Recombinant Bacillus subtilis Flagellar biosynthetic protein FliQ (fliQ)

What is the genetic organization of fliQ in Bacillus subtilis?

FliQ is encoded within the major che-fla operon of B. subtilis. Specifically, nucleotide sequencing has identified fliQ as one of three genes in this operon, alongside fliP and fliZ. The fliQ gene begins at position 1490 in the sequence and has a potential ribosome binding site (AGGGTAGG) 11 nucleotides upstream of the GTG start codon. The downstream partial open reading frame (ORF) encodes for the first three amino acids of B. subtilis FliR, which continues into a 10.9-kb EcoRI fragment .

What homologies exist between B. subtilis FliQ and proteins from other bacterial species?

B. subtilis FliQ shares significant sequence homology with flagellar biosynthetic proteins from several Gram-negative bacteria. Specifically, it is homologous to the FliQ proteins of Escherichia coli and Salmonella typhimurium . Additionally, sequence analyses have revealed that B. subtilis FliQ shares significant homology with the Shigella flexneri Spa9 virulence protein, which is involved in the presentation of surface plasmid antigens. This homology supports the growing hypothesis that a superfamily of proteins exists for the biosynthesis of supramolecular structures that lie external to the cell membrane .

What experimental approaches can be used to study FliQ function in B. subtilis?

To investigate FliQ function in B. subtilis, researchers can employ several methodological approaches:

• Gene knockout studies: Creating fliQ null mutants through insertional mutagenesis using antibiotic resistance markers such as the chloramphenicol acetyltransferase (cat) gene. This approach allows researchers to observe phenotypic changes associated with FliQ absence .

• Complementation assays: Expressing fliQ from a plasmid in null mutants to restore motility, confirming the specific role of FliQ in flagellar assembly .

• Fluorescence microscopy: Using cysteine-reactive dyes to visualize flagellar structures, similar to approaches used for studying FlgE (the hook protein) .

• Motility assays: Employing swimming and swarming assays on semi-solid agar to quantify the effects of fliQ mutations on bacterial motility .

• Electron microscopy: Directly visualizing flagellar structures to determine the impact of fliQ mutations on flagellar assembly .

How does flagellar gene expression regulation occur in B. subtilis?

In B. subtilis, flagellar genes are regulated through a hierarchical system:

• The proteins needed for the hook-basal body (HBB), including FliQ, are transcribed in the 31-gene fla-che operon .

• The penultimate gene of this operon, sigD, encodes the sigma factor σD that activates transcription of the late flagellar genes, including the flagellar filament gene hag, stator genes motA and motB, the anti-sigma factor flgM, and the hook-filament junction genes flgK and flgL .

• In wild-type B. subtilis, while all cells transcribe the fla-che operon, only a subpopulation synthesize flagella due to heterogeneity in sigD transcription, with a threshold level of sigD required for flagellar gene expression .

• The anti-sigma factor FlgM binds to and inhibits σD, preventing premature expression of late flagellar genes. Unlike in Salmonella, FlgM has never been reported to be secreted in B. subtilis, and the coordination of FlgM activity with flagellar assembly remains poorly understood .

What are the specific interactions between FliQ and other flagellar proteins in the type III secretion system of B. subtilis?

The flagellar biosynthesis in B. subtilis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. FliQ's role in this system involves interactions with several other proteins:

• FliP-FliQ-FliR complex: These proteins likely form a membrane-embedded complex that facilitates protein export through the cytoplasmic membrane. While the exact interactions are not fully characterized in B. subtilis, by analogy with Salmonella, this complex forms part of the export gate of the flagellar type III secretion system .

• FlhB interaction: FliQ likely interacts with FlhB, which is involved in substrate specificity switching during flagellar assembly. The B. subtilis FlhB protein shares homology with Shigella flexneri Spa40, further supporting the conservation of these interaction networks .

• Export apparatus assembly: Current evidence suggests that FliQ, along with FliP and FliR, are required for the assembly of the rivet at the earliest stage of flagellar biosynthesis, indicating these proteins function early in the flagellar assembly pathway .

Experimental approaches to study these interactions include:

• Bacterial two-hybrid assays to detect protein-protein interactions

• Co-immunoprecipitation studies

• Cross-linking experiments followed by mass spectrometry

• Site-directed mutagenesis to identify critical interaction residues

How does the structure of FliQ contribute to its function in flagellar assembly?

While the three-dimensional structure of B. subtilis FliQ has not been fully resolved, sequence analysis and comparative studies provide insights into its structural features:

• Membrane topology: FliQ is likely a membrane protein with predicted transmembrane domains that anchor it in the cytoplasmic membrane.

• Conserved domains: The homology between B. subtilis FliQ and its counterparts in other bacteria suggests the presence of conserved domains critical for function.

• Structural prediction: By comparison with the better-studied Salmonella FliQ, B. subtilis FliQ likely forms part of the export gate complex within the basal body of the flagellum.

Methodological approaches to investigate FliQ structure include:

• Protein structure prediction using computational tools

• Membrane topology mapping using reporter fusions

• Cysteine scanning mutagenesis to identify exposed residues

• X-ray crystallography or cryo-electron microscopy of purified protein complexes

What are the differences in flagellar assembly between Gram-positive bacteria like B. subtilis and Gram-negative models such as Salmonella?

The flagellar assembly process differs significantly between Gram-positive B. subtilis and Gram-negative models:

Research approaches to study these differences include:

• Comparative genomics and proteomics

• Heterologous expression of B. subtilis components in Gram-negative models and vice versa

• Creation of chimeric flagellar proteins to identify functional domains

• High-resolution imaging of flagellar structures in both systems

How can recombinant B. subtilis strains with modified fliQ be designed for enhanced flagellar assembly or function?

Designing recombinant B. subtilis strains with modified fliQ requires several strategic considerations:

• Expression system selection: For controlled expression of modified fliQ, researchers can use inducible promoters such as the xylose-inducible system or the IPTG-inducible Pspac promoter. The B. subtilis rrnO promoter system has been successfully used for high-level expression of recombinant proteins .

• Integration strategies: Gene constructs can be integrated into the B. subtilis chromosome at neutral loci such as amyE (amylase) using plasmids like pDG364 or pDL243 that allow double-crossover recombination .

• Protein tagging approaches: FliQ can be modified with epitope tags or fluorescent proteins, positioning these modifications carefully to avoid disrupting function. For instance:

  • C-terminal tags are often less disruptive to membrane protein function

  • Linker sequences between FliQ and tags can reduce steric hindrance

  • Site-specific incorporation of cysteine residues allows for fluorescent labeling with thiol-reactive dyes

• Strain background selection: Using protease-deficient strains (e.g., WB800 derivatives) can improve stability of recombinant proteins. Additionally, selecting strains with intact flagellar operons but specific mutations in genes of interest provides a clean background for complementation studies .

What methods can be used to purify and characterize recombinant FliQ protein for structural studies?

Purification and characterization of membrane proteins like FliQ present unique challenges. The following methodological approaches are recommended:

• Expression systems optimization:

  • Use of E. coli strains specialized for membrane protein expression (C41, C43)

  • Codon optimization of the fliQ gene for the expression host

  • Low-temperature induction to reduce inclusion body formation

  • Addition of fusion partners like maltose-binding protein (MBP) to enhance solubility

• Membrane extraction protocols:

  • Selective extraction using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

  • Sequential extraction with increasing detergent concentrations

  • Use of styrene-maleic acid lipid particles (SMALPs) to extract membrane proteins with their native lipid environment

• Purification strategies:

  • Affinity chromatography using His-tags or other fusion partners

  • Size exclusion chromatography to separate protein-detergent complexes

  • Ion exchange chromatography for further purification

• Structural characterization methods:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Nuclear magnetic resonance (NMR) for solution structure determination

  • X-ray crystallography following successful crystallization trials

  • Cryo-electron microscopy for membrane protein complexes

  • Hydrogen-deuterium exchange mass spectrometry to probe protein dynamics

How can transcriptomic and proteomic approaches be used to understand the regulatory network controlling fliQ expression?

Understanding the regulatory network controlling fliQ expression requires multi-omics approaches:

• Transcriptomic analyses:

  • RNA-Seq to quantify fliQ transcript levels under different conditions

  • Differential expression analysis comparing wild-type and regulatory mutants

  • Transcription start site mapping using 5' RACE or primer extension

  • ChIP-Seq to identify transcription factors binding to the fliQ promoter region

• Proteomic approaches:

  • Quantitative proteomics using stable isotope labeling (SILAC)

  • Targeted proteomics (SRM/MRM) to accurately quantify FliQ protein levels

  • Phosphoproteomics to identify potential post-translational modifications

  • Protein-protein interaction studies using pull-down assays coupled with mass spectrometry

• Data integration strategies:

  • Correlation analysis between transcript and protein abundance

  • Network analysis to identify regulatory hubs

  • Comparison with known flagellar gene regulatory networks in other bacteria

  • Machine learning approaches to predict regulatory interactions

• Validation experiments:

  • Reporter gene assays using fliQ promoter fusions

  • Electrophoretic mobility shift assays (EMSA) to confirm protein-DNA interactions

  • In vitro transcription assays to validate direct regulatory effects

  • CRISPR interference (CRISPRi) to modulate expression of candidate regulators

How can understanding FliQ function contribute to the development of novel antimicrobial strategies?

The essential role of FliQ in flagellar biosynthesis makes it a potential target for antimicrobial development:

• Target validation approaches:

  • Demonstrate that inhibition of FliQ function reduces bacterial virulence in infection models

  • Show that FliQ is essential for colonization using competitive index assays

  • Verify conservation of FliQ across pathogenic species to ensure broad-spectrum potential

• Inhibitor screening strategies:

  • Develop high-throughput assays based on flagellar motility

  • Design peptide mimetics that interfere with FliQ-protein interactions

  • Conduct virtual screening against the predicted FliQ structure

  • Implement fragment-based drug discovery approaches

• Alternative applications:

  • FliQ-based vaccines targeting flagellar assembly

  • Anti-virulence approaches that inhibit motility without killing bacteria, potentially reducing selective pressure for resistance

What are the challenges in expressing and analyzing post-translational modifications of FliQ?

Post-translational modifications (PTMs) of flagellar proteins add complexity to their analysis:

• Detection challenges:

  • Low abundance of membrane proteins like FliQ makes PTM detection difficult

  • Sample preparation methods can remove or modify PTMs

  • Heterogeneity in modification sites complicates analysis

• Analysis approaches:

  • Enrichment strategies for phosphorylated proteins

  • Multiple reaction monitoring (MRM) mass spectrometry for targeted PTM analysis

  • Application of electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation for improved PTM site localization

• Functional validation:

  • Site-directed mutagenesis of modified residues to assess functional impact

  • Creation of phosphomimetic (e.g., Ser/Thr to Asp/Glu) or non-phosphorylatable (e.g., Ser/Thr to Ala) mutations

  • Analysis of protein localization, stability, and interaction partners in PTM mutants

• Comparative analysis:

  • The B. subtilis flagellum has been shown to be regulated by mechanisms not identified in other bacterial species

  • Tyrosine 49 and arginine 60 have been identified as phosphorylation sites in YvyG (FlgN), suggesting that other flagellar proteins like FliQ might also be regulated by phosphorylation

How can researchers effectively design experiments to study FliQ function without polar effects on downstream genes?

Studying FliQ function without affecting downstream genes requires careful experimental design:

• Non-polar mutation strategies:

  • Use of in-frame deletion methods to preserve reading frame

  • Implementation of markerless deletion techniques using counterselectable markers

  • Design of transcriptional terminators followed by promoters to isolate mutations

• Complementation approaches:

  • Expression of fliQ from a separate locus (e.g., amyE) under its native promoter

  • Use of IPTG-inducible systems to titrate expression levels

  • Construction of operon fusions that maintain downstream gene expression

• Validation methods:

  • RT-qPCR to confirm expression of downstream genes

  • Western blotting to verify protein levels

  • Phenotypic assays to ensure complementation restores wild-type behavior

What are the best methods to analyze contradictory data regarding FliQ localization and function?

Resolving contradictory data about FliQ requires systematic approaches:

• Technical validation:

  • Replicate experiments using multiple independent methods

  • Compare results across different strain backgrounds

  • Verify antibody specificity or tag functionality in control experiments

• Biological interpretation:

  • Consider context-dependency of FliQ function

  • Evaluate growth conditions and their impact on flagellar assembly

  • Assess potential functional redundancy with other proteins

• Reconciliation strategies:

  • Develop testable hypotheses that could explain discrepancies

  • Design experiments specifically to address contradictions

  • Consider temporal aspects of FliQ function during flagellar assembly

• Collaborative approaches:

  • Share reagents between labs reporting contradictory results

  • Perform blinded analyses to eliminate confirmation bias

  • Implement standardized protocols across research groups