Recombinant Rhizobium meliloti Flagellar M-ring protein (fliF)

What is the basic structure of R. meliloti FliF protein?

FliF is a ~60 kDa protein embedded in the cytoplasmic membrane through two transmembrane helices. The periplasmic region between these helices consists of three structurally similar domains termed Ring-Building Motifs (RBMs): RBM1, RBM2, and RBM3. These RBMs possess a common fold but adopt different symmetries within the assembled MS-ring structure . The periplasmic domains show structural homology with components of the Type III Secretion System (T3SS), with RBM1 and RBM2 having sequence similarity to the T3SS protein SctJ, while RBM3 shows homology to the SpoIIIAG protein involved in spore formation .

How does the MS-ring form, and what is its role in flagellar assembly?

The MS-ring, formed by oligomerization of FliF proteins, serves as the fundamental scaffold for flagellar structure and assembly. It is one of the first structures to form during flagellar biogenesis and functions as a platform for recruiting the C-ring through interaction with FliG and the export apparatus . The assembled MS-ring exhibits a remarkable symmetry mismatch between its domains: RBM3 forms a 34-mer ring, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring . This structural arrangement is crucial for the subsequent steps of flagellar assembly.

How does FliF differ between R. meliloti and enteric bacteria?

While the general function of FliF is conserved across bacterial species, there are significant differences in structure and assembly mechanisms. Unlike the enterobacterial FliF, which readily forms oligomeric rings when purified in isolation, FliF from non-peritrichous organisms like Vibrio alginolyticus and Helicobacter pylori requires additional factors to trigger efficient MS-ring assembly . This suggests different regulatory mechanisms for flagellar assembly in R. meliloti compared to model organisms like E. coli and Salmonella.

What are effective approaches for producing recombinant R. meliloti FliF?

Based on experimental studies, the following methodology has proven effective:

• Gene cloning: Amplify the fliF gene from R. meliloti genomic DNA using PCR with specific primers targeting the coding region.

• Expression system selection: Express in either E. coli or R. meliloti itself, with E. coli BL21(DE3) being suitable for high-yield production.

• Vector design: Use vectors containing T7 or tac promoters for inducible expression.

• Purification strategy: Employ a two-step purification using affinity chromatography with His-tagged constructs followed by size exclusion chromatography (SEC) .

• Domain-specific constructs: For structural studies, engineer constructs encompassing individual RBM domains or combinations thereof:

  • RBM1-RBM2-RBM3 (residues 50-438)

  • RBM1-RBM2 (residues 50-229)

  • RBM2 (residues 124-229)

  • RBM3 (residues 231-438)

How can MS-ring assembly be visualized and analyzed?

Multiple complementary techniques have been successfully employed:

• Size exclusion chromatography (SEC): Determines the oligomeric state of FliF constructs in solution.

• SEC-MALS: Provides accurate molecular weight measurements of purified protein complexes.

• Negative stain electron microscopy: Reveals the ring-like structures formed by FliF oligomers.

• High-speed atomic force microscopy (HS-AFM): Captures dynamic movements of the ring structures and flexible domains.

• Cryo-electron microscopy: Enables higher-resolution structural analysis of the assembled MS-ring .

What genetic approaches are most effective for studying FliF function in R. meliloti?

Effective genetic approaches include:

• Gene replacement: Using suicide vectors to replace the chromosomal fliF gene with a mutant version.

• Transposon mutagenesis: Mini-Tn5 transposon insertions in fliF to create non-motile mutants .

• Complementation analysis: Expressing wild-type or mutant fliF from plasmids in fliF-deletion strains.

• Domain swapping: Creating chimeric constructs combining domains from FliF proteins of different bacterial species.

• Site-directed mutagenesis: Introducing specific mutations to analyze the contribution of key residues to FliF function.

• Protein-protein interaction studies: Bacterial two-hybrid assays to identify interaction partners of FliF .

How does FliF interact with other flagellar proteins in R. meliloti?

FliF primarily interacts with:

• FliG: The MS-ring directly interacts with FliG to recruit the C-ring components. Studies using fusion proteins connecting FliF and FliG have demonstrated that this interaction is essential for flagellar assembly and function .

• Export apparatus proteins: FliF interacts with components of the flagellar export apparatus (including FliPQR complex), which assembles within the central pore of the MS-ring .

• Stator proteins: Indirectly connected to the MotA/MotB stator system via FliG .

These interactions form the basis of a complex protein network that enables flagellar assembly and function in R. meliloti.

What regulatory mechanisms control fliF expression in R. meliloti?

The expression of fliF in R. meliloti is regulated within a hierarchical flagellar gene expression system:

• Master regulators: VisN and VisR act as global transcription activators of flagellar genes, forming a heterodimer (VisNR) that regulates class II flagellar genes, which likely include fliF .

• Hierarchical organization: Flagellar genes in R. meliloti are organized into three classes: class I (visN and visR), class II (likely including fliF and other structural genes), and class III (flagellin genes) .

• Promoter structure: The fliF gene is likely transcribed from promoters recognized by sigma factors specific for flagellar gene expression.

• Feedback regulation: The assembly state of the flagellum may provide feedback to regulate expression of flagellar genes including fliF.

How do mutations in fliF affect flagellar assembly and bacterial motility?

Mutations in fliF have profound effects on flagellar assembly and bacterial motility:

Deletion of the fliF gene blocks flagellar assembly at an early stage, preventing the formation of the export apparatus and all subsequent flagellar structures, resulting in complete loss of motility .

How does R. meliloti FliF compare structurally with FliF proteins from other bacterial species?

Comparative analysis reveals significant structural and functional differences:

While all FliF proteins contain similar domain organizations, their assembly mechanisms differ significantly between peritrichous bacteria like Salmonella and non-peritrichous organisms like V. alginolyticus and H. pylori. R. meliloti FliF likely shares characteristics with both groups .

How does the genetic organization of flagellar genes differ between R. meliloti and other bacteria?

The genetic organization of flagellar genes in R. meliloti differs significantly from the enterobacterial paradigm:

• Contiguous region: In R. meliloti, flagellar genes are organized as seven operons and six transcription units mapping to a contiguous 45-kb chromosomal region .

• Regulatory genes: The master regulators visN and visR are specific to R. meliloti and related alpha-proteobacteria, differing from the flhDC system in enterobacteria .

• Flagellin genes: R. meliloti contains multiple flagellin genes (flaA, flaB, flaC, flaD) that encode subunits for its complex flagellar filaments, in contrast to the single flagellin genes in many other bacteria .

• Additional motility genes: R. meliloti possesses unique motility genes, including motC, motD (now identified as fliK), and the fliL paralog motF, that are not found in the enterobacterial flagellar system .

How does the unique symmetry mismatch within the FliF MS-ring contribute to flagellar function in R. meliloti?

The MS-ring formed by FliF exhibits an intriguing symmetry mismatch, with RBM3 forming a 34-mer ring while RBM2 forms a 23-mer ring . This structural feature raises several research questions:

• Assembly mechanism: The different symmetries within a single protein suggest a complex folding and assembly pathway. Research indicates that RBM1 may prevent RBM2 oligomerization by occluding its interface, while RBM3 can displace RBM1 to allow RBM2 oligomerization .

• Functional implications: The symmetry mismatch may serve multiple functions in flagellar assembly and rotation. It could facilitate interactions with both the 34-fold symmetric outer membrane components and the lower-symmetry C-ring components.

• Evolutionary significance: This arrangement may represent an evolutionary adaptation specific to the complex flagellar system of R. meliloti and related bacteria.

Advanced structural studies combining cryo-EM, computational modeling, and site-directed mutagenesis would be needed to fully elucidate this phenomenon.

What is the role of FliF in coordinating the assembly of the complex flagellar filaments of R. meliloti?

R. meliloti possesses complex flagellar filaments composed of multiple flagellin proteins (FlaA, FlaB, FlaC, FlaD), unlike the single-flagellin filaments of model organisms . Understanding how FliF coordinates the assembly of these complex filaments involves several research directions:

• Export apparatus recruitment: FliF must correctly position the export apparatus to enable sequential export of flagellar components, including the multiple flagellins.

• Temporal regulation: The assembly of complex filaments may require precise temporal regulation of flagellin export, potentially involving signals transmitted through the MS-ring.

• Structural adaptations: The MS-ring structure may contain specific adaptations to accommodate the complex filament architecture.

Research approaches could include creating chimeric FliF proteins between R. meliloti and single-flagellin species, followed by analysis of filament structure and composition.

How do environmental factors influence FliF assembly and function in R. meliloti during plant-microbe interactions?

R. meliloti is a soil bacterium that forms symbiotic relationships with legume plants. Environmental factors likely influence FliF assembly and function during these interactions:

• Rhizosphere conditions: The plant rhizosphere presents unique chemical and physical conditions that may affect flagellar assembly. Studies have shown that R. meliloti genes involved in rhizosphere colonization can be identified using signature-tagged mutagenesis approaches .

• Host-specific adaptation: The flagellar system may be adapted for specific host interactions. Gene expression in different legume rhizospheres could reveal host-specific adaptations of the flagellar system .

• Nutrient availability: Limited nutrients in the rhizosphere, such as biotin, can affect R. meliloti growth and potentially flagellar assembly . How nutrient limitation affects FliF expression and assembly remains an open question.

• Regulatory cross-talk: Potential cross-talk between symbiosis signaling and flagellar gene regulation could influence FliF function during plant colonization.

Research approaches could include analyses of FliF expression and MS-ring assembly under simulated rhizosphere conditions and in plant-associated bacteria.

What are the molecular mechanisms behind the differential assembly requirements of FliF in peritrichous versus non-peritrichous bacteria?

FliF from peritrichous bacteria like Salmonella readily forms MS-rings when expressed alone, while FliF from non-peritrichous bacteria like V. alginolyticus and H. pylori requires additional factors . Understanding this difference involves:

• Structural determinants: Identifying the specific structural features that enable or prevent spontaneous oligomerization.

• Assembly factors: Characterizing the additional proteins required for MS-ring assembly in non-peritrichous bacteria.

• Evolutionary significance: Investigating whether these differences reflect adaptations to different ecological niches or motility strategies.

• R. meliloti positioning: Determining where R. meliloti FliF falls on this spectrum and what factors influence its assembly.

Research approaches could include domain-swapping experiments between FliF proteins of different bacteria and identification of potential assembly factors in R. meliloti.

What are the challenges in purifying and reconstituting functional R. meliloti FliF in vitro?

Several methodological challenges exist:

• Membrane protein handling: As a membrane protein, FliF presents typical challenges related to solubility and stability.

• Large complex formation: The MS-ring is a large multimeric complex that may be difficult to maintain in vitro.

• Detergent selection: The choice of detergent is critical for maintaining native-like structure during purification. Different detergents (CHAPS, Triton X-100) have been used for isolating basal body components with varying success .

• Reconstitution environment: Reconstituting functional MS-rings may require specific lipid compositions or membrane mimetics.

• Associated proteins: Purification may require co-expression of additional flagellar components to stabilize the complex.

These challenges can be addressed through systematic optimization of purification conditions and potentially co-expression strategies with interacting partners.

How can genetic manipulation in R. meliloti be optimized for studying FliF function?

Genetic manipulation in R. meliloti presents specific challenges that can be addressed through optimized approaches:

• Gene replacement strategies: Use suicide vectors with counter-selectable markers to facilitate efficient allelic exchange .

• Complementation analysis: Employ broad-host-range vectors like pRK290 for controlled expression of wild-type or mutant fliF in deletion backgrounds .

• Expression control: Carefully consider promoter strength, as excessive expression of flagellar genes can disrupt normal regulation and assembly .

• Phenotypic assessment: Combine motility assays, electron microscopy, and fluorescence microscopy to comprehensively evaluate flagellar structure and function.

• Rhizosphere relevance: Consider testing mutants not only in laboratory conditions but also in plant-associated environments to assess ecological relevance .