Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Enoyl-[acyl-carrier-protein] reductase [NADH] FabI (fabI)

What is the genomic context of the fabI gene in Buchnera aphidicola subsp. Baizongia pistaciae?

The fabI gene in Buchnera aphidicola from Baizongia pistaciae is part of the highly reduced genome that characterizes this obligate endosymbiont. Buchnera from B. pistaciae possesses a unique double membrane system, unlike some other Buchnera strains which have three membranes . The genome of Buchnera from B. pistaciae (BBp) contains approximately 618 kb and has a very low GC content (about 25%), with most genes dedicated to essential metabolic functions . The fabI gene has been retained despite extensive genome reduction, indicating its essential role in bacterial survival within the aphid host environment.

How does the FabI protein from Buchnera differ structurally from homologs in free-living bacteria?

The FabI protein from Buchnera aphidicola likely exhibits structural adaptations resulting from the bacterium's long-term symbiotic lifestyle. Due to genomic reduction and the accumulation of mildly deleterious mutations characteristic of Buchnera, the FabI protein may show decreased thermodynamic stability compared to homologs in free-living bacteria . This reduced stability is often compensated by the high expression of chaperones like GroEL, which helps maintain proper protein folding despite potentially destabilizing mutations . Structural analyses would typically reveal a conserved catalytic core maintaining the essential NADH-binding site and enzyme activity while potentially showing more variability in peripheral regions.

What expression systems are most suitable for producing recombinant Buchnera FabI protein?

For optimal recombinant expression of Buchnera aphidicola FabI protein, a codon-optimized synthetic gene approach is recommended due to the low GC content (approximately 25%) of the Buchnera genome . E. coli BL21(DE3) with pET expression systems offers a reliable platform, but expression temperatures should be maintained below 30°C to prevent inclusion body formation. The following protocol typically yields functional protein:

• Clone codon-optimized fabI into pET-28a with N-terminal His-tag

• Transform into E. coli BL21(DE3)

• Grow cultures at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5 mM IPTG

• Shift temperature to 18-25°C for overnight expression

• Purify using nickel affinity chromatography followed by size exclusion

This approach typically yields 2-5 mg of purified protein per liter of culture, with enzyme activity preserved through careful buffer optimization containing 10% glycerol and reducing agents.

What are effective strategies for analyzing Buchnera FabI enzyme kinetics despite its instability?

Analyzing enzyme kinetics of Buchnera FabI requires specialized approaches to address protein instability. A comprehensive methodology includes:

• Stabilization buffer optimization containing:

  • 50 mM HEPES (pH 7.5)

  • 150 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • 0.5 mM EDTA

• Spectrophotometric assay monitoring NADH oxidation at 340 nm using:

  • Crotonyl-CoA substrate (50-500 μM range)

  • NADH (25-250 μM range)

  • Temperature maintained at 25°C

• Data analysis using the Michaelis-Menten equation:
  v₀ = Vₘₐₓ[S]/(Kₘ + [S])

Thermal shift assays to determine stability under various conditions are also valuable, using SYPRO Orange and real-time PCR thermal cyclers to generate melting curves. Researchers should prepare fresh enzyme for each experiment and consider the physiological temperature range of the insect host (typically 15-30°C) when interpreting results .

How can researchers effectively express and purify Buchnera FabI for structural studies?

For structural studies of Buchnera FabI, a multi-step purification strategy is essential:

• Large-scale expression:

  • Use E. coli BL21(DE3) with pET system containing codon-optimized fabI

  • Grow in terrific broth with glucose supplementation

  • Induce at OD₆₀₀ = 0.8 with 0.2 mM IPTG

  • Express at 18°C for 18 hours

• Purification protocol:

  • Lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM PMSF

  • Nickel affinity chromatography with imidazole gradient

  • TEV protease cleavage of His-tag (if crystallography is planned)

  • Ion exchange chromatography (MonoQ)

  • Size exclusion chromatography in final buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT)

• Crystallization screening:

  • Concentrate protein to 10-15 mg/mL

  • Include substrate analogs or NADH for co-crystallization

  • Screen at both 4°C and 18°C

This protocol typically yields >95% pure protein suitable for crystallization trials or cryo-EM studies. Researchers should verify protein activity after each purification step to ensure the retention of native structure .

What methods are most effective for studying the role of FabI in the context of the Buchnera-aphid symbiosis?

To study FabI within the Buchnera-aphid symbiotic context requires an integrated approach:

• Localization studies:

  • Immunogold electron microscopy using anti-FabI antibodies

  • Confocal microscopy with fluorescently labeled antibodies

  • Analysis of bacteriocyte and symbiosomal membranes

• Functional genomics approach:

  • RNA interference (RNAi) targeting host factors that might interact with bacterial FabI

  • Transcriptomic analysis comparing expression levels across different aphid developmental stages

  • Metabolomic profiling to detect fatty acid intermediates

• In silico pathway analysis:

  • Construction of metabolic network models incorporating FabI function

  • Identification of potential metabolic complementation with host pathways

  • Flux balance analysis to predict the impact of FabI inhibition

These methods should be complemented with physiological experiments on aphids, monitoring growth and reproduction when fed with FabI inhibitors through artificial diets . Research has shown that the three-membraned symbiosomal structure in many Buchnera strains plays crucial roles in regulating metabolite exchange, though B. pistaciae has evolved a distinct double-membrane system .

How has the FabI enzyme evolved across different Buchnera strains in various aphid species?

The evolution of FabI across different Buchnera strains reflects the symbiont's adaptation to various aphid hosts. Comparative genomic analysis reveals:

The membrane system differences between strains (particularly BBp's unique double membrane) likely influence the cellular environment in which FabI functions, potentially affecting enzyme kinetics and substrate availability .

What insights can comparative genomics provide about the retention of fabI in Buchnera's reduced genome?

The retention of fabI in Buchnera's highly reduced genome provides critical insights into essential gene functions in obligate endosymbionts:

• The fabI gene has been maintained across diverse Buchnera strains despite significant genome reductions, indicating its essential role in bacterial survival and symbiotic function .

• Comparative genomic analysis reveals that fatty acid biosynthesis genes, including fabI, are among the core genes retained even in the most reduced Buchnera genomes (such as BCc at 416 kb) .

• The selective pressure on fabI appears stronger than on many other metabolic genes, suggesting that its function cannot be complemented by the aphid host's metabolism .

• Analysis of nonsynonymous to synonymous substitution rates (dN/dS) indicates purifying selection on fabI, further emphasizing its functional importance .

These patterns suggest that fatty acid biosynthesis, specifically the reaction catalyzed by FabI, represents a non-replaceable bacterial contribution to the symbiotic relationship, even as other metabolic functions may be lost or complemented by the host or secondary symbionts .

How does the metabolic role of FabI compare between Buchnera aphidicola and free-living bacteria?

The metabolic role of FabI in Buchnera aphidicola differs from free-living bacteria in several key aspects:

• Network context: In free-living bacteria, FabI functions within a complete and redundant metabolic network, whereas in Buchnera, it operates in a highly streamlined network with minimal redundancy . Metabolic network analysis reveals that in Buchnera, fatty acid biosynthesis represents one of the few complete pathways maintained despite extreme genome reduction .

• Regulatory differences: Free-living bacteria typically regulate fabI expression in response to environmental conditions, while Buchnera likely maintains constitutive expression due to the loss of many regulatory elements .

• Substrate limitations: Unlike free-living bacteria that can obtain fatty acid precursors from the environment, Buchnera must synthesize them from a limited set of inputs available from the aphid host . Analysis of transporter repertoires indicates that Buchnera from B. pistaciae has lost many of its outer-membrane integral proteins, affecting how substrates reach FabI .

• Complementarity with host: In Buchnera, FabI's function may be partially integrated with host metabolism, with end products potentially being exported to the aphid, unlike the primarily cell-autonomous role in free-living bacteria .

These differences highlight how endosymbiotic lifestyle has reshaped even conserved metabolic functions like those catalyzed by FabI, optimizing them for the specific constraints of the symbiotic relationship .

What potential inhibitors could target Buchnera FabI without affecting aphid metabolism?

Developing selective inhibitors for Buchnera FabI requires understanding structural and functional differences between bacterial and aphid fatty acid synthesis pathways:

• Natural product derivatives:

  • Triclosan analogs modified to enhance penetration through the symbiosomal membranes

  • Fatty acid thioesters that preferentially target bacterial FabI over eukaryotic homologs

• Structure-based design strategies:

  • Target the NADH-binding pocket unique to bacterial FabI enzymes

  • Focus on inhibitors with high selectivity index (SI > 100) between bacterial and insect enzymes

• Predicted effective compounds:

Effective inhibitors would need to penetrate multiple membrane systems unique to Buchnera in aphid bacteriocytes . The distinct double-membrane system in B. pistaciae Buchnera presents unique challenges and opportunities for inhibitor design compared to the three-membraned systems in other Buchnera strains .

How do mutations in Buchnera fabI affect the aphid host's fitness and development?

Mutations in Buchnera fabI can significantly impact aphid fitness through disruption of essential fatty acid biosynthesis:

• Critical developmental impacts:

  • Reduced nymphal growth rates

  • Delayed time to reproductive maturity

  • Decreased fecundity and offspring viability

  • Altered lipid composition in bacteriocytes

• Physiological mechanisms:

  • Disruption of membrane integrity in both symbiont and host tissues

  • Impaired signaling molecule synthesis dependent on specific fatty acids

  • Altered energy metabolism due to compromised fatty acid availability

• Variable effects across aphid species:

  • Aphids with co-primary symbionts (like C. cedri) may show greater resilience to fabI mutations

  • Specialized aphids with reduced host plant range (like B. pistaciae) demonstrate higher sensitivity to fabI disruption

Research approaches to study these effects include controlled feeding of aphids with sublethal concentrations of FabI inhibitors, followed by comprehensive fitness assessment . The specialized bacteriocyte cells housing Buchnera require intact symbiont metabolism for proper function, making fabI an essential gene for maintaining the symbiotic relationship .

How does temperature stress affect Buchnera FabI function and what are the implications for aphid-symbiont homeostasis?

Temperature stress significantly impacts Buchnera FabI function with cascading effects on symbiotic homeostasis:

• Molecular consequences of temperature stress:

  • Reduced enzymatic efficiency at temperature extremes

  • Protein misfolding and aggregation at elevated temperatures

  • Altered substrate binding kinetics affecting catalytic rates

• Adaptive responses:

  • Increased expression of chaperones (particularly GroEL) to maintain FabI folding under stress

  • Potential induction of alternative metabolic pathways in the aphid host

  • Temporary metabolic depression during extreme temperature events

• Experimental findings on thermal sensitivity:

The temperature sensitivity of Buchnera proteins, including FabI, is particularly relevant given the lack of typical bacterial stress response systems in these reduced genomes . GroEL, which is highly expressed in Buchnera, likely plays a crucial role in maintaining FabI function during temperature fluctuations, preserving the integrity of fatty acid biosynthesis in the symbiont .

What are the challenges in crystallizing Buchnera FabI and how can they be overcome?

Crystallizing Buchnera FabI presents several technical challenges requiring innovative approaches:

• Primary challenges:

  • Inherent protein instability due to accumulation of slightly deleterious mutations

  • Low expression yields in recombinant systems

  • Tendency to form microcrystalline aggregates

• Innovative solutions:

  • Surface entropy reduction (SER) mutagenesis targeting clusters of high-entropy residues

  • Co-crystallization with stabilizing ligands (NADH, substrate analogs, or inhibitors)

  • Fusion protein approaches using crystallization chaperones like T4 lysozyme or BRIL

• Optimized crystallization protocol:

  a. Protein preparation:

  • Extensive buffer screening to identify stabilizing conditions

  • Addition of 10% glycerol and 1 mM TCEP as stabilizing agents

  • Concentration limited to 5-8 mg/mL to prevent aggregation

  b. Crystallization setup:

  • Sitting drop vapor diffusion at 4°C

  • Screening with factorial kits focused on conditions successful for other FabI enzymes

  • Microseeding from initial microcrystals

  • Inclusion of 0.5-1 mM NADH in all conditions

These approaches have successfully resolved technical issues with other challenging bacterial proteins and should be adaptable to Buchnera FabI . The characteristic accumulation of slightly deleterious mutations in endosymbiont proteins requires special attention to protein stability during all crystallization steps .

How can researchers effectively study Buchnera FabI function in vivo given the unculturable nature of the symbiont?

Studying Buchnera FabI function in vivo requires creative approaches to overcome the symbiont's unculturable nature:

• Aphid-based experimental systems:

  • Artificial diet supplementation with FabI inhibitors

  • Microinjection of antisense oligonucleotides targeting fabI mRNA

  • Stable isotope labeling to track fatty acid synthesis and transfer

  • In situ hybridization to monitor fabI expression in different tissues and conditions

• Heterologous expression systems:

  • Complementation studies in E. coli fabI temperature-sensitive mutants

  • Yeast expression systems with inducible promoters

  • Cell-free protein synthesis for functional analysis

• Advanced microscopy approaches:

  • Super-resolution microscopy to localize FabI within bacteriocytes

  • Correlative light and electron microscopy (CLEM) to study enzyme distribution

  • Fluorescence recovery after photobleaching (FRAP) to analyze protein dynamics

The use of bacteriocyte-specific proteomic analysis combined with metabolic labeling can provide insights into FabI function despite the inability to culture Buchnera . The specialized membrane system of Buchnera, particularly the unique double-membrane structure in B. pistaciae, provides an opportunity to study protein function within a naturally minimized system .

What approaches can be used to investigate potential interactions between Buchnera FabI and aphid host proteins?

Investigating interactions between Buchnera FabI and aphid host proteins requires specialized techniques:

• Proteomics-based approaches:

  • Cross-linking mass spectrometry (XL-MS) of isolated bacteriocytes

  • Proximity labeling with BioID or APEX2 fused to FabI expressed in E. coli

  • Co-immunoprecipitation using anti-FabI antibodies followed by mass spectrometry

• Imaging techniques:

  • Fluorescence resonance energy transfer (FRET) with fluorescently tagged proteins

  • Proximity ligation assay (PLA) to detect protein-protein interactions in situ

  • Super-resolution microscopy to visualize potential interaction sites at symbiosomal membranes

• Functional validation methods:

  • Yeast two-hybrid screening with Buchnera FabI against aphid cDNA libraries

  • Bimolecular fluorescence complementation (BiFC) in heterologous systems

  • Surface plasmon resonance (SPR) to measure binding affinities of candidate interactors

The research should focus particularly on potential interactions with symbiosomal membrane proteins that might facilitate metabolite transfer between the symbiont and host . The unique double-membrane system observed in Buchnera from B. pistaciae likely influences these protein-protein interactions differently than the three-membraned systems in other Buchnera strains .

How might synthetic biology approaches be applied to understand and manipulate Buchnera FabI function?

Synthetic biology offers promising approaches to understand and manipulate Buchnera FabI function:

• Minimal system reconstruction:

  • Development of E. coli chassis with Buchnera fabI replacing native fabI

  • Creation of in vitro reconstituted fatty acid synthesis systems

  • Cell-free expression systems optimized for Buchnera proteins

• CRISPR-based technologies:

  • Development of specialized CRISPR systems for targeted genome editing in Buchnera

  • CRISPRi approaches to control fabI expression within the symbiosome

  • CRISPR-based imaging to track FabI localization and dynamics

• Biomimetic systems:

  • Artificial vesicles incorporating purified FabI and related enzymes

  • Microfluidic devices mimicking the bacteriocyte environment

  • 3D-printed microenvironments recreating the symbiotic interface

These approaches could overcome the limitations of studying unculturable endosymbionts like Buchnera . The long-term co-evolution between Buchnera and aphids has created specialized systems that synthetic biology can help dissect and potentially repurpose for biotechnological applications .

What implications does the study of Buchnera FabI have for understanding metabolic complementarity in symbiotic systems?

The study of Buchnera FabI provides key insights into metabolic complementarity in symbiotic systems:

• Theoretical frameworks:

  • Black Queen Hypothesis: Loss of functions that can be complemented by partners

  • Metabolic handoffs: Identification of pathway splitting between symbiont and host

  • Shared selective pressures on complementary enzymes

• Evolutionary implications:

  • Retention of fabI across highly reduced genomes indicates non-replaceable function

  • Comparative analysis with other symbiotic systems reveals convergent patterns

  • Identification of minimal essential gene sets for obligate endosymbionts

• Applications to other systems:

  • Potential insights for human microbiome research

  • Models for designing synthetic microbial communities

  • Understanding metabolic integration in organelle evolution

The fatty acid biosynthesis pathway, including FabI, represents one of the core metabolic functions retained in Buchnera despite extreme genome reduction, indicating its essential role in the symbiotic relationship . Different Buchnera strains show various degrees of genome reduction, with B. pistaciae maintaining a moderate genome size (618 kb) and a unique double-membrane system compared to other strains .

How can understanding Buchnera FabI contribute to new strategies for managing aphid pests in agriculture?

Understanding Buchnera FabI can inform novel approaches to aphid pest management:

• Targeted control strategies:

  • Development of specific FabI inhibitors as symbiont-targeted insecticides

  • RNA interference approaches targeting fabI expression

  • Disruption of symbiont-host metabolic integration

• Resistance management:

  • Identification of potential resistance mechanisms based on fabI mutations

  • Design of inhibitor combinations to address multiple symbiont targets

  • Predictive modeling of evolutionary responses to symbiont-targeting approaches

• Ecological considerations:

  • Specificity to aphid pests without affecting beneficial insects

  • Reduced environmental impact compared to broad-spectrum insecticides

  • Integration with existing integrated pest management strategies

The obligate nature of the Buchnera-aphid symbiosis makes targeting symbiont-specific processes like those catalyzed by FabI particularly attractive for pest management . Different aphid species harbor Buchnera strains with varying genomic features, which necessitates understanding strain-specific characteristics when developing control strategies .