Recombinant Bacillus subtilis Flagellar biosynthetic protein fliZ (fliZ)

What is the molecular structure and function of fliZ protein in Bacillus subtilis?

fliZ is a 219-amino-acid protein with a predicted molecular mass of 24,872 Da encoded by the Bacillus subtilis major che-fla operon . The protein contains a conventional N-terminal signal sequence that does not direct secretion of the protein but appears to target it to the membrane . Current research indicates that fliZ is essential for flagellar assembly, as null mutants lacking fliZ do not produce flagella and consequently lack motility . The protein's membrane localization suggests it may function as part of the flagellar export apparatus, potentially facilitating the transport of flagellar components during assembly.

Two possible models for membrane insertion have been proposed:

How can recombinant fliZ be effectively expressed in laboratory settings?

Expression of recombinant fliZ requires careful consideration of several factors:

• Expression System Selection: While E. coli is commonly used for heterologous protein expression, B. subtilis itself can serve as an excellent host for producing its native proteins . For fliZ expression, using B. subtilis as the host may provide the correct cellular environment for proper folding and membrane insertion.

• Optimization Approach:

  • Clone the fliZ gene into an appropriate expression vector with an inducible promoter

  • Consider genome minimization strategies for B. subtilis to enhance protein secretion capability

  • Remove extracellular proteases, prophages, and genes for spore development to improve production

  • Optimize growth media and culturing conditions for maximum yield

• Monitoring Expression:

  • Use SDS-PAGE and Western blotting to confirm expression

  • Monitor cell motility as a functional readout for properly folded protein

The reintroduction of the fliZ gene via a plasmid has been shown to restore motility in fliZ null mutants, confirming the functionality of recombinant expression .

What methods are effective for detecting and quantifying fliZ protein?

The detection and quantification of fliZ protein can be accomplished through several complementary approaches:

• Immunological Methods:

  • Generate specific antibodies against purified fliZ or synthetic peptides derived from the fliZ sequence

  • Use Western blotting for specific detection in cell lysates

  • Employ ELISA for quantitative measurement

• Fluorescent Tagging:

  • Create fluorescent fusion proteins (e.g., fliZ-GFP) for localization studies

  • Ensure the tag doesn't interfere with membrane targeting or function

• Mass Spectrometry:

  • Use LC-MS/MS for identification and absolute quantification

  • Employ multiple reaction monitoring (MRM) for targeted quantification

• Functional Assays:

  • Measure bacterial motility (swim plates or tracking microscopy)

  • Complementation of fliZ null mutants can serve as a functional readout

The choice of detection method should be guided by the specific research question, with consideration for preserving the membrane-associated properties of the protein.

How does fliZ contribute to bacterial motility in Bacillus subtilis?

fliZ is essential for flagellar assembly in B. subtilis, with null mutants completely lacking flagella . The functional role of fliZ in motility can be assessed through:

• Motility Assays:

  • Swim plate assays: Measuring the diameter of bacterial colonies spreading in soft agar

  • Microscopic tracking: Direct observation of bacterial swimming behavior

• Flagellar Visualization:

  • Negative staining and transmission electron microscopy to directly observe flagellar structures

  • Fluorescence microscopy with flagellar staining dyes or fluorescently tagged flagellar components

• Genetic Complementation:

  • Expression of fliZ from a plasmid can restore motility in null mutants

  • This provides a direct functional assay for testing mutant versions of fliZ

• Comparative Analysis:

  • Unlike some flagellar proteins in Salmonella Typhimurium where functional redundancy exists, B. subtilis appears to have a stricter dependence on specific flagellar proteins

How does the membrane targeting mechanism of fliZ differ from other flagellar proteins?

The membrane targeting of fliZ involves a conventional N-terminal signal sequence, but unlike many secreted proteins, this sequence doesn't direct secretion of the protein . Instead, it appears to specifically target fliZ to the membrane, where it likely functions as part of the flagellar assembly apparatus.

Comparative Analysis with Other Flagellar Proteins:

Research Approaches to Study Membrane Targeting:

• Generate signal sequence mutations to identify critical residues

• Create chimeric proteins with different signal sequences to assess targeting specificity

• Use fluorescently tagged constructs to visualize membrane localization

• Employ fractionation studies to confirm membrane association

Understanding this unique targeting mechanism could provide insights into the specialized type III secretion system that underpins flagellar biosynthesis .

What are the molecular interactions between fliZ and other components of the flagellar export apparatus?

Investigating molecular interactions of fliZ requires sophisticated biochemical and genetic approaches:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with tagged fliZ to identify interaction partners

  • Bacterial two-hybrid systems to screen for direct interactions

  • Cross-linking followed by mass spectrometry (XL-MS) to map interaction surfaces

  • Surface plasmon resonance to measure binding kinetics

• Genetic Interaction Mapping:

  • Synthetic lethality screens to identify genes with functional relationships

  • Suppressor mutation analysis to identify compensatory changes

  • Construct double/triple mutants with other flagellar genes to characterize epistatic relationships

• Structural Studies:

  • Cryo-electron microscopy of the flagellar basal body complex

  • X-ray crystallography of fliZ alone or in complex with interaction partners

  • NMR studies for dynamic interaction analysis

Unlike certain flagellar proteins in S. Typhimurium where overexpression of one component can compensate for the absence of another (e.g., overexpression of flgK compensating for flgN deletion), B. subtilis shows stricter dependence on specific flagellar proteins, suggesting less functional redundancy in its flagellar assembly system .

How do posttranslational modifications affect fliZ function in flagellar assembly?

Posttranslational modifications (PTMs) can significantly influence protein function through various mechanisms. For flagellar proteins in B. subtilis, phosphorylation has been identified as a potential regulatory mechanism, though its functional significance remains under investigation.

Investigation Approaches:

• PTM Identification:

  • Mass spectrometry-based phosphoproteomics to identify modification sites

  • Western blotting with phospho-specific antibodies

  • Radiolabeling with 32P to detect phosphorylation events

• Functional Analysis of PTMs:

  • Site-directed mutagenesis of potential modification sites

  • Creation of phosphomimetic (e.g., serine to aspartate) and phospho-null (serine to alanine) mutations

  • Complementation assays to assess functionality of modified proteins

• Temporal Regulation:

  • Analysis of modification patterns during different growth phases

  • Correlation with flagellar assembly stages

It's worth noting that for some flagellar proteins like FlgN, mutation of tyrosine and arginine phosphorylation sites has been shown to have no effect on B. subtilis motility , raising questions about the biological relevance of some posttranslational modifications identified in global proteomic approaches.

What experimental approaches can resolve the structure-function relationship of fliZ?

Understanding the structure-function relationship of fliZ requires a multifaceted approach:

• Structural Determination:

  • X-ray crystallography of purified fliZ (challenging due to membrane association)

  • NMR spectroscopy for solution structure (if protein size allows)

  • Cryo-electron microscopy for larger complexes

  • Computational structure prediction and molecular dynamics simulations

• Functional Domain Mapping:

  • Generate truncation mutants to identify functional domains

  • Alanine-scanning mutagenesis to identify critical residues

  • Domain swapping with related proteins to create chimeras

• Structure-Guided Functional Analysis:

  • Design mutations based on structural insights

  • Assess impact on:

    • Protein stability and folding

    • Membrane targeting

    • Protein-protein interactions

    • Flagellar assembly and motility

• Evolutionary Analysis:

  • Compare sequences across different bacterial species

  • Identify conserved motifs that may indicate functional importance

This comprehensive approach can help elucidate how the unique structural features of fliZ contribute to its essential role in flagellar assembly, despite its lack of significant homology to other known proteins .

What are effective protocols for generating and characterizing fliZ knockout strains?

Generation of fliZ Knockout Strains:

• Gene Replacement Method:

  • Amplify the upstream and downstream regions of fliZ using PCR

  • Digest fragments with appropriate restriction enzymes (e.g., XbaI, SalI, BamHI)

  • Ligate fragments into a cloning vector (e.g., pUC19)

  • Transfer to an integration vector (e.g., pMAD)

  • Transform B. subtilis and select for integration events

  • Screen for marker excision to identify clean deletions

• CRISPR-Cas9 Approach:

  • Design sgRNAs targeting fliZ

  • Provide repair template with homology arms

  • Transform cells and select for successful editing

Characterization of Knockout Strains:

• Molecular Verification:

  • PCR verification of deletion

  • Sequencing of junction regions

  • RT-PCR to confirm absence of transcript

  • Western blotting to confirm absence of protein

• Phenotypic Analysis:

  • Motility assays using soft agar plates

  • Microscopic observation of swimming behavior

  • Electron microscopy to confirm absence of flagella

  • Growth curve analysis to assess general fitness

• Complementation Testing:

  • Reintroduce fliZ gene via plasmid

  • Confirm restoration of motility

  • Use inducible promoters to control expression levels

Based on previous research, null mutants in fliZ do not produce flagella, resulting in non-motile bacteria, but motility can be restored by expression of fliZ from a plasmid .

How can recombinant fliZ be optimally purified for structural and biochemical studies?

Purification of membrane-associated proteins like fliZ presents unique challenges that require specialized approaches:

Purification Strategy:

• Expression Optimization:

  • Use B. subtilis as expression host for native environment

  • Consider a genome-minimized strain lacking proteases

  • Add appropriate affinity tags (His, FLAG, etc.)

  • Optimize induction conditions and harvest time

• Membrane Extraction:

  • Cell lysis via sonication or French press

  • Differential centrifugation to isolate membrane fractions

  • Solubilization using appropriate detergents:

    • Mild detergents (DDM, CHAPS) to maintain native structure

    • Test multiple detergents for optimal solubilization

• Chromatography Steps:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as final polishing step

• Quality Control:

  • SDS-PAGE and Western blotting to assess purity

  • Mass spectrometry for identity confirmation

  • Dynamic light scattering for homogeneity assessment

  • Circular dichroism to verify proper folding

For functional studies, it's critical to verify that the purified protein maintains its native conformation and activity, which can be assessed through complementation assays or in vitro reconstitution experiments.

What imaging techniques are most effective for studying fliZ localization and dynamics?

Understanding the spatial and temporal dynamics of fliZ requires advanced imaging approaches:

Fixed-Cell Imaging:

• Immunofluorescence Microscopy:

  • Fix cells with paraformaldehyde or methanol

  • Permeabilize and immunostain with anti-fliZ antibodies

  • Use super-resolution techniques (STED, STORM) for detailed localization

• Electron Microscopy:

  • Immunogold labeling for transmission electron microscopy

  • Cryo-electron tomography for 3D visualization

  • Correlative light and electron microscopy (CLEM)

Live-Cell Imaging:

• Fluorescent Fusion Proteins:

  • Create fliZ-fluorescent protein fusions (GFP, mCherry)

  • Verify functionality through complementation assays

  • Time-lapse imaging to track dynamics during flagellar assembly

• Advanced Techniques:

  • Fluorescence recovery after photobleaching (FRAP) to assess protein mobility

  • Single-molecule tracking for detailed dynamics

  • Förster resonance energy transfer (FRET) to detect protein-protein interactions

These imaging approaches can reveal crucial information about how fliZ integrates into the membrane and interacts with other flagellar components during the assembly process, providing insights into its essential role in flagellar biogenesis.

Why might recombinant fliZ expression yield low protein levels or inactive protein?

Membrane proteins like fliZ present several challenges for recombinant expression. Common issues and solutions include:

Expression Challenges and Solutions:

For B. subtilis specifically, genome-minimized strains lacking extracellular proteases, prophages, and genes for spore development have shown significantly improved protein production capabilities . These optimized chassis strains have demonstrated over 3000-fold increased secretion of active proteins compared to parental reference strains .

How can contradictory results in fliZ functional assays be reconciled?

Contradictory results in functional studies of fliZ may arise from several factors. A systematic approach to reconciliation includes:

• Strain Background Differences:

  • Different B. subtilis strains may have genetic variations affecting flagellar assembly

  • Document complete strain lineage and confirm genetic background

  • Consider performing experiments in multiple strain backgrounds

• Experimental Condition Variations:

  • Motility assays are sensitive to media composition, temperature, and agar concentration

  • Standardize growth conditions (pH, temperature, media)

  • Use response surface methodology (RSM) to optimize conditions

• Expression Level Variations:

  • Variations in protein expression can affect complementation results

  • Quantify protein levels in each experimental condition

  • Use inducible promoters with varying inducer concentrations to establish dose-response relationships

• Protein Modification Differences:

  • Post-translational modifications may vary between conditions

  • Analyze protein modifications using mass spectrometry

  • Test the impact of specific modifications through site-directed mutagenesis

Unlike flagellar systems in organisms like Salmonella, where some functional redundancy exists, B. subtilis shows a stricter dependence on specific flagellar proteins , potentially making the system more sensitive to experimental variations.

What controls are essential when studying interactions between fliZ and other flagellar proteins?

Rigorous control experiments are crucial for reliable protein interaction studies:

Essential Controls for Interaction Studies:

• Negative Controls:

  • Non-specific binding controls (unrelated proteins with similar properties)

  • Empty vector controls for genetic assays

  • Isotype controls for immunoprecipitation

  • Competition with unlabeled proteins to verify specificity

• Positive Controls:

  • Known interacting protein pairs (e.g., FliS with flagellin )

  • Artificially forced interactions (protein fusions)

• Validation Through Multiple Methods:

  • Confirm interactions using orthogonal techniques:

    • Co-immunoprecipitation

    • Bacterial two-hybrid

    • Fluorescence resonance energy transfer (FRET)

    • Bimolecular fluorescence complementation (BiFC)

• Functional Validation:

  • Assess the impact of mutations that disrupt putative interactions

  • Synthetic lethal analysis for genetic interactions

  • Suppressor mutations to identify compensatory changes

For membrane proteins like fliZ, special attention must be paid to maintaining the native membrane environment during interaction studies, possibly through the use of membrane-mimetic systems such as nanodiscs or liposomes.

How might systems biology approaches enhance our understanding of fliZ's role in flagellar assembly?

Systems biology offers powerful approaches to contextualize fliZ function within the broader flagellar system:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map temporal changes during flagellar assembly

  • Identify regulatory networks controlling fliZ expression

• Computational Modeling:

  • Create kinetic models of flagellar assembly

  • Simulate the impact of fliZ perturbations

  • Predict system behaviors under various conditions

• Network Analysis:

  • Construct protein-protein interaction networks

  • Identify hub proteins and bottlenecks

  • Compare flagellar networks across bacterial species

• High-throughput Genetic Screens:

  • Transposon mutagenesis to identify synthetic interactions

  • CRISPRi screens for partial loss-of-function phenotypes

  • Suppressor screens to identify functional relationships

These approaches can reveal emergent properties of the flagellar system that might not be apparent from studying individual components in isolation, potentially identifying new roles for fliZ beyond its known function in flagellar assembly.

What are the evolutionary implications of fliZ's unique characteristics in Bacillus subtilis?

The evolutionary aspects of fliZ provide intriguing research directions:

• Comparative Genomics:

  • Analyze fliZ homologs across bacterial species

  • Identify conserved motifs and divergent regions

  • Reconstruct the evolutionary history of fliZ

• Structure-Function Evolution:

  • Compare membrane targeting mechanisms across species

  • Identify selective pressures on different protein domains

  • Assess the impact of horizontal gene transfer

• Functional Divergence:

  • Test complementation with fliZ homologs from other species

  • Map species-specific protein interactions

  • Identify adaptive changes related to different ecological niches

• Regulatory Evolution:

  • Compare expression patterns and regulation across species

  • Analyze promoter evolution and transcription factor binding sites

  • Investigate co-evolution with interacting partners

The observation that fliZ lacks significant homology to known proteins suggests a unique evolutionary history that may provide insights into the diversification of bacterial motility systems.

How can structural biology techniques be optimized for membrane-associated proteins like fliZ?

Structural characterization of membrane proteins presents unique challenges that require specialized approaches: