Recombinant Buchnera aphidicola subsp. Acyrthosiphon pisum Flagellar biosynthetic protein FliQ (fliQ)

Basic Research Questions

• What is Buchnera aphidicola FliQ protein and what is its fundamental role in flagellar biosynthesis?

  FliQ is a flagellar biosynthetic protein found in Buchnera aphidicola (strain APS), an endosymbiotic bacterium of the pea aphid (Acyrthosiphon pisum). This small membrane protein (89 amino acids) is a component of the flagellar type III export apparatus, which is essential for the assembly of flagellar structures. The amino acid sequence is: MTSEYVMELFYNAMKVALIIASPLLLAALISGLIISILQAATQVNEQTLSFIPKIISVLGVISILGPWMLGVMLDYMHNLFNNIILIK .

  Despite Buchnera being nonmotile, it maintains 26 flagellar genes arranged in five operons, clustered in three regions of its genome . FliQ is part of a conserved set of proteins that form the flagellar basal body and export apparatus, suggesting its evolutionary repurposing beyond motility function.

• Why does Buchnera aphidicola maintain and express flagellar genes despite being nonmotile?

  Buchnera aphidicola has undergone extensive genome reduction (only 600 kbps) during its evolution as an obligate endosymbiont, retaining only genes relevant to its symbiotic lifestyle . The retention of flagellar genes despite the lack of motility suggests these structures have been repurposed for functions essential to the aphid-Buchnera symbiosis.

  Research indicates that these flagellar structures likely function as a type III secretion system (T3SS), potentially serving to:

  • Export proteins to signal to the aphid host

  • Act as surface recognition molecules during vertical transmission to new aphid embryos

  • Facilitate the transport of nutrients across membranes

  Notably, transcriptome analyses of pea aphid lines with different Buchnera titers reveal differential expression of flagellar genes. In aphid lines with low Buchnera populations, the endosymbionts show elevated expression of flagellar secretion genes (fliP, fliQ, and fliR), while those in aphid lines with high Buchnera numbers show increased expression of structural proteins .

• What is the relationship between FliQ and other flagellar proteins in Buchnera aphidicola?

  FliQ functions as part of an integrated flagellar protein complex. Research has shown that Buchnera's flagellar proteins maintain high levels of sequence homology with those in well-studied systems like Salmonella, particularly among the core components of the type III secretion system.

  Protein	Function	Sequence Homology to Salmonella
  FlgG	Rod protein	75%
  FliJ	Export apparatus component	20.8%
  FliQ	Export apparatus component	Moderate (~40%)
  FlhA	Export apparatus component	~40%
  FlhB	Export apparatus component	~40%
  FliI	Export apparatus ATPase	~40%
  FliP	Export apparatus component	~40%
  FliR	Export apparatus component	~40%

  Mass spectrometry analysis of isolated Buchnera basal bodies indicates that FliQ functions alongside other flagellar proteins like FliF, FlgI, FlgE, FlhA, and FlgF, which are significantly enriched during isolation procedures .

Experimental Design and Methodology

• What are the optimal conditions for soluble expression of recombinant FliQ in E. coli systems?

  Based on experimental design approaches for similar recombinant proteins, the following protocol is recommended for optimal soluble expression of FliQ:

  1. Expression system: Use E. coli as the host organism with a suitable expression vector containing the fliQ gene from Buchnera aphidicola .

  2. Culture conditions: For optimal expression, grow E. coli until an OD600 of 0.8 in a medium containing:

     • 5 g/L yeast extract

     • 5 g/L tryptone

     • 10 g/L NaCl

     • 1 g/L glucose

     • 30 μg/mL of appropriate antibiotic (e.g., kanamycin)

  3. Induction parameters:

     • IPTG concentration: 0.1 mM

     • Temperature: 25°C (critical for proper folding)

     • Induction time: 4 hours

  4. Protein extraction and purification:

     • Use a Tris-based buffer with 50% glycerol for stability

     • Store at -20°C for short-term use or -80°C for extended storage

     • Avoid repeated freeze-thaw cycles

  This methodology has been shown to yield high levels (up to 250 mg/L) of soluble recombinant protein with proper folding and functionality .

• How can researchers effectively isolate and characterize flagellar basal body complexes containing FliQ from Buchnera?

  A successful protocol for isolating flagellar basal body complexes from Buchnera has been developed and validated through mass spectrometry. The procedure involves:

  1. Preparation of Buchnera cells:

     • Surgically isolate bacteriocytes from aphids

     • Homogenize to release Buchnera cells

     • Purify cells through differential centrifugation

  2. Basal body isolation:

     • Lyse cells and adjust pH to 10

     • Perform sequential centrifugation: first at 5000g (multiple times)

     • Final centrifugation at 30,000g

     • Incubate overnight in TET buffer

  3. Verification methods:

     • SDS-PAGE analysis should reveal approximately 16 bands corresponding to flagellar proteins

     • Protein identification through mass spectrometry

     • Electron microscopy to visualize hook-basal body structures

  This procedure has been proven to successfully enrich flagellar proteins, with FliQ alongside structural proteins like FliF, FlgI, FlgE, FlhA, and FlgF showing 3-fold or greater enrichment compared to whole-cell samples .

• What molecular techniques can be employed to study FliQ expression and its regulation in Buchnera aphidicola?

  Despite the challenges of working with an unculturable endosymbiont, several methodological approaches can be employed:

  1. Transcriptional analysis:

     • RT-PCR using primers specific for the fliQ gene (design primers based on conserved regions)

     • RNA-Seq to identify transcriptional patterns and potential regulatory elements

     • Single-cell approaches to detect stochastic expression patterns

  2. Protein expression analysis:

     • Proteomic analysis through peptide mass fingerprinting

     • Western blotting using antibodies against recombinant FliQ

     • Two-dimensional PAGE to identify post-translational modifications

  3. Regulatory studies:

     • Investigation of transcriptional pulsing patterns seen in flagellar gene expression

     • Analysis of coordinated expression within specific classes of flagellar genes

     • Examination of potential post-translational regulatory mechanisms

  Recent research on flagellar biosynthesis in Escherichia coli has revealed that flagellar promoters are stochastically activated in pulses, with coordinated patterns within specific classes of promoters. Similar mechanisms might exist in Buchnera and could be investigated using single-cell approaches and statistical analysis of expression patterns .

Advanced Research Questions

• How might FliQ and other flagellar proteins function in the type III secretion system of Buchnera aphidicola?

  The flagellar apparatus in Buchnera is hypothesized to function as a type III secretion system (T3SS), despite the absence of known pathogenicity proteins or secreted effectors in the Buchnera genome.

  Methodological approach to investigate this function:

  1. Structural analysis:

     • Cryo-electron microscopy of isolated basal bodies

     • Comparison with known T3SS structures from other bacteria

     • Identification of potential adaptations specific to symbiotic function

  2. Functional characterization:

     • Identify potential substrates for the T3SS through bioinformatic prediction

     • Develop in vitro secretion assays using isolated complexes

     • Use heterologous expression systems to test export capabilities

  3. Host interaction studies:

     • Investigate differential expression of fliQ and other flagellar genes in different aphid lines

     • Examine potential correlations between flagellar gene expression and symbiotic efficiency

     • Analyze host cell responses to isolated basal body complexes

  The conserved nature of FliQ and other core T3SS components (~40% sequence homology with Salmonella) suggests functional preservation of secretion capability, while adaptation for symbiosis-specific roles .

• What evolutionary processes have shaped the retention and function of FliQ in Buchnera aphidicola?

  Investigating the evolutionary history of FliQ requires a comparative genomic approach:

  1. Sequence analysis:

     • Compare FliQ sequences across different Buchnera lineages

     • Analyze selection pressures using dN/dS ratios

     • Identify conserved domains essential for function

  2. Comparative genomics:

     • Examine flagellar gene retention patterns across different Buchnera strains

     • Compare with free-living relatives and other endosymbionts

     • Map gene losses and retentions on phylogenetic trees

  3. Functional prediction:

     • Correlate sequence conservation with predicted functional domains

     • Model structural changes compared to ancestral proteins

     • Identify potential neofunctionalization signatures

  Buchnera lineages vary in their set of retained flagellar genes, but all have lost genes encoding flagellin and motor proteins, indicating a functional shift away from motility. This pattern suggests strong selective pressure to maintain the export apparatus (including FliQ) while eliminating components solely required for motility .

• How do environmental factors influence the expression and function of FliQ in the aphid-Buchnera symbiosis?

  The expression of flagellar genes in Buchnera appears to respond to ecological conditions:

  1. Host physiological influences:

     • Investigate how aphid nutritional status affects fliQ expression

     • Examine developmental stage-specific patterns

     • Study the impact of host stress on flagellar gene expression

  2. Population dynamics:

     • Compare fliQ expression between high and low Buchnera titer aphid lines

     • Examine density-dependent regulatory mechanisms

     • Investigate potential quorum sensing-like systems

  3. Experimental manipulation:

     • Develop aphid feeding assays to manipulate nutrient availability

     • Use RNA interference in aphids to alter host signaling

     • Apply external stressors to study symbiotic resilience

  Research has shown that in aphid lines with low Buchnera populations, flagellar secretion genes (fliP, fliQ, and fliR) show elevated expression, suggesting these components may be particularly important under conditions where symbiont efficiency is critical .

Data Analysis and Interpretation

• How should researchers interpret mass spectrometry data when analyzing flagellar protein complexes containing FliQ?

  Mass spectrometry analysis of Buchnera flagellar complexes requires specialized approaches:

  1. Data processing methodology:

     • Use spectral counting to assess relative protein abundance

     • Compare enrichment patterns before and after isolation procedures

     • Normalize data against whole proteome references

  2. Enrichment analysis:

     • Calculate fold-enrichment for each flagellar protein

     • Assess stoichiometric relationships between components

     • Identify potentially associated non-flagellar proteins

  3. Interpretation framework:

     Protein	Role in Complex	Expected Enrichment	Interpretation of Low Signal
     FliQ	Export apparatus	2-4 fold	Potential isolation issue or low abundance
     FliF	MS ring	>3 fold	Central structural component
     FlgI	P ring	>3 fold	Outer membrane component
     FlhA	Export apparatus	>3 fold	Central to type III secretion
     FlgK	Hook-filament junction	Variable	Peripheral location may affect isolation

  4. Validation approaches:

     • Western blot confirmation of key proteins

     • Electron microscopy correlation

     • Functional assays of isolated complexes

  Based on published data, core structural proteins and export apparatus components (including FliQ) should show significant enrichment (3-fold or greater) in properly isolated basal body preparations, while peripheral components may show variable results .

• What bioinformatic approaches are most effective for analyzing FliQ sequence conservation and predicting functional domains?

  Multiple computational approaches can yield insights into FliQ structure and function:

  1. Sequence analysis pipeline:

     • Multiple sequence alignment of FliQ homologs from diverse bacteria

     • Phylogenetic analysis to map evolutionary relationships

     • Identification of conserved residues across bacterial species

  2. Structural prediction methods:

     • Use of algorithms like AlphaFold2 for 3D structure prediction

     • Transmembrane domain prediction (FliQ is a membrane protein)

     • Molecular dynamics simulations to study conformational states

  3. Functional domain analysis:

     • Conservation mapping to predicted structures

     • Protein-protein interaction interface prediction

     • Identification of potentially repurposed functional regions

  4. Integrative approaches:

     • Correlation of sequence conservation with expression data

     • Assessment of co-evolution patterns with interacting partners

     • Comparative analysis across different symbiotic systems

  For membrane proteins like FliQ, special attention should be paid to hydrophobic domains and potential interaction sites with other flagellar export apparatus components (FlhA, FlhB, FliP, and FliR) .

• How can researchers design experiments to test hypotheses about FliQ's role in symbiotic interactions?

  Testing functional hypotheses about FliQ requires creative experimental approaches given the challenges of working with an uncultivable endosymbiont:

  1. Heterologous expression systems:

     • Express Buchnera FliQ in model bacteria with manipulable flagellar systems

     • Create chimeric proteins to test domain-specific functions

     • Use bacterial two-hybrid systems to identify interaction partners

  2. Ex vivo approaches:

     • Develop cell-free assays for testing secretion capabilities

     • Reconstitute minimal export complexes in liposomes

     • Use fluorescently labeled proteins to track potential substrates

  3. In vivo correlation studies:

     • Measure FliQ expression levels across different aphid developmental stages

     • Correlate expression patterns with symbiotic efficiency markers

     • Use microscopy to study FliQ localization during vertical transmission

  4. Statistical design considerations:

     • Apply factorial design approaches to test multiple variables

     • Use appropriate controls for environmental factors

     • Develop clear metrics for symbiotic function assessment

  These methodological approaches provide a framework for hypothesis testing despite the experimental limitations of the Buchnera-aphid system, allowing researchers to gain insights into the functional role of FliQ in this important symbiotic relationship.