Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Flagellar assembly protein fliH (fliH)

What is Buchnera aphidicola and why is its subspecies from Baizongia pistaciae significant for research?

Buchnera aphidicola is an obligate symbiotic bacterium that sustains the physiology of aphids by complementing their exclusive phloem sap diet. It has established a symbiotic relationship with aphids dating back 160-280 million years . The subspecies from Baizongia pistaciae (the Pistacia horn gall aphid) represents one of several distinct strains that have co-evolved with different aphid hosts. This particular subspecies is significant because it offers a unique model for studying genome reduction in obligate symbionts and the evolution of protein transport mechanisms in bacteria with highly specialized ecological niches .

Baizongia pistaciae itself is distributed across the Mediterranean, Middle East, parts of Asia, and North Africa, forming distinctive horn-shaped galls on Pistacia trees as part of its two-year holocyclic life cycle . The Buchnera strain within this aphid has adapted to this specific host environment, making it valuable for comparative genomic and proteomic studies.

What is the functional role of the flagellar assembly protein FliH in Buchnera aphidicola?

Despite being nonmotile, Buchnera aphidicola retains clusters of flagellar genes, including those encoding components of the hook-basal body (HBB) complex. The flagellar assembly protein FliH plays a crucial role in the type III secretion system (T3SS) that forms part of the flagellar export apparatus . In Buchnera, the flagellar proteins are actually transcribed, translated, and assembled into functional HBB complexes on the cell surface .

Unlike in motile bacteria where FliH contributes to flagellar motility, in Buchnera this protein appears to have been repurposed. The hundreds of HBB complexes covering the Buchnera cell surface suggest these structures function as protein transporters rather than motility apparatus . FliH likely facilitates the export of flagellar components and potentially other proteins necessary for maintaining the symbiotic relationship with the aphid host.

How has the FliH protein evolved in Buchnera compared to free-living bacteria?

The FliH protein in Buchnera aphidicola represents an example of functional repurposing during reductive evolution. Unlike free-living bacteria where flagellar proteins primarily serve motility functions, Buchnera has retained only those flagellar genes necessary for building the HBB complex, while genes for flagellar filaments and motor proteins have been lost through genome reduction .

This selective retention suggests strong evolutionary pressure to maintain the protein transport function of the flagellar apparatus even as the motility function was lost. The Buchnera FliH protein likely maintains its core role in coordinating protein export through the flagellar T3SS, but may have evolved specific adaptations for transporting proteins involved in the symbiotic relationship with its aphid host. This functional shift demonstrates how bacterial proteins can be repurposed when organisms transition from free-living to endosymbiotic lifestyles.

What are the optimal methods for expressing and purifying recombinant Buchnera aphidicola FliH protein?

For expressing recombinant Buchnera aphidicola FliH protein, a heterologous expression system using E. coli is generally most effective due to the challenging nature of culturing Buchnera directly. The recommended methodology involves:

• Gene synthesis or PCR amplification of the fliH gene from Buchnera aphidicola subsp. Baizongia pistaciae genomic DNA

• Cloning into an expression vector with an appropriate tag (e.g., His6, GST) for purification

• Transformation into an E. coli expression strain optimized for potentially toxic proteins (BL21-AI or C43(DE3))

• Induction with IPTG at lower temperatures (16-20°C) to enhance solubility

• Cell lysis under native conditions

• Affinity chromatography followed by size exclusion chromatography

Since FliH is part of the flagellar T3SS and may interact with membranes, the addition of mild detergents (0.05% DDM or 0.5% CHAPS) during purification can improve yield and stability. For structural studies, protein buffer optimization through thermal shift assays is recommended to identify conditions that maximize protein stability.

What challenges are associated with studying the structure-function relationship of FliH in Buchnera aphidicola?

Studying the structure-function relationship of FliH in Buchnera aphidicola presents several unique challenges:

• Genetic intractability: As an obligate endosymbiont, Buchnera cannot be cultured independently, making direct genetic manipulation extremely difficult .

• Protein interactions: FliH functions as part of a complex protein interaction network within the flagellar apparatus. In Buchnera, these interactions may be modified due to the repurposing of the flagellar structures for protein transport rather than motility .

• Structural determination challenges: The membrane-associated nature of the FliH protein within the flagellar export apparatus makes it difficult to crystallize. Cryo-electron microscopy approaches may be more suitable but require stable protein complexes.

• Functional assays: Developing assays to measure the protein transport activity of the FliH-containing flagellar apparatus in Buchnera is complicated by the need to maintain the symbiotic environment.

To overcome these challenges, researchers typically employ comparative analysis with better-characterized flagellar systems, heterologous expression systems, and in vitro reconstitution of the protein transport function using purified components.

How can researchers effectively study FliH-protein interactions in the context of the flagellar export apparatus?

To effectively study FliH-protein interactions within the flagellar export apparatus, researchers should employ a multi-faceted approach:

• Yeast two-hybrid or bacterial two-hybrid assays: These can identify direct protein-protein interactions between FliH and other flagellar components like FliI (ATPase) and FliJ (chaperone).

• Pull-down assays with co-expressed proteins: Co-expression of His-tagged FliH with potential interacting partners followed by affinity purification can identify stable complexes.

• Cross-linking coupled with mass spectrometry: This approach can capture transient interactions and identify the specific residues involved in protein-protein contacts.

• Surface plasmon resonance or isothermal titration calorimetry: These techniques provide quantitative measurements of binding affinities between FliH and its interacting partners.

• Cryo-electron microscopy: For structural studies of the assembled HBB complex, cryo-EM can visualize the arrangement of FliH within the native context of the flagellar apparatus.

Since Buchnera has hundreds of HBB complexes per cell , immunoelectron microscopy using antibodies against recombinant FliH can confirm its localization within these structures and provide insights into its arrangement and abundance.

How does the flagellar apparatus in Buchnera aphidicola contribute to symbiosis with its aphid host?

The flagellar apparatus in Buchnera aphidicola, despite lacking motility function, appears to have been repurposed as a critical component of the endosymbiotic relationship with aphids. The hundreds of hook-basal body (HBB) complexes covering the Buchnera cell surface likely function as specialized protein transporters . This system may facilitate:

• Nutrient exchange: The flagellar type III secretion system (T3SS) could export essential amino acids and vitamins synthesized by Buchnera into the bacteriocyte cytoplasm for uptake by the aphid host .

• Signaling molecules: The HBB complexes may secrete proteins that modulate host cell processes or communicate nutritional status.

• Maintaining symbiotic homeostasis: The flagellar apparatus could regulate the exchange of metabolites between symbiont and host, helping maintain the proper balance in this obligate relationship.

The high density of these structures on the Buchnera cell surface (hundreds per cell) suggests they play a crucial role in the symbiotic interface . This represents an elegant example of how bacterial structures can be repurposed during the evolution of endosymbiosis.

What is known about the expression regulation of fliH and other flagellar genes in Buchnera aphidicola?

The regulation of flagellar gene expression in Buchnera aphidicola differs significantly from free-living bacteria due to genome reduction and adaptation to the endosymbiotic lifestyle. Key aspects include:

• Loss of master regulators: Buchnera has lost many of the regulatory genes that control flagellar expression in free-living bacteria, suggesting constitutive expression of the remaining flagellar genes .

• Nutritional influence: Expression of Buchnera genes, including flagellar genes, may be influenced by the nutritional status of the aphid host. Studies have shown that interactions with other symbionts can affect the expression of Buchnera genes involved in amino acid synthesis .

• Transcriptional coupling: The remaining flagellar genes in Buchnera, including fliH, are likely organized in operons that ensure coordinated expression of the proteins required for HBB assembly.

• Post-transcriptional regulation: Given the limited transcriptional regulatory capacity of Buchnera, post-transcriptional mechanisms may play an important role in controlling protein levels.

Research methodologies to study fliH regulation include RT-qPCR to measure transcript levels under different conditions, RNA-seq to identify co-regulated genes, and proteomics to correlate transcript abundance with protein levels.

How does the flagellar assembly protein FliH interact with other symbiotic systems in the aphid host?

The interaction between the Buchnera flagellar system and other symbiotic systems in the aphid represents an emerging area of research. Evidence suggests several important connections:

• Co-localization with other symbionts: Buchnera coexists with facultative symbionts like Arsenophonus within aphid bacteriocytes, creating opportunities for molecular cross-talk between different bacterial species .

• Metabolic integration: The protein transport function of the flagellar apparatus may complement metabolic pathways shared between Buchnera and other symbionts. For example, Buchnera from some aphid species shares the tryptophan biosynthesis pathway with Serratia symbiotica .

• Influence on gene expression: The presence of facultative symbionts has been shown to affect the relative abundance of Buchnera and the expression of Buchnera genes . This suggests that the regulation of flagellar genes, including fliH, might be influenced by the presence of other symbionts.

• Immune system interactions: The flagellar apparatus proteins may interact with the aphid immune system, potentially helping to maintain the symbiotic relationship by preventing immune responses against beneficial symbionts.

Methodological approaches to study these interactions include fluorescence microscopy to visualize spatial relationships, transcriptomics to identify coordinated gene expression patterns, and metabolomics to trace the flow of nutrients between different components of the symbiotic system.

How does FliH from Buchnera aphidicola subsp. Baizongia pistaciae compare to FliH proteins from other Buchnera strains?

Comparative analysis of FliH proteins across different Buchnera strains reveals important evolutionary patterns related to host specialization. The table below summarizes key comparative features:

Methodologically, such comparisons require careful phylogenetic analysis using maximum likelihood methods, structural modeling to predict functional consequences of sequence differences, and potentially heterologous complementation experiments to test functional equivalence.

What insights can be gained from comparing the flagellar apparatus of Buchnera with those of free-living bacteria?

Comparing the flagellar apparatus of Buchnera with those of free-living bacteria provides valuable insights into both functional adaptation and evolutionary processes:

• Structural simplification: Buchnera has retained only the hook-basal body (HBB) complexes without the external filament or motor proteins present in motile bacteria . This simplification reflects adaptation to a non-motile lifestyle.

• Functional repurposing: While the flagellar apparatus in free-living bacteria primarily serves motility, in Buchnera it appears to function as a protein export system . This demonstrates how complex bacterial structures can be repurposed during adaptation to specialized niches.

• Quantitative differences: Buchnera possesses hundreds of HBB complexes per cell , far more than typical motile bacteria. This abundance suggests a critical role in maintaining the symbiotic relationship.

• Regulatory simplification: Free-living bacteria have complex regulatory networks controlling flagellar gene expression, while Buchnera has lost many of these regulators, likely leading to constitutive expression.

• Evolutionary conservation: Despite extensive genome reduction, Buchnera has retained the genes encoding the export apparatus components, including fliH, suggesting strong selective pressure to maintain this function.

Research methodologies to explore these comparisons include electron microscopy to visualize structural differences, comparative genomics to identify retained and lost components, and heterologous expression experiments to test functional conservation.

How can protein structure prediction tools be applied to understand FliH function in the absence of crystallographic data?

In the absence of crystallographic data for Buchnera FliH, protein structure prediction tools offer valuable approaches for understanding its function:

The methodological workflow should include:

• Multiple sequence alignment of FliH homologs

• Secondary structure prediction

• Tertiary structure modeling using multiple approaches

• Model validation using metrics like QMEAN or MolProbity

• Refinement of models based on known biochemical data

• Functional annotation based on predicted structural features

These computational approaches can generate testable hypotheses about FliH function and guide the design of targeted experimental studies.

How might genetic engineering of recombinant FliH protein be utilized to investigate the protein transport function of Buchnera's flagellar apparatus?

Genetic engineering of recombinant FliH offers sophisticated approaches to investigate the protein transport function of Buchnera's flagellar apparatus:

• Domain swap experiments: Replacing domains of Buchnera FliH with corresponding regions from motile bacteria can identify adaptations specific to its protein transport function. For example, creating chimeric proteins with the C-terminal domain from Buchnera FliH and the N-terminal domain from E. coli FliH.

• Site-directed mutagenesis: Introducing specific mutations at conserved residues can identify amino acids critical for protein-protein interactions within the flagellar export apparatus. Particularly valuable targets include:

  • The FliI binding interface residues

  • Putative substrate recognition sites

  • Oligomerization domains

• Fluorescent protein fusions: Creating FliH fusions with fluorescent proteins can enable real-time tracking of protein localization and assembly in heterologous systems.

• Cargo protein engineering: Designing reporter proteins fused with putative export signals can test the substrate specificity of the Buchnera flagellar export system when co-expressed with recombinant FliH and other export apparatus components.

• Reconstitution experiments: In vitro reconstitution of the minimal flagellar export apparatus using purified components, including recombinant FliH, can directly test transport function and specificity.

These approaches can provide mechanistic insights into how Buchnera utilizes its flagellar apparatus for protein transport in the context of its symbiotic relationship with aphids.

What experimental approaches can determine if the Buchnera flagellar apparatus transports specific proteins between the symbiont and host?

Determining if the Buchnera flagellar apparatus transports specific proteins between symbiont and host requires sophisticated experimental approaches:

• Protein secretion assays: Developing reporter systems where potential secreted proteins are fused to detectable tags (e.g., luciferase or alkaline phosphatase) can identify proteins exported via the flagellar apparatus.

• Proteomics of the bacteriocyte interface: Using spatially resolved proteomics techniques to analyze proteins at the interface between Buchnera cells and the aphid bacteriocyte can identify candidates for flagellar-mediated transport.

• Immunolocalization studies: Generating antibodies against putative transported proteins and using immunogold electron microscopy can visualize their association with the HBB complexes.

• In vitro transport assays: Developing cell-free systems with purified HBB complexes to test the translocation of candidate proteins across membranes.

• Heterologous expression systems: Reconstituting the Buchnera flagellar export apparatus in E. coli and testing its ability to secrete aphid-symbiont interface proteins.

• Crosslinking coupled with mass spectrometry: This approach can capture transient interactions between the flagellar apparatus and proteins being transported.

These methods can be complemented by comparative genomic and transcriptomic analyses to identify proteins uniquely present at the symbiont-host interface that might represent transported substrates.

How can systems biology approaches integrate data on FliH to model the symbiotic interface in the Buchnera-aphid system?

Systems biology approaches offer powerful frameworks for integrating diverse data on FliH and the flagellar apparatus within the broader context of the Buchnera-aphid symbiosis:

The resulting integrated models can generate testable predictions about how perturbations to FliH function might impact the broader symbiotic system, guiding experimental design and interpretation within a holistic framework of symbiont-host interactions.