Recombinant Buchnera aphidicola subsp. Acyrthosiphon pisum Thymidylate synthase (thyA)

What is Buchnera aphidicola and why is it important for aphid biology?

Buchnera aphidicola is an obligate bacterial endosymbiont that has coevolved with aphids for over 100 million years, as evidenced by congruent phylogenies of Buchnera and aphids . This intracellular symbiont resides in specialized cells called bacteriocytes within almost all aphid species . Its primary importance lies in synthesizing essential amino acids that are absent or rare in the phloem sap diet of aphids, enabling aphids to thrive on nutritionally imbalanced diets .

Despite having a highly reduced genome (typically 412-646 kb), Buchnera retains genes involved in the biosynthesis of the ten amino acids essential to animals . This genomic specialization demonstrates the evolutionary adaptation to its symbiotic role, making Buchnera critical for aphid survival and reproduction. The symbiotic relationship is transmitted maternally from aphid mother to offspring, ensuring continuity across generations .

How does the Buchnera-aphid symbiotic relationship function at the molecular level?

The Buchnera-aphid symbiosis represents a classic example of obligate mutualism that functions through several key molecular mechanisms:

• Nutritional exchange: Genome analysis reveals extensive metabolite exchange between aphids and Buchnera, including sharing of amino acid biosynthesis pathways . The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera .

• Genomic complementarity: Through reductive evolution, Buchnera has lost many genes but retained those essential for its symbiotic function, particularly those involved in amino acid biosynthesis . Interestingly, some metabolic pathways are distributed between the genomes of the pea aphid and Buchnera, creating genetic interdependence .

• Metabolic integration: Research on dTTP synthesis shows it functions as a key metabolic input in several host insect/obligate symbiont systems . In studies with other symbiont systems, suppressing dTTP production significantly reduced symbiont density and affected essential amino acid synthesis gene expression .

• Specialized cellular environment: Aphids house Buchnera in bacteriocytes, providing a protected intracellular niche that facilitates metabolite exchange . This specialized environment likely influences the expression of genes like thyA.

• Regulatory coordination: Despite having lost many genes involved in transcriptional regulation, Buchnera maintains essential metabolic functions, suggesting that gene expression may be coordinated with host signaling .

How has genome reduction in Buchnera aphidicola affected thyA evolution compared to free-living bacteria?

Genome reduction in Buchnera aphidicola has had several specific effects on thyA evolution:

• Selective retention despite massive gene loss: While Buchnera has lost approximately 80-90% of the genes found in free-living bacterial relatives, the thyA gene has been retained in all sequenced strains . This underscores the essential nature of thyA for DNA synthesis and bacterial survival.

• Altered regulatory context: Buchnera has lost many genes involved in transcriptional regulation , suggesting that thyA expression lacks the sophisticated regulation seen in free-living bacteria. Instead, it likely operates under simplified, possibly constitutive expression patterns.

• Conservation of genomic location: Despite extensive genomic reduction, thyA has maintained a stable chromosomal location in Buchnera, unlike some amino acid biosynthesis genes that have moved between plasmids and chromosomes . This positional conservation suggests the importance of its genomic context.

• Sequence evolution under symbiotic constraints: Compared to free-living bacteria where thyA evolves in response to diverse environmental pressures, in Buchnera it has evolved within the relatively stable environment of aphid bacteriocytes, leading to different selective pressures.

• Integration with host metabolism: The thyA pathway in Buchnera functions within a reduced metabolic network that is integrated with host metabolism . This has likely shaped its evolution to optimize function within this interdependent system.

The reconstruction of the most recent shared Buchnera ancestor revealed a genome containing 616 protein-coding genes, already dramatically reduced compared to free-living relatives . This indicates that major genome reduction, including streamlining of thyA-related pathways, occurred early in Buchnera evolution, prior to the diversification of aphid lineages.

What evidence exists for positive selection in thyA across different Buchnera strains?

Analysis of Buchnera aphidicola genomes provides several lines of evidence regarding selection on the thyA gene:

While direct evidence for positive diversifying selection specifically in thyA is limited in current research, the broader pattern of selective gene retention and the essential nature of thyA suggest it has been subject to strong selective constraints throughout Buchnera evolution.

How does metabolic exchange between aphids and Buchnera aphidicola influence thyA function?

The metabolic exchange between aphids and Buchnera aphidicola creates a complex interdependency that affects thyA function in several ways:

• Nutrient provisioning and precursor availability: Within bacteriocytes, aphids provide Buchnera with nutrients that likely include precursors needed for thyA function, such as folate derivatives and nucleotide precursors essential for thymidylate synthesis.

• dTTP as a key metabolic currency: Research has revealed that deoxythymidine triphosphate (dTTP), produced through the pathway involving thyA, serves as a metabolic input in host insect/obligate symbiont systems . This suggests thyA function is critical not only for the symbiont's DNA synthesis but also as part of the metabolic exchange with the host.

• Impact on symbiont population dynamics: The dTTP synthesis pathway influences symbiont density, as demonstrated in the B. tabaci/Portiera system where suppressing dTTP production significantly reduced symbiont density . By extension, thyA function in Buchnera likely affects its population within aphid bacteriocytes.

• Influence on amino acid synthesis: Research showed that suppressing dTTP production significantly repressed the expression of horizontally transferred essential amino acid synthesis-related genes in B. tabaci . This suggests interconnections between nucleotide metabolism (including thyA function) and amino acid synthesis, the primary symbiotic function of Buchnera.

• Metabolic pathway distribution: Genomic analyses reveal that some metabolic pathways are distributed between aphid and Buchnera genomes . This suggests that thyA may function within an integrated metabolic network spanning both organisms, with precursors or products being exchanged.

The inventory of metabolic genes in the pea aphid genome confirms extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis pathways . This integration likely shapes the expression and evolutionary trajectory of the thyA gene in ways distinct from free-living bacteria.

What does comparative genomics reveal about thyA conservation across different Buchnera strains?

Comparative genomics analyses of Buchnera strains provide several key insights about thyA conservation:

• Universal retention across lineages: The thyA gene has been retained in all 39 sequenced Buchnera strains examined, including those from diverse aphid hosts . This universal conservation underscores the essential nature of thymidylate synthase for the endosymbiont's survival.

• High sequence conservation: As a key enzyme in DNA synthesis, thyA shows significant sequence conservation across Buchnera strains, particularly in catalytic domains . This conservation is especially remarkable given the accelerated sequence evolution often observed in endosymbiont genomes.

• Ancestral presence confirmed: Reconstruction of the genome of the most recent shared Buchnera ancestor revealed that thyA was present in the ancestral Buchnera (which contained 616 protein-coding genes) before aphid diversification . This indicates thyA has been conserved throughout the entire evolutionary history of the Buchnera-aphid symbiosis.

• Synteny conservation: Buchnera genomes exhibit extraordinary stability in gene order, with almost perfect synteny conservation for over 100 million years . This genomic stability extends to the context of thyA, which has maintained a consistent position within the chromosome across different strains.

• Selective pressures: While many Buchnera genes show evidence of genetic drift due to population bottlenecks during vertical transmission, essential genes like thyA are under strong purifying selection to maintain function .

The high degree of conservation of thyA across Buchnera lineages stands in contrast to the differential loss of many other genes, highlighting the non-random nature of gene loss during endosymbiont genome reduction. Among the genes tested across Buchnera strains, thyA belongs to the core set that has been conserved despite extensive genome reduction in some lineages.

How do horizontal gene transfers in aphids relate to thyA function in Buchnera?

The relationship between horizontal gene transfers (HGTs) in aphids and thyA function in Buchnera reveals interesting aspects of this co-evolved symbiotic system:

• Complementary metabolic functions: While thyA in Buchnera is essential for DNA synthesis, other metabolic pathways in aphids have been acquired through horizontal gene transfer from microorganisms . For example, pea aphids have acquired genes for carotenoid biosynthesis through HGT . These horizontally transferred genes often complement Buchnera's metabolic capabilities.

• Maintenance of thyA in symbiont genome: Unlike some metabolic functions transferred to the aphid genome, thyA function has been retained in Buchnera rather than transferred to the host . The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria, and Buchnera's reduced gene count does not reflect gene transfer to the host genome .

• Regulatory relationships: Research on B. tabaci demonstrated that suppressing dTTP production (involving thyA) significantly reduced symbiont density and repressed expression of horizontally transferred essential amino acid synthesis genes . This suggests a regulatory relationship between thyA function in the symbiont and expression of horizontally transferred genes in the host.

• Differential evolutionary patterns: While horizontally transferred genes in aphids have undergone duplications and functional diversification (as seen with carotenoid synthesis genes in pea aphids ), thyA in Buchnera has remained highly conserved . This reflects distinct selective pressures on symbiont and host genes.

• Integrated metabolic networks: Rather than replacing symbiont functions through HGT, aphids have often acquired complementary functions, creating an integrated metabolic network. For instance, carotenoid biosynthesis in aphids relies on geranylgeranyl diphosphate synthase (GGPPS) and horizontally acquired carotenoid synthesis genes , while Buchnera maintains essential pathways including thyA-dependent DNA synthesis.

This relationship illustrates how host-symbiont systems can evolve complementary metabolic capabilities through different mechanisms (vertical inheritance, reductive evolution, and horizontal gene transfer) to create an integrated functional unit.

What are the optimal approaches for expressing and purifying recombinant Buchnera aphidicola thyA?

Based on available research, the following methodology is recommended for expressing and purifying recombinant Buchnera aphidicola thyA:

• Expression system selection: Yeast has been successfully used as an expression system for Buchnera aphidicola thyA . This eukaryotic system can provide appropriate post-translational modifications and yield properly folded protein. For bacterial expression, E. coli systems with optimization for low-GC content genes may be appropriate.

• Protein tagging strategy: For purification purposes, adding a purification tag is recommended. The optimal tag type should be determined based on the protein's characteristics . Common options include:

  • His-tag for metal affinity chromatography

  • GST-tag for glutathione affinity purification

  • MBP-tag for improved solubility and amylose affinity purification

• Purification protocol:

  • Initial capture using affinity chromatography based on the selected tag

  • Secondary purification using size exclusion or ion exchange chromatography

  • Aim for >85% purity as assessed by SDS-PAGE

• Storage conditions:

  • For liquid form: -20°C/-80°C with shelf life of approximately 6 months

  • For lyophilized form: -20°C/-80°C with shelf life of approximately 12 months

  • Avoid repeated freezing and thawing cycles

• Reconstitution protocol:

  • Briefly centrifuge vial prior to opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Aliquot to avoid freeze-thaw cycles

• Quality control assessment:

  • SDS-PAGE for purity verification

  • Spectrophotometric methods for concentration determination

  • Enzymatic activity assay measuring conversion of dUMP to dTMP

  • Structural characterization (circular dichroism, thermal shift assays)

The recombinant protein's high confidence structural model (pLDDT global: 97.63) suggests it should form a stable, properly folded structure when expressed under appropriate conditions.

What experimental protocols can researchers use to study thyA function in the Buchnera-aphid system?

Researchers can employ several complementary approaches to study thyA function in the Buchnera-aphid symbiosis:

• RNA interference (RNAi) approaches:

  • Dietary RNAi to suppress genes in the dTTP synthesis pathway

  • This approach reveals effects on symbiont density, gene expression, and host reproduction

  • Protocol: Design dsRNA targeting thyA or related genes, deliver through artificial diet or plant-mediated feeding

• Plant-mediated feeding experiments:

  • For delivering chemicals affecting thyA function or dTTP synthesis

  • Protocol: Place a razor-cut stem of a leaf in a microfuge tube containing the test chemical, allowing it to migrate to leaves through the plant vascular system and be ingested by aphids

  • This technique enables testing of thyA inhibitors or substrate analogs

• Visualization and microscopy techniques:

  • Immunocytochemistry to visualize effects on Buchnera distribution

  • Protocol: Dissect adult aphids, remove germarium/ovariole structures, fix in glutaraldehyde, wash with PBS, incubate with appropriate antibodies, and visualize using fluorescence microscopy

• Molecular quantification methods:

  • qPCR to quantify Buchnera density and gene expression levels

  • Digital droplet PCR for absolute quantification

  • RNA-Seq to assess transcriptome-wide responses to thyA manipulation

• Controlled environment studies:

  • Atmospheric simulators (like Atmosim 2100) to create defined conditions

  • Protocol: Maintain aphids in chambers with controlled CO₂ levels (e.g., 400 ppm vs. 900 ppm) to study environmental effects on thyA function and symbiont dynamics

• Reproductive and developmental assessment:

  • Monitor effects on aphid reproduction, embryo development, and progeny survival

  • Protocol: Place adult aphids on treated plants for defined periods, then transfer to fresh plants and assess reproductive parameters over time

• Proteomics approaches:

  • iTRAQ-based comparative proteomics to assess protein expression changes

  • Protocol: Extract proteins, digest using FASP procedure, label with iTRAQ reagents, analyze via LC-MS/MS

  • These approaches provide insights into thyA abundance and related protein networks

The integration of these complementary approaches allows researchers to understand thyA function at multiple levels, from molecular mechanisms to physiological outcomes.

How can proteomics approaches be applied to study thyA in the Buchnera-aphid system?

Proteomics offers powerful tools for studying thyA in the Buchnera-aphid system, with several specific applications:

• iTRAQ-based comparative proteomics workflow:

  • Sample preparation: For each sample, solubilize 100 μg of proteins in reducing reagent (37°C, 60 min)

  • Protein processing: Add cysteine-blocking reagent (room temperature, 30 min), wash with dissolution buffer

  • Digestion: Process with trypsin (protein/trypsin ratio of 50:1) at 37°C overnight

  • Separation: Using liquid chromatography with the following gradient conditions:

    • 6–9% buffer B (0–8 min)

    • 9–14% buffer B (8–24 min)

    • 14–30% buffer B (24–60 min)

    • 30–40% buffer B (60–75 min)

    • 40–95% buffer B (75–78 min)

    • 95% buffer B (78–85 min)

  • Mass spectrometry analysis: Using search parameters including precursor ion mass tolerance ±15ppm, MS/MS tolerance ±20 mmu

• Protein identification parameters:

  • Search against Buchnera aphidicola proteome databases

  • Peptide score threshold ≥ 10

  • False discovery rate (FDR) < 0.01

  • Require at least one unique peptide for protein identification

• Quantitative applications:

  • Measure thyA abundance changes in response to environmental stresses

  • Compare thyA levels across different aphid developmental stages

  • Assess changes following experimental manipulations (e.g., RNAi targeting related pathways)

• Functional proteomics approaches:

  • Protein-protein interaction studies via co-immunoprecipitation and mass spectrometry

  • Post-translational modification analysis to identify regulatory modifications

  • Subcellular fractionation to confirm thyA localization within Buchnera cells

• Structural proteomics:

  • Limited proteolysis combined with mass spectrometry to probe protein structure

  • Hydrogen-deuterium exchange mass spectrometry to examine structural dynamics

  • Cross-linking mass spectrometry to map interaction interfaces

These proteomic approaches provide valuable tools for understanding thyA at the protein level, complementing genomic and functional studies to build a comprehensive picture of its role in the Buchnera-aphid symbiosis.