Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Flagellar biosynthetic protein FliQ (fliQ)

What is Buchnera aphidicola and why is its flagellar system significant to researchers?

Buchnera aphidicola is an intracellular bacterial symbiont of aphids that maintains a remarkably small genome of approximately 600 kilobase pairs (kbp) . Despite this reduced genome, which typically retains only genes essential for symbiosis with its aphid host, Buchnera curiously maintains gene clusters coding for flagellum basal body structural proteins and flagellum type III export machinery . This preservation of flagellar genes in an otherwise streamlined genome suggests their potential importance in the symbiotic relationship, making them particularly interesting for evolutionary and functional studies.

The significance lies in the fact that these flagellar structures have been demonstrated to be highly expressed and present in large numbers on Buchnera cells, despite the absence of recognizable pathogenicity factors or secreted proteins in the genome . This raises fundamental questions about the role of these structures in maintaining symbiosis, potentially revealing novel functions for flagellar components in bacterial-host interactions beyond motility.

What is the known function of FliQ in bacterial flagellar systems?

FliQ is a critical component of the flagellar type III secretion system (T3SS), which functions as a molecular machine responsible for the export of flagellar proteins from the cytoplasm to the cell exterior during flagellar assembly. As indicated by comparative studies of flagellar proteins in Listeria monocytogenes, flagellar biosynthetic proteins like FliQ are essential for the proper assembly and function of the flagellar apparatus .

In bacterial systems where it has been characterized, FliQ serves as a membrane component of the flagellar export apparatus, interacting with other flagellar proteins to facilitate protein transport across the cell membrane. Similar to the roles demonstrated for other flagellar proteins (FlhB, FliM, and FliY) in Listeria, FliQ likely participates in both structural assembly and regulatory functions within the flagellar system . The precise mechanisms through which FliQ contributes to flagellar assembly in Buchnera specifically remain an active area of research.

What are the optimal storage and handling conditions for recombinant Buchnera aphidicola FliQ?

Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Flagellar biosynthetic protein FliQ is typically supplied in liquid form containing glycerol, and proper storage is critical for maintaining protein integrity . The recommended storage protocol involves:

• Short-term storage (up to one week): Store working aliquots at 4°C

• Long-term storage: Store at -20°C

• Extended long-term storage: Conserve at -20°C or -80°C

For optimal protein stability, repeated freezing and thawing cycles should be strictly avoided as they can lead to protein denaturation and loss of structural integrity . When working with the protein, it is advisable to thaw aliquots quickly at room temperature or in a water bath at 37°C, followed by immediate use or return to appropriate storage conditions. Working with the protein on ice during experiments can help preserve its activity.

What methodologies are most effective for isolating flagellar basal body complexes containing FliQ from Buchnera aphidicola?

Isolation of flagellar basal body complexes from Buchnera aphidicola requires specialized techniques due to the intracellular nature of this symbiont. Researchers have successfully employed methods to isolate these complexes from the cellular membrane of Buchnera, confirming the enrichment of flagellum basal body proteins relative to other proteins in the Buchnera proteome .

The methodology involves:

• Initial separation of Buchnera cells from host tissues: This typically involves gentle homogenization of aphid tissues followed by differential centrifugation to isolate bacterial cells.

• Membrane fraction isolation: The bacterial cells are lysed using detergent treatments, followed by ultracentrifugation to separate membrane fractions.

• Selective solubilization: Membrane proteins are solubilized using mild detergents (e.g., n-dodecyl β-D-maltoside) that preserve protein-protein interactions within the flagellar complex.

• Affinity purification or density gradient centrifugation: These techniques are employed to isolate intact flagellar basal body complexes.

• Verification of isolated complexes: Mass spectrometry analysis can confirm the presence of flagellar proteins including FliQ in the isolated fractions .

A detailed step-by-step protocol for this isolation procedure has been made publicly available at dx.doi.org/10.17504/protocols.io.bs5wng7e, and mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD024664) .

How can researchers assess the functional properties of recombinant FliQ in experimental systems?

Assessing the functional properties of recombinant FliQ requires multiple complementary approaches:

• Complementation assays: Researchers can utilize FliQ deletion mutants to test whether the recombinant protein restores flagellar function. Similar approaches with flagellar proteins in Listeria have demonstrated that complemented strains (CΔ strains) fully restore motility and flagella synthesis .

• Protein-protein interaction studies: Co-immunoprecipitation, bacterial two-hybrid assays, or surface plasmon resonance can identify interaction partners of FliQ within the flagellar apparatus. These methods can reveal whether recombinant FliQ maintains proper binding capacity to other flagellar components.

• Structural integrity assessment: Circular dichroism spectroscopy can be used to evaluate whether the recombinant protein maintains proper secondary structure, while thermal shift assays can assess protein stability.

• Functional reconstitution: In vitro reconstitution of partial flagellar export complexes can test the ability of recombinant FliQ to participate in protein export.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry analysis can provide information about the spatial arrangement of FliQ relative to other flagellar proteins in membrane complexes.

What effect does deletion or mutation of fliQ have on flagellar gene expression and protein synthesis in bacterial systems?

Studies in model organisms like Listeria monocytogenes provide valuable insights into the regulatory consequences of flagellar gene deletions. While specific data on FliQ in Buchnera is limited, research on related flagellar genes suggests that deletion of essential flagellar components leads to significant downstream effects:

• Transcriptional downregulation: Deletion of flagellar genes like flhB, fliM, or fliY in Listeria results in significant downregulation of most flagellar-associated genes . For example, in Listeria, deletion of flhB causes downregulation of flaA, fliY, fliS, motA, lmo0695, and lmo0698 .

• Protein expression changes: Western blot analysis in Listeria has shown that deletion of flagellar genes can abolish the expression of major flagellar proteins (like FlaA) and reduce the expression of cytoplasmic flagellar proteins . The table below summarizes these effects based on findings in Listeria:

• Phenotypic consequences: In Listeria, deletion of flagellar genes leads to complete abolishment of flagella synthesis and motility . Similar effects would be expected for deletion of fliQ in systems where it plays an essential role in flagellar assembly.

These findings suggest that FliQ, like other flagellar proteins, likely plays both structural and regulatory roles within the flagellar synthesis network. Its deletion would potentially disrupt the expression and assembly of multiple flagellar components through regulatory feedback mechanisms.

What are the critical factors to consider when designing experiments to study protein-protein interactions involving FliQ in Buchnera aphidicola?

When designing experiments to study protein-protein interactions involving FliQ in Buchnera aphidicola, researchers should consider several critical factors:

How can researchers overcome challenges in expressing and purifying functional recombinant FliQ protein?

Expressing and purifying functional recombinant FliQ presents several challenges due to its membrane-associated nature. Researchers can implement the following strategies to maximize success:

• Optimization of expression systems:

  • Use of specialized E. coli strains designed for membrane protein expression (e.g., C41(DE3), C43(DE3))

  • Decreasing expression temperature (16-20°C) to allow proper folding

  • Using weaker promoters to prevent formation of inclusion bodies

  • Codon optimization for the expression host

• Solubilization strategies:

  • Systematic screening of detergents for optimal solubilization

  • Use of amphipols or nanodiscs to maintain membrane protein stability

  • Fusion with solubility-enhancing tags (MBP, NusA, or SUMO)

• Purification approaches:

  • Multi-step purification combining affinity chromatography with size exclusion chromatography

  • On-column detergent exchange during purification

  • Inclusion of stabilizing agents (specific lipids, glycerol) in all buffers

• Quality control assessments:

  • Size exclusion chromatography to confirm monodispersity

  • Thermal shift assays to assess stability

  • Circular dichroism spectroscopy to verify secondary structure

  • Limited proteolysis to evaluate folding quality

• Co-expression strategies:

  • Co-expressing FliQ with interacting partners from the flagellar system

  • Co-expressing with chaperones to aid proper folding

The table below outlines a systematic approach to optimizing expression conditions:

What insights can comparative genomics provide about the evolution of FliQ in Buchnera aphidicola compared to free-living bacteria?

Comparative genomic analysis of FliQ in Buchnera aphidicola versus free-living bacteria reveals several important evolutionary insights:

• Selective retention in a reduced genome: The retention of fliQ and other flagellar genes in the highly reduced Buchnera genome (approximately 600 kbp) strongly suggests important functionality beyond motility, as these symbionts are non-motile within host cells . This selective retention occurred despite extensive gene loss during adaptation to the intracellular lifestyle.

• Sequence conservation patterns: Alignment of FliQ sequences from Buchnera strains with those from free-living relatives can identify:

  • Highly conserved residues essential for core functions

  • Buchnera-specific substitutions that might reflect adaptation to the symbiotic lifestyle

  • Potential relaxation of selective constraints on portions of the protein no longer needed for motility

• Gene neighborhood changes: While free-living bacteria typically maintain flagellar genes in large, well-organized operons, endosymbionts like Buchnera often show genomic rearrangements. Analysis of gene synteny around fliQ can provide insights into the evolutionary forces reshaping the genome.

• Selection pressure analysis: Calculating dN/dS ratios (ratio of non-synonymous to synonymous substitutions) for fliQ across different bacterial lineages can reveal whether the gene is under purifying selection, positive selection, or neutral evolution in Buchnera compared to free-living bacteria.

• Structural prediction comparisons: Using homology modeling to predict the structure of Buchnera FliQ compared to well-characterized FliQ proteins can identify structural adaptations specific to the symbiotic lifestyle.

These comparative approaches can help elucidate why flagellar genes have been retained in Buchnera and how their functions may have been repurposed in the context of symbiosis.

How might the study of FliQ contribute to understanding the symbiotic relationship between Buchnera aphidicola and its aphid host?

The study of FliQ and other flagellar proteins in Buchnera aphidicola offers unique insights into the symbiotic relationship with aphids:

• Potential repurposed functions: Research suggests that flagellar basal body complexes in Buchnera may serve functions beyond motility, potentially related to:

  • Mediating nutrient exchange between symbiont and host

  • Functioning as a conduit for signaling molecules

  • Anchoring the bacterium within specialized host cells (bacteriocytes)

  • Participating in the transfer of Buchnera cells during aphid reproduction

• Evolutionary constraint evidence: The maintenance of flagellar genes despite genome reduction indicates strong selective pressure for their retention, suggesting they play crucial roles in maintaining the symbiotic relationship. Studying FliQ's interactions could reveal key aspects of this essential functionality.

• Host-microbe interface: The flagellar apparatus may represent an important interface between Buchnera and its aphid host. Characterizing FliQ's role in this apparatus could reveal mechanisms by which the symbiont interacts with host cells.

• Comparative insights across aphid species: Comparing FliQ and flagellar structures across Buchnera strains from different aphid species can identify conserved functions essential to the core symbiosis versus adaptations specific to particular host relationships.

• Implications for symbiosis establishment: Understanding the role of flagellar proteins may reveal mechanisms involved in the initial establishment of symbiosis during aphid development and the vertical transmission of symbionts to offspring.

The flagellar apparatus has been demonstrated to be highly expressed and present in large numbers on Buchnera cells, suggesting it serves important functions despite the absence of motility requirements . Elucidating FliQ's role within this context may reveal fundamental principles about how bacterial structures can be repurposed during the evolution of symbiotic relationships.

What emerging technologies could advance our understanding of FliQ function in Buchnera aphidicola?

Several cutting-edge technologies hold promise for deepening our understanding of FliQ function in Buchnera aphidicola:

• Cryo-electron microscopy (cryo-EM): This technique could reveal the detailed structure of the entire flagellar basal body complex in Buchnera at near-atomic resolution, including FliQ's position and interactions within the complex . Recent advances in cryo-EM have made it possible to resolve membrane protein complexes without crystallization.

• In situ cryo-electron tomography: This approach could visualize flagellar complexes directly within intact Buchnera cells in their native context, providing insights into their spatial organization and potential interfaces with host structures.

• Proximity labeling methods: Techniques like BioID or APEX2 could identify proteins in close proximity to FliQ within living cells, helping map the protein interaction network of FliQ in its native environment.

• Single-molecule fluorescence techniques: These methods could track the dynamics of individual FliQ molecules within the flagellar complex, revealing information about assembly processes and protein exchange.

• Microfluidic aphid culture systems: These systems could enable real-time imaging of Buchnera within living aphid hosts while manipulating conditions to study flagellar complex dynamics.

• Gene editing in aphid hosts: CRISPR-based approaches to modify aphid genes that interact with Buchnera could reveal host factors that influence flagellar complex function.

• Systems biology approaches: Multi-omics integration (combining proteomics, transcriptomics, and metabolomics) could place FliQ function within the broader context of symbiont-host metabolic integration.

These technologies, especially when used in combination, could overcome the current limitations in studying obligate intracellular symbionts and reveal the full functional significance of FliQ in the Buchnera-aphid symbiosis.

What are the most promising experimental approaches for understanding the regulatory networks controlling fliQ expression in Buchnera aphidicola?

Understanding the regulatory networks controlling fliQ expression in Buchnera aphidicola presents unique challenges due to the symbiont's obligate intracellular lifestyle. The most promising experimental approaches include:

• Transcriptome analysis under varying conditions:

  • RNA-seq of Buchnera during different stages of aphid development

  • Differential expression analysis in response to environmental stressors

  • Single-cell RNA-seq to capture cell-to-cell variation in expression

• Promoter analysis and regulatory element identification:

  • Identification of conserved motifs upstream of fliQ and other flagellar genes

  • Reporter gene assays using Buchnera promoters in surrogate bacterial hosts

  • DNA-protein interaction studies (e.g., electrophoretic mobility shift assays) to identify transcription factors that bind to the fliQ promoter region

• Comparative genomics approaches:

  • Analysis of regulatory regions across different Buchnera strains

  • Identification of conserved regulatory networks by comparison with free-living relatives

  • Assessment of selection pressure on regulatory regions

• Experimental manipulation approaches:

  • RNA interference in aphids targeting host factors that might influence Buchnera gene expression

  • Nutritional manipulation of aphids to observe effects on Buchnera flagellar gene expression

  • Development of cell-free transcription systems using Buchnera components

• Integration with host regulatory systems:

  • Investigation of potential host-derived signals that influence fliQ expression

  • Analysis of temporal coordination between host developmental stages and changes in flagellar gene expression

By applying these approaches systematically, researchers can begin to unravel the complex regulatory networks controlling fliQ expression in this highly specialized symbiotic bacterium, potentially revealing novel mechanisms of gene regulation that have evolved in the context of long-term intracellular symbiosis.