Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Flagellar M-ring protein (fliF)

What is Buchnera aphidicola and why is it significant for research?

Buchnera aphidicola is an obligate symbiotic bacterium that sustains aphid physiology by complementing their exclusive phloem sap diet . This symbiotic relationship has been remarkably successful, with almost all members of the Aphididae family harboring this gamma-proteobacterium . The symbiosis between Buchnera and primitive aphids has led to strict co-speciation, resulting in different strains associated with different aphid species, including Acyrthosiphon pisum (BAp), Schizaphis graminum (BSg), Baizongia pistaciae (BBp), and Cinara cedri (BCc) .

Buchnera is characterized by its small genome, having lost many genes essential for autogenous life while retaining those necessary for obtaining nutrients from the host . This genomic reduction makes Buchnera an excellent model for studying the evolution of symbiotic relationships and the minimal genetic requirements for bacterial life, providing insights into fundamental biological processes and evolutionary adaptation mechanisms.

What is the flagellar M-ring protein (FliF) and what is its standard function in bacteria?

The flagellar M-ring protein (FliF) is a critical structural component of bacterial flagella that forms the M-ring structure embedded in the membrane at the base of the flagellum . It serves as the initial structure required for flagellum assembly and comprises multiple molecules of a two-transmembrane protein . In motile bacteria, FliF forms part of the MS-ring complex, which acts as a rotor and serves as a foundation for the assembly of other flagellar components .

The standard function of FliF in motile bacteria is to anchor the flagellar structure to the cell membrane and provide a platform for the assembly of the C-ring, which is formed by FliG, FliM, and FliN proteins just below the MS-ring . This complex structure is essential for bacterial motility, enabling the cell to respond to environmental stimuli through directed movement.

Why would researchers study flagellar proteins in a non-motile bacterium like Buchnera?

Although Buchnera cells are nonmotile, they surprisingly retain clusters of flagellar genes, which are actually transcribed and translated . This apparent paradox presents an intriguing research question: why would a non-motile bacterium maintain and express genes traditionally associated with motility?

Researchers study flagellar proteins in Buchnera because these structures appear to have been repurposed for alternative functions essential to the symbiotic relationship. The Buchnera cell surface is covered with hundreds of hook-basal-body (HBB) complexes, suggesting a role other than motility . Current evidence indicates that these structures may function as protein transporters, not only for flagellar proteins but also for other proteins necessary to maintain the symbiotic system .

This functional repurposing of flagellar structures represents a fascinating example of evolutionary adaptation and provides insights into how protein secretion systems can evolve new functions in the context of symbiotic relationships.

How does the FliF protein in Buchnera aphidicola compare to that in free-living bacteria?

For FliF specifically, while the search results don't provide the exact sequence homology percentage with Salmonella, we can see that other flagellar proteins in Buchnera show homology ranging from as high as 75% for FlgG to as low as 20.8% for FliJ . This pattern of conservation suggests that selective pressure has maintained the structural integrity of certain flagellar components, including FliF, even as their functional roles may have shifted in the context of symbiosis.

The conservation of FliF structure, despite the loss of motility, strongly supports the hypothesis that this protein serves an important alternative function in Buchnera, likely related to protein transport or maintaining the symbiotic relationship with the aphid host.

What is known about the genetic organization of flagellar genes in Buchnera?

The flagellar genes in Buchnera aphidicola are arranged in five operons, which are clustered in three regions of the genome . This organization is remarkably similar to that found in free-living bacteria like Salmonella, with the order of genes within operons being highly conserved . This conservation in genetic organization suggests that the regulatory mechanisms governing flagellar gene expression may also be preserved.

Importantly, while Buchnera retains many flagellar genes, it lacks the late genes necessary for motility, including the flagellin gene . This selective retention of early and middle flagellar genes, but not those required for the final assembly of a functional motile flagellum, supports the hypothesis that in Buchnera, these genes serve purposes other than motility.

The genomic analysis of Buchnera aphidicola strain APS has revealed that it retains flagellar genes coding for proteins of the hook and the basal body, but not for the filament . This pattern of gene retention and loss provides important clues about the functional adaptation of the flagellar apparatus in this obligate symbiont.

What expression systems are most effective for producing recombinant Buchnera FliF?

Based on research with similar flagellar proteins, heterologous expression systems using E. coli are likely the most effective for producing recombinant Buchnera FliF. Studies have shown successful expression of fusion proteins connecting FliF and FliG from other bacterial species in E. coli systems . These fusion proteins were able to form MS-rings efficiently, suggesting that E. coli can properly express and fold these complex membrane proteins .

When designing an expression system for Buchnera FliF, researchers should consider several factors:

• Codon optimization: Due to the different codon usage between Buchnera and expression hosts like E. coli, codon optimization may improve expression levels.

• Membrane protein expression tags: Since FliF is a membrane protein with two transmembrane domains, expression may be improved using tags specifically designed for membrane proteins, such as fusion partners that assist in membrane insertion.

• Induction conditions: Lower temperatures (16-25°C) and reduced inducer concentrations often improve the proper folding of complex membrane proteins.

• Host strain selection: E. coli strains specifically designed for membrane protein expression, such as C41(DE3) or C43(DE3), may provide better results than standard laboratory strains.

What challenges might researchers face when working with recombinant FliF?

Working with recombinant FliF presents several significant challenges due to its nature as a membrane protein and its role in complex protein assemblies:

• Membrane protein solubility: As a membrane-embedded protein, FliF is hydrophobic and may form inclusion bodies when overexpressed. Researchers may need to optimize solubilization conditions using appropriate detergents.

• Maintaining native structure: Ensuring that recombinant FliF maintains its native conformation is crucial, especially if functional studies are planned. The protein's ability to form the characteristic ring structure should be verified.

• Protein-protein interactions: FliF normally interacts with other flagellar proteins, particularly FliG. Studies have shown that the N-terminal region of FliG interacts with the C-terminal region of FliF . These interaction domains may affect protein folding and stability when FliF is expressed alone.

• Functional assessment: Since Buchnera FliF likely serves a non-motility function, traditional flagellar function assays may not be applicable. Researchers will need to develop appropriate assays to evaluate the protein's activity in protein transport or other symbiosis-related functions.

How can researchers verify the correct assembly of recombinant FliF?

Verifying the correct assembly of recombinant FliF requires multiple complementary approaches:

• Electron microscopy: Transmission electron microscopy can be used to visualize the characteristic ring structures formed by properly assembled FliF proteins. Research has demonstrated that this approach can effectively identify MS-rings formed by FliF and FliF-FliG fusion proteins .

• High-speed atomic force microscopy (HS-AFM): This technique has been successfully used to observe the formation and structure of MS-rings by FliF-FliG fusion proteins, revealing both the stable ring structure and the flexible movements at the FliG region .

• Size exclusion chromatography: This method can help determine whether FliF has assembled into the expected high-molecular-weight complexes characteristic of MS-rings.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can verify that FliF monomers are interacting correctly to form the expected oligomeric structures.

• Functional reconstitution: For the most rigorous verification, researchers might attempt to reconstitute partial flagellar structures by combining recombinant FliF with other flagellar proteins and observing whether they assemble correctly, similar to studies that have shown that FliFG fusion proteins can form ring structures in E. coli .

How can recombinant FliF be used to study protein transport mechanisms in Buchnera-aphid symbiosis?

Recombinant FliF can serve as a valuable tool for investigating the protein transport systems that are crucial for maintaining the Buchnera-aphid symbiotic relationship. Since the flagellar apparatus in Buchnera appears to function primarily as a protein transporter rather than for motility, recombinant FliF can be used in several experimental approaches:

• Protein interaction studies: Pull-down assays, co-immunoprecipitation, or yeast two-hybrid screening using recombinant FliF as bait can identify aphid or Buchnera proteins that interact with the flagellar apparatus, revealing potential cargo for this transport system.

• Reconstitution of transport systems: Recombinant FliF could be used to reconstruct minimal versions of the transport apparatus in liposomes or membrane vesicles, allowing researchers to test the transport of specific candidate proteins.

• Structural studies: Cryo-electron microscopy of recombinant FliF assembled into rings, alone or with other flagellar components, can provide insights into structural adaptations that may facilitate protein transport rather than motility.

• Mutational analysis: By creating specific mutations in recombinant FliF based on comparisons with motile bacterial homologs, researchers can identify regions crucial for protein transport functions versus those needed for motility.

• In vivo studies: Transgenic expression of modified versions of FliF in Buchnera (though technically challenging) could help evaluate how alterations in this protein affect the symbiotic relationship with the aphid host.

What techniques are most suitable for analyzing interactions between FliF and other flagellar proteins?

Several complementary techniques are suitable for analyzing the interactions between FliF and other flagellar proteins, particularly FliG, which is known to interact directly with FliF:

• Yeast two-hybrid (Y2H) analysis: This can be used to map the interaction domains between FliF and other flagellar proteins. Previous studies have shown that the N-terminal region of FliG interacts with the C-terminal region of FliF .

• Bacterial two-hybrid systems: These may be more appropriate than Y2H for membrane proteins like FliF and can provide insights into interactions in a bacterial cellular environment.

• Surface plasmon resonance (SPR): This technique can measure the binding kinetics and affinity between purified recombinant FliF and other flagellar proteins.

• Co-expression and co-purification: Co-expressing FliF with potential interaction partners and attempting co-purification can provide evidence of stable complex formation. The successful formation of MS-rings by FliF-FliG fusion proteins supports the feasibility of this approach .

• Cross-linking mass spectrometry: This technique can identify specific residues involved in protein-protein interactions, providing detailed structural information about how FliF interacts with other flagellar components.

• Fluorescence resonance energy transfer (FRET): By tagging FliF and potential interaction partners with appropriate fluorophores, FRET can detect interactions in real-time and potentially in living cells.

How might researchers investigate the evolutionary adaptation of FliF in different Buchnera strains?

Investigating the evolutionary adaptation of FliF across different Buchnera strains can provide insights into how this protein has been repurposed for symbiosis-specific functions. Several approaches can be employed:

• Comparative genomics: Analyzing FliF sequences from multiple Buchnera strains from different aphid hosts (such as those from Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistaciae, and Cinara cedri ) can reveal patterns of conservation and divergence that might correlate with specific functional adaptations.

• Selection analysis: Calculating the ratio of nonsynonymous to synonymous substitutions (dN/dS) across FliF sequences can identify regions under positive, negative, or relaxed selection, providing clues about functional constraints.

• Structural modeling: Using homology modeling based on crystallographic structures of FliF from model organisms to predict the structures of FliF from different Buchnera strains can highlight structural adaptations that might relate to functional changes.

• Functional complementation studies: Testing whether FliF from different Buchnera strains can functionally substitute for each other or for FliF in free-living bacteria can reveal functional divergence.

• Host-specific adaptation analysis: Correlating variations in FliF sequence or expression with differences in aphid host biology or ecology could identify adaptations related to specific symbiotic relationships. Research has shown that Buchnera population size is significantly affected by the host plant but varies with aphid genotype interactions , suggesting that bacterial adaptations might be influenced by both the aphid host and its diet.

How does the flagellar type III secretion system function in Buchnera despite genome reduction?

The functioning of the flagellar type III secretion system (T3SS) in Buchnera despite its reduced genome represents a fascinating research question. The search results indicate that the six core proteins of the T3SS (FlhA, FlhB, FliI, FliP, FliQ, and FliR) are highly conserved in Buchnera, with approximately 40% sequence homology compared to those of Salmonella . This conservation suggests strong selective pressure to maintain this secretion machinery.

A detailed analysis of the sequence conservation of these T3SS components in Buchnera compared to Salmonella reveals varying degrees of homology:

The notably high conservation of FliI (55.6%), FliQ (63.6%), and FlhA (62.1%) suggests that these components are particularly crucial for the functioning of the secretion system . FliI is known to be an ATPase that provides energy for the secretion process, while FliQ and FlhA are integral components of the export apparatus.

What is the role of FliF in maintaining the symbiotic relationship between Buchnera and aphids?

The role of FliF in maintaining the symbiotic relationship between Buchnera and aphids likely centers on its function as a structural component of the protein transport apparatus. As the foundation of the MS-ring, FliF provides the architectural platform upon which the rest of the flagellar basal body is assembled . In Buchnera, this structure appears to have been repurposed primarily for protein secretion rather than motility .

Several lines of evidence suggest potential roles for FliF in symbiosis:

• Nutrient exchange: The flagellar apparatus may facilitate the transport of amino acids and other nutrients from Buchnera to the aphid host, complementing the aphid's nutrient-poor phloem sap diet .

• Developmental signaling: Secreted proteins might serve as signals that coordinate the development or reproduction of both partners in the symbiosis.

• Maintenance of bacteriocytes: The specialized cells (bacteriocytes) that house Buchnera within the aphid require specific conditions for proper function. Research has confirmed that these bacteriocytes contain Buchnera using PCR amplification of the dnaK gene . The flagellar apparatus might participate in maintaining the appropriate microenvironment within these cells.

• Adaptation to host plant variation: Studies have shown that Buchnera population size is significantly affected by the aphid's host plant . The protein transport system based on the flagellar apparatus might help Buchnera adapt to changing nutrient availability as the aphid feeds on different plant species.

How might the study of Buchnera FliF contribute to our understanding of protein secretion system evolution?

The study of Buchnera FliF offers unique insights into the evolution of protein secretion systems for several reasons:

• Repurposing of motility structures: Buchnera provides a clear example of how structures originally evolved for motility can be repurposed for protein secretion, offering insights into the evolutionary plasticity of bacterial cellular machinery.

• Minimalist approach to secretion: With its reduced genome, Buchnera has likely retained only the essential components needed for protein secretion. Studying this streamlined system can help identify the core components required for functional type III secretion.

• Host-adaptation signatures: Comparing FliF and other flagellar proteins across different Buchnera strains associated with different aphid hosts may reveal how secretion systems adapt to specific symbiotic relationships. This could provide a model for studying host-specific adaptation of bacterial secretion systems.

• Evolutionary trade-offs: The maintenance of flagellar genes despite genome reduction suggests strong selective pressure for their retention, highlighting the crucial nature of protein secretion in symbiotic relationships. Studying the genetic and structural features that have been conserved versus those that have been lost can illuminate the evolutionary trade-offs involved in adapting secretion systems to new functions.

• Convergent evolution: Comparing the Buchnera flagellar apparatus with other protein secretion systems that have evolved from different ancestral structures may reveal patterns of convergent evolution, providing insights into the fundamental principles governing the evolution of protein transport mechanisms.

What are the broader implications of Buchnera FliF research for understanding symbiotic systems?

Research on Buchnera FliF and its associated flagellar structures has several broader implications for understanding symbiotic systems:

• The repurposing of flagellar structures for protein transport in Buchnera demonstrates how ancient cellular machinery can be adapted for new functions in the context of symbiosis. This example of exaptation (the repurposing of structures for new functions) provides insights into the molecular mechanisms underlying the evolution of symbiotic relationships.

• The conservation of FliF and other flagellar components despite genome reduction highlights the essential nature of protein transport in maintaining symbiotic relationships. This suggests that similar transport mechanisms might be crucial in other symbiotic systems, even those with more extensive genome reduction.

• The study of Buchnera FliF contributes to our understanding of how bacteria and hosts communicate and exchange resources, a fundamental aspect of all symbiotic relationships. The protein transport mechanisms facilitated by the flagellar apparatus may represent a common strategy for maintaining stable and mutually beneficial interactions between partners.

• The influence of host plant on Buchnera population size suggests that environmental factors affecting the host also impact the symbiont. Understanding how the flagellar apparatus and its protein transport function respond to these changing conditions may provide insights into the resilience and adaptability of symbiotic systems in general.

What future research directions are most promising for advancing our understanding of Buchnera FliF?

Several promising research directions could significantly advance our understanding of Buchnera FliF:

• Structural biology approaches: Cryo-electron microscopy and X-ray crystallography of Buchnera FliF, both alone and in complex with other flagellar proteins, would provide detailed insights into structural adaptations for its specialized function in symbiosis.

• Comparative genomics and proteomics: Expanding the analysis to include more Buchnera strains from diverse aphid hosts could reveal patterns of conservation and adaptation in FliF and other flagellar proteins that correlate with specific symbiotic relationships.

• Functional studies: Developing in vitro reconstitution systems for the Buchnera flagellar apparatus would allow direct testing of its protein transport capabilities and the identification of transported substrates.

• Systems biology approaches: Integrating transcriptomic, proteomic, and metabolomic data to understand how the flagellar apparatus functions within the broader context of Buchnera-aphid symbiosis could reveal regulatory networks and metabolic dependencies.

• Experimental evolution: While challenging due to the obligate nature of the symbiosis, experimental evolution studies with aphids and their Buchnera symbionts under different conditions might reveal adaptations in the flagellar apparatus that respond to changing selective pressures.

• Comparative studies with other symbiotic systems: Comparing the Buchnera flagellar apparatus with similar structures in other endosymbionts could identify common principles of adaptation and reveal how different symbiotic systems have solved similar challenges.