Recombinant Pseudomonas syringae pv. tomato Flagellar hook-basal body complex protein FliE (fliE)

What is FliE in Pseudomonas syringae pv. tomato and what is its structural organization?

FliE is a critical protein component of the bacterial flagellar assembly in Pseudomonas syringae pv. tomato. Structurally, FliE consists of three α-helices (α1, α2, and α3) that form the most proximal part of the flagellar rod. The α1 helix binds to the inner wall of the MS-ring, while the α2 and α3 helices form domain D0 in a manner similar to other rod proteins. Six copies of FliE assemble together at the base of the flagellar rod within the MS-ring . This structural organization enables FliE to serve as an interface between the MS-ring and the rod complex, facilitating critical interactions with other basal body proteins including FliP, FliR, FlgB, and FlgC .

Research shows that FliE's position in the flagellar assembly is crucial for proper flagellar function as it serves as both the final component of the flagellar type III secretion system (fT3SS) and as the base of the rod structure, making it essential for motility and pathogenicity .

How is the fliE gene organized in the P. syringae genome?

The fliE gene in Pseudomonas syringae is part of the flagellar gene cluster. Based on genomic analyses, flagellar genes in Pseudomonas syringae and related bacteria are typically organized into several regions or operons. In bacteria like Salmonella, which serves as a model system for flagellar studies, the flagellar genes are organized into the flg, flh, fli, and flj regions .

The fliE gene specifically belongs to the fli operon, which encodes proteins necessary for the assembly of the flagellar basal body and export apparatus. This gene organization reflects the sequential assembly process of the flagellum, where FliE is expressed early in the assembly pathway . The flagellar gene organization in P. syringae pv. tomato DC3000 has been extensively studied as this strain is one of the most intensively researched bacterial plant pathogens .

How is FliE evolutionarily conserved across bacterial species?

FliE shows significant conservation across flagellated bacteria, although with some structural variations reflecting adaptation to different ecological niches. Comparative analysis between FliE from Pseudomonas syringae and other bacterial species like Escherichia coli and Salmonella reveals conserved functional domains that are critical for flagellar assembly.

The consistent presence of FliE across diverse bacterial species underscores its fundamental role in flagellar assembly and bacterial motility. This evolutionary conservation makes FliE an interesting target for comparative genomic studies and potentially for broad-spectrum antimicrobial development.

What expression systems are optimal for producing recombinant FliE protein?

For successful recombinant expression of FliE from Pseudomonas syringae pv. tomato, bacterial expression systems have proven most effective. Based on published research, E. coli-based expression systems are commonly used due to their high yield and relative simplicity. The following approach has been demonstrated to yield purified, functional recombinant FliE protein:

Expression System Components:

• Vector: pET-based expression vectors with a His-tag for purification

• Host: E. coli BL21(DE3) or similar strains optimized for protein expression

• Induction: IPTG induction at 0.5-1 mM concentration

• Culture conditions: 25-30°C post-induction to minimize inclusion body formation

Research on recombinant flagellar proteins, including FliE, has demonstrated successful purification using SDS-PAGE analysis and His-tag detection . The resulting proteins can be confirmed by LC-ESI-MS/MS methodology to verify protein identity and integrity.

When working with FliE specifically, it's critical to optimize expression conditions to maintain proper protein folding, as improper folding can affect functional studies of this structural protein. Some studies suggest co-expression with chaperone proteins may enhance proper folding of flagellar proteins.

How can site-directed mutagenesis be used to study FliE function?

Site-directed mutagenesis of FliE provides valuable insights into structure-function relationships within the flagellar assembly. An effective methodology involves:

• Plasmid Construction for Mutagenesis:

  • PCR amplification of fliE gene from P. syringae genomic DNA

  • Introduction of specific mutations using overlap extension PCR or commercial mutagenesis kits

  • Cloning into appropriate vectors (e.g., pENTR/D-TOPO followed by Gateway cloning into expression vectors)

• Transformation and Selection:

  • Electroporation into E. coli S17-1 for conjugal transfer

  • Selection of P. syringae transconjugants on media containing appropriate antibiotics

  • Confirmation of mutations by PCR and sequencing

• Functional Assessment:

  • Motility assays to assess the impact of mutations on swimming behavior

  • Electron microscopy to examine flagellar structure

  • Export assays to evaluate secretion system functionality

Previous research has demonstrated that mutations in the fliE gene can significantly affect flagellar assembly and function. For instance, in Salmonella, a deletion mutant lacking amino acids 18-31 of FliE showed impaired motility. Interestingly, motile revertants emerged through tandem duplications of fliE sequences flanking the deleted region, restoring the protein length to near wild-type, which highlights the importance of FliE's structural integrity .

What methods are available for studying FliE-protein interactions in the flagellar assembly?

Several methodological approaches can be employed to investigate FliE interactions with other flagellar proteins:

• Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged FliE in P. syringae

  • Crosslink protein complexes in vivo

  • Isolate complexes using antibodies against the tag

  • Identify interacting partners through mass spectrometry

• Bacterial Two-Hybrid Assays:

  • Clone fliE and potential interacting partners into two-hybrid vectors

  • Co-transform into reporter strains

  • Measure reporter activity to detect protein-protein interactions

• In vitro Binding Assays:

  • Purify recombinant FliE and potential binding partners

  • Perform pull-down assays using affinity tags

  • Detect interactions through Western blotting

• Cryo-electron Microscopy:

  • Recent advances in cryo-EM have enabled visualization of the flagellar basal body at 2.2-3.7 Å resolution

  • These techniques reveal how FliE interacts with the MS-ring and other proximal rod proteins

Research has shown that FliE interacts with several proteins including FliP, FliR, FlgB, and FlgC in the basal body . Understanding these interactions is crucial for determining how FliE contributes to both flagellar structure and the type III secretion system functionality.

How does FliE contribute to bacterial motility and virulence in P. syringae?

FliE plays a critical dual role in P. syringae motility and virulence through its functions in flagellar assembly:

Contribution to Motility:
FliE forms the base of the flagellar rod and is essential for complete flagellar assembly. Without functional FliE, the flagellum cannot be properly constructed, resulting in impaired motility. Motility is crucial for P. syringae to move toward favorable environments, colonize plant surfaces, and locate entry points for infection .

Link to Virulence:
Flagellar-mediated motility is a significant virulence factor in P. syringae. Recent research has revealed that P. syringae coordinates the production of motility and virulence factors, with flagellar expression showing phenotypic heterogeneity both in vitro and during plant colonization . This suggests bacterial subpopulations with different roles during infection:

• Flagella-expressing bacteria (with functional FliE) may be critical for:

  • Invasion and initial colonization

  • Dispersal from infected tissues before necrosis

  • Movement between infection sites

The flagellum can also trigger plant immune responses as a pathogen-associated molecular pattern (PAMP), making the regulation of flagellar expression (including FliE) crucial for immune evasion strategies. Studies have shown that P. syringae mutants lacking functional flagella (which would include fliE mutants) show altered virulence profiles in plant infection models .

How can recombineering techniques be applied to study FliE function in P. syringae?

Recombineering offers powerful approaches for precise genetic manipulation of P. syringae to study FliE function. Based on established methodologies, the following recombineering protocol can be implemented:

Protocol for FliE Recombineering in P. syringae:

• Expression of Recombination Proteins:

  • Construct a plasmid expressing RecT from P. syringae pv. syringae B728a (for ssDNA recombination) or RecTE (for dsDNA recombination)

  • Transform this plasmid into P. syringae pv. tomato DC3000

  • Express recombination proteins under appropriate promoter control

• Design of Targeting DNA:

  • For point mutations in fliE: Design 60-80 bp oligonucleotides centered on the desired mutation

  • For gene replacement/tagging: Design dsDNA PCR products with 50-100 bp homology arms flanking the target sequence

• Transformation and Selection:

  • Introduce DNA by electroporation into RecT/RecTE-expressing cells

  • For selectable modifications, plate on appropriate selective media

  • For non-selectable modifications, screen by PCR or phenotypic assays

Recombineering efficiency data from similar systems suggests:

• RecT-mediated ssDNA recombination: ~4,000 recombinants/10⁸ viable cells (25-fold higher than control)

• RecTE-mediated dsDNA recombination: Approximately 17-fold increased efficiency compared to RecT alone

This approach allows various modifications to study FliE function:

• Introduction of point mutations to assess specific residues

• In-frame deletions to study domain function

• Epitope tagging for interaction studies

• Fusion to fluorescent reporters to monitor expression and localization

The RecTE system from P. syringae has demonstrated robust performance in generating targeted mutations, making it an ideal tool for detailed functional analysis of FliE .

What is the role of FliE in the flagellar type III secretion system of P. syringae?

FliE plays a crucial role in the flagellar type III secretion system (fT3SS) of P. syringae, serving as both a structural component and facilitating secretion:

Structural Role in fT3SS:
FliE forms the most proximal component of the rod structure, creating a critical junction between the MS-ring (FliF) and the rest of the export apparatus. Studies in related systems show that six copies of FliE assemble into a ring-like structure, creating a passage for secreted proteins .

Secretion Functions:

• Substrate Export: FliE is essential for the export of flagellar proteins including hook (FlgE) and hook-capping (FlgD) proteins. Research in Salmonella has shown an 8-fold reduction in hook protein secretion in fliE null strains .

• Self-secretion Mechanism: Interestingly, FliE facilitates its own secretion. Studies using FliE-Bla fusion proteins demonstrated significant ampicillin resistance levels (6.25 μg/ml compared to <1 μg/ml for controls), indicating efficient secretion into the periplasm. This creates a "causality dilemma" where the first FliE subunit must be secreted by an apparatus lacking FliE .

Integration with Export Apparatus:
Molecular studies reveal that FliE interacts directly with the export apparatus components FliP and FliR, creating a continuous channel from the cytoplasm to the growing flagellar structure. These interactions create a functional secretion system that recognizes and exports flagellar proteins in a highly ordered manner .

Understanding FliE's role in the fT3SS has significant implications beyond flagellar assembly, as the flagellar export system shares evolutionary origins with virulence-associated type III secretion systems that deliver effector proteins into host cells.

How does the structure of recombinant FliE compare to native FliE in terms of folding and functionality?

The structural comparison between recombinant and native FliE reveals important considerations for functional studies:

Structural Characteristics:

Functional Assessment Methods:

• Complementation Assays: Testing if recombinant FliE can restore motility in fliE deletion mutants

• In vitro Assembly: Assessing if recombinant FliE can incorporate into partially assembled flagellar structures

• Protein-Protein Interaction Analysis: Comparing binding affinities with partner proteins

Critical Factors Affecting Recombinant FliE Functionality:

• Expression temperature (lower temperatures often yield better folding)

• Solubilization conditions (detergent selection critical for membrane-proximal proteins)

• Purification method (native versus denaturing conditions)

• Refolding protocols when necessary

Advanced structural studies have confirmed that properly produced recombinant FliE can maintain functional conformation, particularly when expression conditions are optimized to prevent inclusion body formation. Cryo-EM studies at 2.2-3.7 Å resolution have provided detailed insights into FliE structure within the intact flagellar basal body, establishing a benchmark for evaluating recombinant protein quality .

What is the potential of FliE as a target for plant protection against P. syringae infection?

FliE presents several advantages as a potential target for developing strategies against P. syringae infection in plants:

Targeting Rationale:

• Essential for Motility: FliE is critical for flagellar assembly and bacterial motility, which is essential for P. syringae virulence and host colonization .

• Surface Accessibility: As part of the flagellar structure, epitopes of FliE may be accessible to antibodies or other binding molecules.

• Immunogenic Properties: Studies in other bacterial systems have shown that recombinant flagellar proteins, including FliE, can be immunogenic. Research with Campylobacter jejuni FliE demonstrated reactivity with various antibodies, suggesting immunological relevance .

Intervention Strategies:

Efficacy Considerations:
The effectiveness of FliE-targeting approaches depends on several factors:

• Timing relative to infection cycle (most effective before widespread colonization)

• Delivery method to ensure contact with bacteria

• Potential for bacterial adaptation or resistance

• Impact on beneficial microbiota

Recent research showing that P. syringae populations coordinate flagellar expression during infection suggests that targeting FliE could disrupt the specialized motile subpopulation critical for bacterial dispersal and infection progression .

How can transcriptional regulation of fliE be studied in P. syringae under different environmental conditions?

Understanding the transcriptional regulation of fliE in P. syringae under varying environmental conditions requires sophisticated experimental approaches:

Experimental Design for fliE Transcriptional Analysis:

• Reporter Fusion Construction:

  • Create transcriptional fusions between the fliE promoter and reporter genes (GFP, luciferase)

  • Clone the promoter region (250-300 bp upstream of fliE) into reporter vectors like pPROBE-GT

  • Transform constructs into P. syringae by electroporation

• Environmental Condition Testing:

  • Plant apoplast-mimicking medium vs. rich laboratory medium

  • Various temperatures (18°C, 22°C, 28°C)

  • pH variations (5.5-7.5)

  • Nutrient limitations

  • Plant extract exposure

  • Presence of plant defense compounds

• Single-Cell Expression Analysis:

  • Flow cytometry to quantify reporter expression at the single-cell level

  • Fluorescence microscopy to visualize expression in microcolonies

  • Time-lapse imaging to track expression dynamics

• Regulatory Network Analysis:

  • Construct mutations in known flagellar regulators (FleQ, FliA)

  • Examine fliE expression in regulatory mutants

  • ChIP-seq to identify direct binding of regulators to the fliE promoter

Recent research has revealed that flagellar gene expression in P. syringae displays significant phenotypic heterogeneity both in vitro and during plant colonization . The fleQ gene has been identified as a key regulator of flagellar genes in Pseudomonas species. Experimental approaches have successfully used allelic exchange to generate fleQ deletion mutants by replacing the ORF with a kanamycin cassette, allowing for detailed assessment of its regulatory impact .