Recombinant Blochmannia pennsylvanicus ATP synthase subunit c (atpE)

What is Blochmannia pennsylvanicus ATP synthase subunit c (atpE)?

Blochmannia pennsylvanicus ATP synthase subunit c (atpE) is a membrane protein component of the F-type ATP synthase complex in the obligate endosymbiotic bacterium Blochmannia pennsylvanicus. This protein is also known as ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, and lipid-binding protein. The protein is encoded by the atpE gene (locus BPEN_003) and consists of 79 amino acids with the following sequence: MEHLNFDMLYIAAAIMMGLAAIGAAIGIGILGSKFLEGAARQPDLIPILRTQFFIVMGLVDAIPMITVGLGLYVMFSAV . The recombinant version is produced through heterologous expression systems to enable structural and functional studies of this protein.

What is the biological context of Blochmannia pennsylvanicus?

Blochmannia pennsylvanicus is an obligate endosymbiont that lives within specialized cells of Camponotus pennsylvanicus ants (carpenter ants). The bacterium has a 792-kb genome that has undergone significant reduction, a common characteristic of obligate endosymbionts . Blochmannia provides important nutritional functions to its ant host, including biosynthesis of all essential amino acids except arginine . The symbiotic relationship between Blochmannia and Camponotus ants is thought to have evolved over millions of years, with the divergence between B. pennsylvanicus and B. floridanus estimated at approximately 16-20 million years ago .

What role does ATP synthase play in Blochmannia physiology?

ATP synthase is a critical enzyme for energy production in all living cells. In Blochmannia pennsylvanicus, the ATP synthase complex, including the c subunit, is involved in generating ATP through the process of oxidative phosphorylation. Given the nutritional support role of Blochmannia in ant hosts, ATP production is likely essential for powering biosynthetic pathways involved in producing amino acids and other nutrients for the host. The maintenance of ATP synthase genes despite significant genome reduction highlights the critical importance of energy metabolism in sustaining the endosymbiotic relationship.

What are optimal expression systems for producing recombinant Blochmannia pennsylvanicus ATP synthase subunit c?

Recombinant expression of Blochmannia pennsylvanicus ATP synthase subunit c can be achieved using Escherichia coli expression systems. Based on research with similar ATP synthase c subunits, successful protocols involve:

• Cloning the atpE gene into a suitable expression vector with an inducible promoter

• Transforming the construct into E. coli expression strains

• Inducing expression and extracting the membrane-embedded protein using appropriate detergents

• Purifying the protein through column chromatography methods

What is the predicted structure and stoichiometry of the c-ring in Blochmannia pennsylvanicus ATP synthase?

While the specific structure of Blochmannia pennsylvanicus ATP synthase c-ring has not been directly determined in the available literature, insights can be drawn from related bacterial systems. Research on sodium F-ATP synthases from I. tartaricus and P. modestum revealed "membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11" .

Atomic force microscopy (AFM) studies of reconstituted c-rings showed that:

• c-ring assemblies had identical diameters

• Most rings represented completely assembled undecameric complexes (c11)

• Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed

Based on the conserved nature of ATP synthase structure across bacterial species, it is reasonable to hypothesize that Blochmannia pennsylvanicus ATP synthase may form similar c11 ring structures, though experimental confirmation would be required.

What methodological approaches are recommended for studying c-ring assembly and structure?

For researchers investigating the structure and assembly of recombinant Blochmannia pennsylvanicus ATP synthase subunit c, the following methodological approaches are recommended:

• SDS-PAGE analysis: To assess the distribution between monomeric c subunits and assembled c-rings. Research on similar systems showed that "subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers" .

• Atomic Force Microscopy (AFM): For high-resolution structural analysis of c-rings. AFM "topographs of c rings reconstituted into lipid bilayers" can reveal ring diameter, subunit stoichiometry, and structural variations .

• Membrane reconstitution: Properly reconstituting c-rings into lipid bilayers is essential for structural studies and can be achieved through established protocols for membrane protein reconstitution.

• Mass spectrometry: For precise molecular weight determination of assembled c-rings and verification of protein modifications.

• Cross-linking studies: To stabilize c-ring assemblies and analyze subunit interactions.

How does genome reduction in Blochmannia affect ATP synthase genes compared to other cellular functions?

Comparative genomic analyses between Blochmannia species provide insights into differential gene loss patterns while maintaining essential functions:

Despite significant genome reduction to approximately 792 kb, Blochmannia pennsylvanicus retains genes encoding ATP synthase components, suggesting strong selective pressure to maintain energy production capabilities . This contrasts with differential loss in other functional categories, reflecting the critical nature of ATP synthesis even in highly reduced endosymbiont genomes.

What experimental approaches can determine the functional impact of ATP synthase variations in Blochmannia?

Investigating the functional implications of ATP synthase variations in Blochmannia presents unique challenges due to its obligate endosymbiotic lifestyle. Researchers can employ these experimental approaches:

• Site-directed mutagenesis: Introducing specific mutations in the recombinant atpE gene to assess their impact on c-ring assembly and ATP synthase function.

• In vitro reconstitution assays: Reconstituting the ATP synthase complex with native or modified c subunits to measure ATP synthesis/hydrolysis activities.

• Comparative biochemical analysis: Comparing the properties of ATP synthase components from different Blochmannia species to correlate sequence variations with functional differences.

• Structural biology approaches: Using X-ray crystallography or cryo-electron microscopy to determine the atomic structure of the c-ring and identify species-specific features.

• Computational modeling: Employing molecular dynamics simulations to predict how sequence variations might affect c-ring structure and function.

How does the Blochmannia ATP synthase complex contribute to the endosymbiotic relationship with Camponotus ants?

The ATP synthase complex in Blochmannia likely plays a critical role in the endosymbiotic relationship by:

• Generating ATP to power biosynthetic pathways for essential amino acids that benefit the ant host. Genomic analysis reveals that "Blochmannia retains several key nutritional functions that may benefit the ant host, including biosynthesis of all essential amino acids except arginine" .

• Supporting nitrogen recycling pathways. In Blochmannia, "urease converts urea to carbon dioxide and ammonia, the latter of which is then assimilated into glutamine by glutamine synthetase" . This process requires energy in the form of ATP.

• Maintaining cellular viability of the endosymbiont through energy production, which is essential for the continuity of the symbiotic relationship.

• Potentially adapting to the specific energetic requirements of the intracellular environment of host cells.

Research suggests that the endosymbiotic relationship between Blochmannia and Camponotus ants has been maintained for millions of years, with each partner adapting to optimize the symbiosis. ATP synthase functionality would be essential for sustaining the metabolic contributions of Blochmannia to its host.

What protein purification strategies are most effective for recombinant Blochmannia pennsylvanicus ATP synthase subunit c?

Based on studies with similar membrane proteins, the following purification strategy is recommended:

• Membrane fraction isolation: After expression in E. coli, harvest cells and disrupt by sonication or French press to isolate membrane fractions by differential centrifugation.

• Detergent solubilization: Carefully select detergents for solubilization—mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin are often effective for maintaining native structure of membrane proteins.

• Affinity chromatography: If the recombinant protein includes a tag (His-tag, FLAG-tag, etc.), use appropriate affinity chromatography methods for initial purification.

• Size exclusion chromatography: For separating properly assembled c-rings from monomeric subunits, as "SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers" .

• Buffer optimization: Use buffers containing glycerol for stability, similar to the "Tris-based buffer, 50% glycerol, optimized for this protein" described in product information .

• Storage considerations: Following purification, store at -20°C or -80°C for extended storage, with the caution that "repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week" .

What analytical methods can confirm the functional integrity of purified recombinant ATP synthase subunit c?

To verify that purified recombinant Blochmannia pennsylvanicus ATP synthase subunit c retains its structural and functional integrity, researchers should employ multiple complementary analytical approaches:

• Circular dichroism (CD) spectroscopy: To assess secondary structure content and confirm proper folding of the protein.

• Fluorescence spectroscopy: To monitor tertiary structure and ligand binding properties.

• Native gel electrophoresis: To analyze the oligomeric state and assembly of c-rings under non-denaturing conditions.

• ATP synthesis/hydrolysis assays: When reconstituted with other ATP synthase subunits, functional assays can determine if the recombinant c subunit supports catalytic activity.

• Liposome reconstitution: Reconstitution into lipid vesicles allows assessment of membrane integration and proton/ion translocation capabilities.

• Binding assays: To test interaction with inhibitors and other ATP synthase subunits as a measure of structural integrity.

How can researchers investigate the evolutionary adaptations of ATP synthase in obligate endosymbionts?

Investigating evolutionary adaptations of ATP synthase in obligate endosymbionts like Blochmannia requires a multifaceted approach:

• Comparative genomics: Analyze ATP synthase genes across diverse bacterial lineages, paying special attention to:

  • Sequence conservation/divergence patterns

  • Selection pressures (dN/dS ratios)

  • Gene order and synteny

• Structural comparisons: Model the structure of Blochmannia ATP synthase components based on known structures and identify endosymbiont-specific adaptations.

• Biochemical characterization: Compare biochemical properties (pH optima, ion specificity, catalytic efficiency) of ATP synthases from free-living relatives and endosymbionts.

• Molecular clock analyses: Estimate evolutionary rates of ATP synthase components in Blochmannia compared to other bacteria. Research has shown "10- to 50-fold faster amino acid substitution rates in Blochmannia" compared to free-living bacteria .

• Experimental evolution: While challenging with obligate endosymbionts, laboratory evolution experiments with more tractable systems can provide insights into adaptation patterns.

What are the major challenges in expressing and purifying membrane proteins from endosymbionts?

Researchers face several specific challenges when working with membrane proteins like ATP synthase subunit c from obligate endosymbionts:

• Codon usage bias: Endosymbiont genes often have different codon usage patterns than expression hosts like E. coli, potentially leading to translation inefficiencies. Solution: Codon optimization of the synthetic gene for the expression host.

• Toxicity to expression host: Overexpression of membrane proteins can be toxic to host cells. Solution: Use tightly regulated expression systems and consider lower induction levels.

• Improper membrane insertion: Membrane proteins may not properly insert into host membranes. Solution: Co-expression with chaperones or membrane-insertion facilitating proteins.

• Protein aggregation: Membrane proteins tend to aggregate when overexpressed. Solution: Expression at lower temperatures (16-25°C) and use of solubility-enhancing fusion tags.

• Detergent selection: Finding the optimal detergent for extraction while maintaining native structure is challenging. Solution: Screen multiple detergents and detergent combinations.

• Low yields: Membrane protein expression often results in low yields. Solution: Scale up culture volumes and optimize expression conditions through factorial experimental designs.

How can researchers overcome the limitations of working with obligate endosymbionts that cannot be cultured independently?

Working with proteins from unculturable organisms like Blochmannia presents unique challenges that can be addressed through these approaches:

• Heterologous expression systems: Utilize recombinant expression in tractable hosts like E. coli, as demonstrated in studies where "subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene" .

• Synthetic biology approaches: Synthesize genes based on genomic sequence data rather than requiring direct isolation from the endosymbiont.

• Co-culture systems: Develop co-culture systems that mimic the natural host environment to maintain endosymbionts in laboratory settings.

• Host tissue extraction: For certain studies, extract proteins directly from host tissues containing the endosymbiont, followed by careful separation methods.

• Comparative studies with closely related culturable species: Gain insights by studying homologous proteins from culturable relatives and extrapolating findings.

• Computational predictions: Employ bioinformatic approaches to predict protein properties, interactions, and functions based on sequence data.

What techniques are recommended for functional characterization of ATP synthase c subunit in lipid environments?

For functional characterization of the ATP synthase c subunit in lipid environments that mimic its native membrane context:

• Proteoliposome reconstitution: Incorporate purified c subunits or c-rings into liposomes of defined lipid composition to study membrane integration and function.

• Solid-state NMR spectroscopy: Analyze protein structure and dynamics within lipid bilayers at atomic resolution.

• Planar lipid bilayer electrophysiology: Measure ion conductance through reconstituted c-rings under various conditions.

• Fluorescence-based proton flux assays: Monitor proton/ion translocation using pH-sensitive fluorescent dyes in proteoliposomes.

• Atomic Force Microscopy: Visualize c-ring assembly and organization in supported lipid bilayers, following methods where "AFM topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters" .

• Native mass spectrometry: Analyze intact membrane protein complexes with associated lipids to understand lipid-protein interactions.

What emerging technologies could advance our understanding of ATP synthase function in endosymbionts?

Several cutting-edge technologies hold promise for deeper insights into ATP synthase structure and function in endosymbiotic bacteria:

• Cryo-electron microscopy (cryo-EM): Advances in cryo-EM now enable near-atomic resolution structures of membrane protein complexes without crystallization, ideal for visualizing the complete ATP synthase complex.

• Single-molecule techniques: Methods like single-molecule FRET can reveal conformational dynamics of ATP synthase components during catalysis.

• CRISPR-based approaches: Development of genetic tools for previously unculturable organisms may eventually allow direct genetic manipulation of endosymbionts.

• In situ structural biology: Techniques like cryo-electron tomography can visualize ATP synthase structure directly within host cells.

• Artificial cell systems: Synthetic biology approaches to create minimal cells could serve as platforms to study endosymbiont proteins in controlled environments.

• Advanced computational methods: Integration of molecular dynamics simulations with machine learning approaches to predict functional properties from sequence data.

How might understanding ATP synthase from obligate endosymbionts contribute to our knowledge of prokaryotic-eukaryotic co-evolution?

Research on ATP synthase from obligate endosymbionts like Blochmannia can provide unique insights into co-evolutionary processes:

• Molecular adaptation mechanisms: Reveals how essential cellular machinery adapts to the intracellular environment while maintaining core functionality.

• Rates and patterns of protein evolution: Studies showing "10- to 50-fold faster amino acid substitution rates in Blochmannia" help understand evolutionary rate variation in different symbiotic contexts.

• Genome streamlining processes: Illuminates how organisms prioritize gene retention during extreme genome reduction, as evidenced by the conservation of ATP synthase genes despite significant gene loss.

• Host-symbiont metabolic integration: Clarifies how energy production in endosymbionts becomes integrated with host metabolism over evolutionary time.

• Transition from free-living to obligate lifestyles: Provides insights into the molecular changes accompanying the evolution of obligate endosymbiosis.

• Parallel evolution in diverse symbiotic systems: Comparison of ATP synthase adaptations across different endosymbiont lineages can reveal convergent evolutionary patterns.

Understanding these processes has broader implications for evolutionary biology, including insights into the ancient endosymbiotic events that gave rise to mitochondria and chloroplasts.