressure to maintain these features for proper flagellar assembly and function.

The presence of similar secretion mechanisms and conserved recognition motifs between flagellar and virulence-associated T3S systems also suggests evolutionary relationships between these systems . The study of FlgB and its interactions can therefore provide insights into the evolution of bacterial secretion systems and potential targets for antimicrobial development.

How can we effectively express and purify recombinant FlgB for structural studies?

To express and purify recombinant FlgB for structural studies, researchers typically employ the following methodology:

• Cloning Strategy: The flgB gene can be amplified from bacterial genomic DNA (commonly Salmonella typhimurium) using PCR with specific primers that incorporate appropriate restriction sites. The amplified gene is then cloned into an expression vector, such as pET-series vectors, which contain a strong T7 promoter and a His-tag for purification .

• Expression System: The recombinant plasmid is transformed into an E. coli expression strain such as BL21(DE3). For optimal expression, growth conditions should be optimized with respect to temperature, induction time, and IPTG concentration. Since FlgB is a relatively small protein (138 amino acids), it generally expresses well in E. coli systems .

• Purification Protocol:

  • Harvest cells by centrifugation and lyse using either sonication or a French press in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and protease inhibitors

  • Clarify the lysate by centrifugation at 20,000 × g for 30 minutes

  • Purify the His-tagged FlgB using nickel affinity chromatography

  • Further purify using size exclusion chromatography

  • Verify protein purity using SDS-PAGE and Western blotting

• Structural Analysis: For structural studies, purified FlgB can be subjected to X-ray crystallography or cryo-electron microscopy. Given the propensity of FlgB to interact with other rod proteins, co-crystallization with its binding partners might be necessary to stabilize its native conformation .

For functional studies, it's important to verify that the recombinant protein maintains its native properties by testing its ability to interact with known binding partners like FlhB using pull-down assays or surface plasmon resonance .

What methods are effective for studying FlgB secretion in vivo?

Several effective methodologies have been developed to study FlgB secretion in vivo:

• β-lactamase Fusion Reporter System: This is a powerful approach where β-lactamase (Bla) lacking its Sec-dependent secretion signal is fused to the C-terminus of FlgB . This system provides both a positive selection for secretion (ampicillin resistance) and a means to quantify secreted protein levels. When FlgB-Bla is secreted into the periplasm, it confers resistance to ampicillin (Ap^R) . The level of resistance correlates with the amount of secreted protein, allowing for quantitative assessment.

• Lac Reporter System: Using a lac operon fusion to a σ^28-dependent flagellar class 3 promoter (fljB::MudJ) as an indicator for functional flagellar T3S apparatus . A defective apparatus would be unable to secrete the anti-σ^28 factor FlgM, resulting in a Lac^- phenotype on lactose indicator medium .

• Deletion Mutagenesis: In-frame deletions of specific regions of FlgB can be created to identify domains important for secretion . This approach has successfully identified regions between amino acids 9-18 and 39-58 as critical for FlgB secretion .

• Site-Directed Mutagenesis: PCR-directed mutagenesis can be used to introduce specific amino acid changes, as demonstrated by the identification of F45 as a critical residue for FlgB secretion .

• Doped Oligonucleotide Mutagenesis: This technique allows for the creation of a library of mutations within a specific region, facilitating the identification of multiple residues important for function .

• Western Blot Analysis: To directly detect secreted FlgB in the periplasmic fraction versus the cytoplasmic fraction, allowing for quantification of secretion efficiency .

How can we investigate the interaction between FlgB and FlhB?

The interaction between FlgB and FlhB can be investigated using several complementary approaches:

• Genetic Suppressor Analysis: This involves identifying mutations in FlhB that suppress secretion defects caused by mutations in FlgB . For example, the FlhB L344E substitution was found to permit secretion of FlgB-Bla F45R, suggesting a specific interaction between these residues .

• Co-Immunoprecipitation (Co-IP): Using antibodies against FlhB to precipitate protein complexes from bacterial lysates, followed by Western blot analysis to detect co-precipitated FlgB. This method can confirm physical interaction between the proteins in vivo .

• Bacterial Two-Hybrid System: This system can be adapted to test direct interactions between FlgB and FlhB in a cellular context. The interaction leads to the reconstitution of a functional transcriptional activator that drives the expression of a reporter gene .

• Surface Plasmon Resonance (SPR): This technique can measure the binding affinity and kinetics between purified FlgB and FlhB. By immobilizing one protein on a sensor chip and flowing the other protein over it, the interaction can be quantified in real-time .

• Cross-linking Studies: Chemical cross-linking followed by mass spectrometry can identify specific residues involved in the interaction between FlgB and FlhB .

• Structural Studies: X-ray crystallography or cryo-electron microscopy of co-crystallized FlgB and FlhB can provide atomic-level details of their interaction .

• Mutational Analysis: Systematic mutation of residues in both FlgB and FlhB followed by functional analysis can identify specific interaction interfaces . This approach has successfully identified F45 of FlgB and the hydrophobic pocket in FlhB as critical for their interaction .

How should researchers analyze conflicting data regarding FlgB secretion mechanisms?

When analyzing conflicting data regarding FlgB secretion mechanisms, researchers should consider the following methodological approach:

When specific conflicts arise, such as differences in the reported importance of certain amino acid residues, researchers should design experiments that directly address the discrepancy, ideally using multiple complementary methods within the same study to control for methodological variations.

What statistical approaches are appropriate for analyzing FlgB mutation effects on flagellar assembly?

Several statistical approaches are appropriate for analyzing the effects of FlgB mutations on flagellar assembly:

• Binary Outcome Analysis: For qualitative phenotypes (e.g., motile/non-motile, secretion-competent/secretion-defective), Fisher's exact test or chi-square analysis can determine if the distribution of phenotypes differs significantly between wild-type and mutant populations .

• Quantitative Phenotype Analysis: For quantitative measurements such as ampicillin resistance levels or β-galactosidase activity from lac reporter fusions, appropriate statistical tests include:

  • Student's t-test for comparing two groups (e.g., wild-type vs. specific mutant)

  • ANOVA with post-hoc tests for comparing multiple groups (e.g., wild-type vs. multiple different mutations)

  • Regression analysis to identify correlations between amino acid properties and functional outcomes

• Multiple Testing Correction: When testing numerous mutations, as in the comprehensive analysis of all possible substitutions at position F45, applying corrections for multiple testing (e.g., Bonferroni, False Discovery Rate) is essential to control type I error rates .

• Structure-Based Statistical Analysis: Methods that incorporate structural information, such as:

  • Principal Component Analysis (PCA) to identify patterns in how mutations at different structural positions affect function

  • Clustering analyses to group mutations with similar effects

  • Statistical coupling analysis to identify coevolving residues that might function together

• Bayesian Approaches: These can be particularly useful when integrating prior knowledge about protein structure with new experimental data on mutation effects .

An example of appropriate statistical analysis from the research is the systematic evaluation of all possible amino acid substitutions at position 45 in FlgB . The data showed that only phenylalanine (wild-type), tryptophan, and tyrosine permitted significant secretion, suggesting the importance of an aromatic residue at this position . Statistical significance of these differences was established through replicated experiments and appropriate controls .

How can researchers integrate structural and functional data to understand FlgB's role in flagellar assembly?

Integrating structural and functional data to understand FlgB's role in flagellar assembly requires a multidisciplinary approach:

• Structure-Guided Mutagenesis: Use structural information to design targeted mutations at:

  • Residues at protein interfaces (for studying protein-protein interactions)

  • Conserved motifs (for studying functional domains)

  • Surface-exposed vs. buried residues (to distinguish structural from functional roles)

  The identification of F45 as critical for FlgB secretion exemplifies this approach, as it targeted a residue predicted to be important based on sequence conservation .

• Correlative Analysis: Establish correlations between structural features and functional outcomes by:

  • Mapping mutation effects onto structural models

  • Identifying clusters of functionally important residues

  • Correlating amino acid properties (hydrophobicity, charge, size) with functional effects

  Research has shown that the hydrophobic nature of F45 in FlgB is critical for its interaction with a hydrophobic pocket in FlhB, demonstrating how amino acid properties correlate with function .

• Integrative Modeling: Combine data from multiple experimental approaches:

  • X-ray crystallography or cryo-EM for high-resolution structures

  • Crosslinking studies for interaction interfaces

  • Mutagenesis data for functional importance

  • Evolutionary conservation for identifying critical domains

  This approach has been used to develop models of the flagellar rod structure incorporating FlgB, FlgC, FlgF, and FlgG in specific arrangements based on their structural similarities and functional relationships .

• Molecular Dynamics Simulations: Use computational approaches to:

  • Predict the effects of mutations on protein stability and interactions

  • Model dynamic processes like protein export and assembly

  • Test hypotheses about mechanism before experimental validation

• Data Visualization Tools: Employ specialized software to:

  • Create integrated visualizations of structural and functional data

  • Generate interactive models that incorporate mutation effects

  • Facilitate communication of complex structure-function relationships

The research on FlgB and FlhB interactions provides an excellent example of integrating structural and functional data . The identification of a hydrophobic pocket in FlhB that interacts with F45 of FlgB, supported by both structural predictions and genetic suppressor analysis, demonstrates how structural insights can explain functional observations and vice versa .

What are the emerging techniques for studying flagellar protein interactions in real-time?

Several cutting-edge techniques are emerging for studying flagellar protein interactions in real-time:

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET): This technique allows researchers to observe conformational changes and interactions between individual protein molecules in real-time. By labeling FlgB and its interaction partners with appropriate fluorophores, researchers can monitor binding events and structural changes at the single-molecule level .

• Live-Cell Super-Resolution Microscopy: Techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) can achieve nanometer resolution, allowing visualization of flagellar assembly processes in living bacterial cells. These approaches could potentially track the incorporation of fluorescently tagged FlgB into growing flagellar structures .

• Cryo-Electron Tomography (Cryo-ET): This technique allows visualization of cellular structures in their native state at molecular resolution. By capturing snapshots of flagellar assembly at different stages, researchers can create 3D reconstructions of the process, potentially revealing the exact position and orientation of FlgB within the growing structure .

• In Situ Structural Analysis: Combining fluorescence microscopy with cryo-electron microscopy (CLEM) allows correlation of functional states with structural details. This approach could help identify transient interactions during flagellar assembly .

• Optogenetic Tools: By engineering light-sensitive domains into flagellar proteins, researchers can use light to trigger interactions or conformational changes, allowing precise temporal control over assembly processes .

• Microfluidics-Based Approaches: These systems allow precise control of the cellular environment while performing real-time imaging, facilitating studies of how environmental changes affect flagellar assembly and FlgB incorporation .

• Mass Spectrometry-Based Footprinting: Techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify protein interaction surfaces and conformational changes in solution, providing complementary data to crystallographic approaches .

These emerging techniques promise to provide unprecedented insights into the dynamic processes of flagellar assembly and the specific role of FlgB within this complex system.

How might understanding FlgB function contribute to anti-bacterial therapeutic development?

Understanding FlgB function could contribute significantly to anti-bacterial therapeutic development through several mechanisms:

• Targeted Inhibition of Flagellar Assembly: As FlgB is essential for flagellar assembly and bacterial motility, compounds that specifically inhibit FlgB secretion or incorporation into the rod structure could impair bacterial motility . This would not directly kill bacteria but could reduce virulence and make bacteria more susceptible to host immune defenses or conventional antibiotics.

• Disruption of Type III Secretion: The similarities between flagellar T3S and virulence-associated T3S systems suggest that inhibitors targeting FlgB-FlhB interactions might also affect virulence factor secretion in pathogenic bacteria . The critical F45 residue in FlgB and its interaction with the hydrophobic pocket in FlhB represent specific targets for inhibitor design .

• Vaccine Development: Recombinant FlgB could potentially serve as an antigen in vaccine formulations. As a conserved flagellar protein, antibodies against FlgB might provide cross-protection against multiple bacterial species that use similar flagellar systems .

• Diagnostic Applications: Understanding the specific sequence and structural features of FlgB across different bacterial species could aid in developing diagnostic tools for detecting and identifying motile pathogens .

• Drug Delivery Systems: Knowledge of the flagellar export pathway, including how FlgB is recognized and secreted, could inform the development of novel drug delivery systems that exploit bacterial secretion machinery to deliver antimicrobial agents .

• Combinatorial Approaches: Inhibitors of FlgB function could be used in combination with conventional antibiotics to enhance efficacy through synergistic effects. For example, impairing bacterial motility through FlgB inhibition might increase the effectiveness of antibiotics that require active uptake .

The high conservation of flagellar components across many bacterial species suggests that therapeutics targeting FlgB could have broad-spectrum potential, while the absence of homologous structures in human cells suggests the possibility of developing highly selective antimicrobials with minimal host toxicity .