Recombinant Pectobacterium carotovorum subsp. carotovorum Flagellar biosynthetic protein FliQ (fliQ)

What is the role of FliQ protein in Pectobacterium carotovorum flagellar assembly?

FliQ is a critical component of the flagellar export apparatus within the Pcc flagellar system. As part of the fliLMNOPQR operon, FliQ functions in the type III secretion system that exports flagellar proteins across the cytoplasmic membrane. While not a structural component of the mature flagellum, FliQ is essential for flagellar biosynthesis and the resulting bacterial motility. Mutational studies of related flagellar export apparatus proteins have demonstrated that disruption of these components completely abolishes flagellation and motility .

How does FliQ relate to other flagellar proteins in Pectobacterium carotovorum?

FliQ operates in conjunction with FliO, FliP, and FliR proteins within the flagellar export apparatus. These proteins are encoded by contiguous genes in the fliLMNOPQR operon and collectively form a membrane-embedded complex essential for flagellar protein export. FliQ interacts with other flagellar components including the C-terminal domain of FlhB (FlhBC), which is involved in substrate specificity switching during flagellar assembly . The coordinated expression and function of these proteins is regulated by flagellar master regulators like FlhD .

What is the molecular structure and characteristics of FliQ?

FliQ is a small, highly hydrophobic membrane protein with a predicted molecular mass of approximately 9.6 kDa (based on Salmonella typhimurium homolog studies) . The protein contains multiple transmembrane domains that anchor it within the bacterial inner membrane. Its high hydrophobic residue content causes it to segregate with the membrane fraction during cell fractionation experiments. Due to its membrane localization and small size, structural characterization of FliQ has been challenging, with most insights derived from genetic and functional studies rather than direct structural analysis .

What genetic approaches are most effective for studying fliQ function?

To study fliQ function, researchers should employ targeted gene deletion using homologous recombination strategies, similar to methods used for other flagellar genes. The process involves:

• Designing primers to amplify upstream and downstream regions flanking fliQ

• Creating a deletion construct with a selectable marker (e.g., antibiotic resistance)

• Transforming Pcc with the construct and selecting for recombinants

• Confirming deletion through PCR and sequencing

• Complementing the deletion with an intact copy of fliQ to verify phenotype restoration

For multiple gene deletions in flagellar operons, consider using the FLP/FRT recombination system to enable sequential gene deletions with marker recycling, as demonstrated in other pathosystems . This approach is particularly valuable when analyzing redundant or related flagellar genes within the same operon .

How can researchers effectively analyze motility phenotypes in fliQ mutants?

Researchers should implement a multi-faceted approach to assess motility phenotypes in fliQ mutants:

• Swimming motility assays: Culture bacteria on soft agar plates (0.3% agar) and measure colony diameter after 24-48 hours incubation. Wild-type strains typically show extended migration zones compared to non-motile mutants.

• Swarming motility assays: Use semi-solid agar plates (0.6% agar) to assess collective cell movement. Document colony morphology and diameter after 24-48 hours .

• Microscopic visualization: Employ negative staining with transmission electron microscopy (TEM) to directly observe the presence/absence of flagella. Also implement fluorescence microscopy of GFP-tagged cells to analyze bacterial movement patterns .

• Quantitative motility measurements: Track individual bacterial cell movement using time-lapse microscopy and calculate parameters such as swimming velocity and run/tumble frequency.

These complementary approaches provide comprehensive characterization of motility defects resulting from fliQ mutation .

What methodological approaches effectively link fliQ expression to biofilm formation?

To investigate the relationship between fliQ expression and biofilm formation in Pcc, researchers should employ the following methodologies:

• Air-liquid (AL) biofilm assays: Culture bacteria in glass test tubes containing appropriate medium (e.g., SOBG) and quantify biofilm formation at the air-liquid interface after appropriate incubation periods.

• Surface-attached liquid (SAL) biofilm assays: Use microtiter plates (polyvinyl chloride) containing suitable medium to assess biofilm formation on surfaces.

• Calcofluor binding assays: Measure cellulose production by examining the binding of the cellulose-specific dye Calcofluor to bacterial colonies or biofilms. Quantify fluorescence intensity to determine relative cellulose levels.

• Gene expression analysis: Perform quantitative reverse transcription-PCR to measure expression levels of fliQ alongside known biofilm-related genes (e.g., bcsA, bcsE, and adrA for cellulose production).

• Environmental stress testing: Evaluate biofilm formation under various environmental conditions (temperature, pH, osmolarity) to determine how these factors affect fliQ expression and subsequent biofilm development .

This comprehensive approach allows researchers to establish direct links between fliQ expression, flagellar assembly, and biofilm formation phenomena .

What expression systems are optimal for recombinant production of Pcc FliQ?

For recombinant production of Pcc FliQ, the following expression systems should be considered, each with specific advantages:

E. coli-based expression systems:

• BL21(DE3): Standard system for cytoplasmic expression

• C41(DE3) or C43(DE3): Specialized for membrane protein expression

• Rosetta strains: Provides rare codons that may be present in Pcc genes

Expression vectors:

• pET vectors with T7 promoter for high-level expression

• pBAD vectors for arabinose-inducible, tunable expression

• Vectors containing solubility tags (MBP, SUMO, TrxA) to enhance solubility

Optimization parameters (determined through experimental design approach):

• Temperature: Lower temperatures (16-25°C) often improve membrane protein folding

• Inducer concentration: Optimize IPTG (0.1-1.0 mM) or arabinose concentration

• Expression duration: 4-24 hours depending on protein stability

• Medium composition: Consider enriched media like Terrific Broth

For membrane proteins like FliQ, maintaining the native structure is challenging. Consider using detergent solubilization approaches or expressing the protein with fusion partners that enhance solubility .

What purification strategies overcome challenges associated with the hydrophobic nature of FliQ?

Purifying hydrophobic membrane proteins like FliQ requires specialized approaches:

• Membrane fraction isolation:

  • Disrupt cells using sonication, French press, or mechanical disruption

  • Separate membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

  • Wash membranes with high-salt buffer to remove peripheral proteins

• Detergent solubilization:

  • Screen detergents (DDM, LDAO, OG, FC-12) at concentrations above their CMC

  • Optimize solubilization conditions (temperature, time, buffer composition)

  • Centrifuge to remove insoluble material

• Affinity chromatography:

  • Use His-tagged FliQ for IMAC purification

  • Include detergent in all purification buffers

  • Consider mild elution conditions to preserve protein structure

• Size exclusion chromatography:

  • Further purify protein and assess oligomeric state

  • Remove aggregated protein

• Protein stability assessment:

  • Monitor protein stability using techniques like differential scanning fluorimetry

  • Optimize buffer conditions for long-term storage

Table 1: Detergent screening for FliQ solubilization efficiency

Note: Values are representative based on similar membrane proteins; specific optimization is required for FliQ .

How can researchers evaluate the functional integrity of recombinant FliQ?

To ensure recombinant FliQ maintains its native functional properties:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Thermal stability assays to determine protein folding

  • Size exclusion chromatography to assess oligomeric state

• Protein-protein interaction studies:

  • In vitro binding assays with other flagellar components (FliO, FliP, FliR)

  • Pull-down assays using tagged proteins to verify interactions

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

• Complementation assays:

  • Express recombinant FliQ in fliQ deletion mutants

  • Assess restoration of motility and flagella formation

  • Quantify complementation efficiency by motility measurements

• Membrane integration analysis:

  • Protease protection assays to verify correct membrane topology

  • Fluorescent labeling to track membrane localization

  • Liposome reconstitution to evaluate membrane insertion properties

• Functional export assays:

  • In vitro reconstitution of flagellar export apparatus

  • Measure protein export using labeled flagellar substrates

These approaches collectively ensure that the recombinant FliQ retains properties similar to its native form .

How does FliQ contribute to type III secretion system function in Pcc?

FliQ is an integral component of the flagellar type III secretion system (T3SS) in Pcc, which shares homology with virulence-associated T3SS. Research should investigate:

• Protein export analysis:

  • Compare secretion profiles between wild-type and fliQ mutants using SDS-PAGE and western blot

  • Quantify export of flagellar proteins (e.g., FlaA, FlgD) into culture supernatants

  • Assess timing and hierarchy of protein export during flagellar assembly

• Interaction with export apparatus components:

  • Analyze interactions between FliQ and other export apparatus proteins (FliP, FliR, FlhB)

  • Investigate substrate recognition mechanisms through crosslinking studies

  • Determine the role of FliQ in substrate specificity switching

• Energy coupling:

  • Evaluate the relationship between FliQ function and proton motive force

  • Investigate ATP requirements for FliQ-mediated export

  • Assess protein-protein interactions with energy supply components

Studies in related systems suggest FliQ forms part of a membrane-embedded export gate that enables the passage of flagellar proteins across the cytoplasmic membrane in an ordered manner, with FliQ potentially involved in substrate recognition or channel formation .

What molecular techniques can uncover the relationship between FliQ and virulence in plant pathogens?

To investigate FliQ's role in virulence:

• Pathogenicity assays:

  • Compare wild-type, fliQ deletion mutants, and complemented strains in plant infection models

  • Assess disease development using multiple inoculation methods:

    • Stem injection (100 μL of 10^8 CFU/mL bacterial suspension)

    • Soil drenching (500 mL of 10^8 CFU/mL bacterial suspension)

    • Potato slice maceration assays (measured using ImageJ)

  • Quantify disease progression through pathogenicity index calculations

• Virulence factor expression analysis:

  • Measure expression of plant cell wall degrading enzymes (PCWDEs) like pectinases, cellulases, and proteases

  • Quantify enzyme activities using plate assays (PGA plates for pectinase, CMC plates for cellulase)

  • Perform qRT-PCR to analyze expression of virulence genes in fliQ mutants compared to wild-type

• Colonization studies:

  • Track bacterial populations in planta using fluorescently labeled strains

  • Assess attachment to plant surfaces and biofilm formation

  • Measure bacterial survival under plant defense-related stress conditions

• Transcriptome analysis:

  • Perform RNA-seq comparing wild-type and fliQ mutants during infection

  • Identify virulence-associated pathways affected by fliQ mutation

  • Validate findings with targeted gene expression studies

These approaches can establish direct connections between flagellar motility, type III secretion, and virulence mechanisms in plant pathogens .

How do environmental conditions modulate fliQ expression and function?

To comprehensively investigate environmental regulation of fliQ:

• Gene expression analysis under varying conditions:

  • Temperature (15°C, 25°C, 30°C, 37°C)

  • pH (5.0, 6.0, 7.0, 8.0)

  • Osmolarity (0, 100, 200, 300 mM NaCl)

  • Nutrient availability (minimal vs. rich media)

  • Plant extract exposure

• Promoter activity studies:

  • Generate transcriptional fusions between fliQ promoter and reporter genes (GFP, luciferase)

  • Monitor promoter activity under different environmental conditions

  • Identify environmental cues that trigger expression changes

• Regulatory network analysis:

  • Investigate roles of known regulators (FlhD, CytR homolog) in controlling fliQ expression

  • Perform chromatin immunoprecipitation (ChIP) to identify direct regulators

  • Construct genetic networks connecting environmental sensing to flagellar gene expression

• Protein functionality assessment:

  • Evaluate FliQ function and stability across environmental conditions

  • Determine critical thresholds for functional export apparatus assembly

  • Assess posttranslational modifications affecting FliQ activity

Table 2: Environmental modulation of fliQ expression relative to housekeeping genes

Note: Values are representative based on similar flagellar genes; specific data would need to be experimentally determined for fliQ in Pcc .

How does the expression of recombinant FliQ affect other flagellar gene expression in heterologous systems?

When expressing recombinant FliQ in heterologous systems, researchers should investigate potential feedback mechanisms and regulatory perturbations:

• Transcriptome analysis:

  • Perform RNA-seq comparing host cells with and without recombinant FliQ expression

  • Focus on changes in endogenous flagellar gene expression

  • Identify potential regulatory cross-talk between foreign and host flagellar systems

• Promoter activity studies:

  • Generate reporter fusions with endogenous flagellar gene promoters

  • Measure activity in the presence/absence of recombinant FliQ

  • Investigate dose-dependent effects by modulating FliQ expression levels

• Protein-protein interaction studies:

  • Perform pull-down assays to identify interactions between recombinant FliQ and host proteins

  • Use bacterial two-hybrid systems to screen for novel interactions

  • Validate interactions using co-immunoprecipitation and FRET analysis

• Flagellar assembly assessment:

  • Examine flagellar morphology and function in host cells expressing recombinant FliQ

  • Determine if recombinant FliQ incorporates into host flagellar structures

  • Assess potential dominant-negative effects on flagellar assembly

What advanced experimental designs can resolve contradictory findings about FliQ function across different bacterial species?

To address contradictory findings about FliQ function:

• Meta-analysis approach:

  • Systematically compile and analyze data from different studies

  • Identify experimental variables contributing to contradictory results

  • Develop standardized protocols for cross-species comparison

• Structured experimental design:

  • Implement factorial design experiments to simultaneously test multiple variables

  • Use multivariate statistical approaches to identify significant factors

  • Employ ANOVA and regression analysis to build predictive models

• Cross-species complementation studies:

  • Express FliQ from different species in Pcc fliQ mutants

  • Assess functional complementation through motility restoration

  • Identify conserved and species-specific functional domains

• Domain swapping experiments:

  • Create chimeric FliQ proteins with domains from different species

  • Map functional regions responsible for species-specific differences

  • Correlate structural features with functional outcomes

• Contradiction detection and resolution framework:

  • Apply formalized contradiction detection methodology similar to dialogue modeling frameworks

  • Identify precise points of contradiction between studies

  • Design targeted experiments specifically addressing contradictory findings

This systematic approach helps resolve apparent contradictions and builds a unified understanding of FliQ function across bacterial species .

How can researchers develop recombinant FliQ as a tool for studying bacterial flagellar assembly dynamics?

Advanced applications of recombinant FliQ include:

• FliQ-based biosensors:

  • Engineer fluorescently labeled FliQ variants to track flagellar assembly in real-time

  • Develop FRET-based sensors to monitor protein-protein interactions during assembly

  • Create split-GFP complementation systems to visualize export apparatus formation

• In vitro reconstitution systems:

  • Purify components of the flagellar export apparatus including FliQ

  • Reconstitute functional export apparatus in artificial membrane systems

  • Measure protein export kinetics and energetics in controlled environments

• Cryo-EM structural studies:

  • Use recombinant FliQ to facilitate structural determination of the export apparatus

  • Generate stable subcomplexes for high-resolution structural analysis

  • Map conformational changes during different stages of export

• Single-molecule tracking:

  • Develop methods to track individual FliQ molecules during flagellar assembly

  • Measure residence times and exchange rates within the export apparatus

  • Determine stoichiometry of FliQ in functional export complexes

• Synthetic biology applications:

  • Engineer modified flagellar export systems with altered specificity

  • Develop protein secretion tools based on flagellar export principles

  • Create minimal synthetic flagellar systems to study essential components

These advanced approaches represent the frontier of flagellar research and provide powerful tools for understanding complex macromolecular assembly processes .