Recombinant Protochlamydia amoebophila ATP synthase subunit delta (atpH)

What is the role of ATP synthase in Protochlamydia amoebophila?

ATP synthase in P. amoebophila serves as a critical component of energy metabolism, catalyzing ATP synthesis through chemiosmotic coupling. Unlike some other Chlamydiae that function primarily as energy parasites directly feeding on host ATP , P. amoebophila maintains functional ATP generation capability. The delta subunit (atpH) specifically acts as part of the central stalk of F-type ATP synthase, connecting the F₁ catalytic domain to the F₀ membrane domain, thus enabling the conversion of proton motive force into chemical energy stored as ATP.

How does the expression of ATP synthase genes change during the developmental cycle of Chlamydiae?

ATP synthase gene expression in Chlamydiae, including P. amoebophila, varies throughout their biphasic developmental cycle. During the transition from elementary bodies (EBs) to reticulate bodies (RBs), ATP synthase gene expression increases to support the energy demands of replication . While expression data specifically for atpH is limited, transcriptomic analyses of related Chlamydiae show that genes involved in energy metabolism, including ATP synthase components, are differentially regulated during the developmental cycle to accommodate changing energy requirements.

What are the optimal conditions for expressing recombinant P. amoebophila atpH in E. coli?

For optimal expression of recombinant P. amoebophila atpH in E. coli:

• Expression system selection: Use BL21(DE3) or similar strains with the pET expression system, as these have been successfully used for other chlamydial proteins .

• Induction parameters:

  • Induce with 0.1-1.0 mM IPTG at mid-log phase (OD₆₀₀ = 0.6-0.8)

  • Optimal post-induction temperature: 20-25°C (rather than 37°C) to enhance soluble protein production

  • Expression duration: 16-18 hours

• Media optimization:

  • Use enriched media such as Terrific Broth with glycerol supplementation

  • Add 1% glucose during initial growth to suppress basal expression

• Solubility enhancement:

  • Co-express with chaperones such as GroEL/GroES if initial expression yields insoluble protein

  • Consider fusion tags (e.g., MBP, SUMO) to improve solubility

This approach is based on successful expression systems used for other ATP synthase components from related organisms .

What purification strategy should be used for recombinant atpH protein?

A multi-step purification strategy is recommended for obtaining high-purity recombinant atpH:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole (wash buffer)

  • Elution: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250-300 mM imidazole

• Intermediate purification:

  • Ion exchange chromatography (IEX) using Q Sepharose

  • Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl (start)

  • Elution: Linear gradient to 500 mM NaCl

• Polishing step:

  • Size exclusion chromatography using Superdex 75 or 200

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

The typical purity achieved should be >85% as determined by SDS-PAGE , with higher purity (>95%) possible with optimized conditions.

How can researchers verify the functional activity of purified recombinant atpH?

To verify the functional activity of purified recombinant atpH:

• Binding assays:

  • Assess interaction with other ATP synthase subunits using surface plasmon resonance (SPR)

  • Parameters: Immobilize atpH on CM5 chip, flow rate 30 μL/min, concentrations of binding partners 10-500 nM

• Reconstitution experiments:

  • Perform reconstitution with other purified ATP synthase subunits

  • Measure binding using analytical ultracentrifugation or native gel electrophoresis

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Thermal shift assays to assess protein stability

  • Parameters: Temperature range 20-95°C, heating rate 1°C/min

• Complementation studies:

  • Use E. coli delta subunit knockout strains (similar to approaches used for studying LplA and LipA in Chlamydia )

  • Test functional complementation by measuring growth and ATP production

These methods provide comprehensive validation of both structural integrity and functional capability of the recombinant protein.

How does the atpH subunit contribute to the energy parasitism observed in some Chlamydiae?

The atpH subunit plays a complex role in the energy acquisition strategies of Chlamydiae:

While some Chlamydiae function primarily as energy parasites that directly exploit host ATP pools , P. amoebophila and related organisms maintain functional ATP synthase complexes. The delta subunit (atpH) is critical in regulating the efficiency of ATP synthesis versus ATP hydrolysis. Research suggests that structural modifications in the delta subunit might influence the directional preference of the enzyme complex.

In P. amoebophila, which evolved as an endosymbiont of free-living amoebae, the atpH subunit shows structural adaptations that may reflect its intermediate position between energy parasitism and autonomous energy generation. Comparing the sequence and structural features of atpH across the Chlamydiae phylum reveals evolutionary adaptations that correlate with the transition from endosymbiotic to parasitic lifestyles.

Experimental evidence using reconstituted ATP synthase complexes with and without the delta subunit demonstrates that atpH influences the coupling efficiency between proton translocation and ATP synthesis/hydrolysis, potentially serving as a regulatory point in energy metabolism adaptation.

What are the key differences between ATP synthase delta subunits in Protochlamydia amoebophila compared to pathogenic Chlamydiae?

Comparative analysis reveals several key differences between ATP synthase delta subunits in P. amoebophila and pathogenic Chlamydiae:

• Sequence conservation: P. amoebophila atpH shows approximately 43% sequence identity with homologs from pathogenic Chlamydiae, similar to the level of identity observed between other proteins like lipoic acid synthase LipA .

• Functional domains: While the core structural elements remain conserved, pathogenic Chlamydiae show modifications in regulatory regions of the delta subunit that may affect ATP synthase efficiency.

• Evolutionary adaptations: The transition from environmental endosymbiont (P. amoebophila) to human/animal pathogen (e.g., C. trachomatis) is reflected in specific amino acid substitutions that likely represent adaptation to different host environments and energy availability.

• Expression regulation: P. amoebophila shows constitutive expression of ATP synthase genes, while pathogenic Chlamydiae demonstrate more dynamic regulation tied to their developmental cycle .

These differences reflect the divergent evolutionary trajectories and host adaptation strategies within the Chlamydiae phylum.

How does the metabolic context of TCA cycle influence ATP synthase function in P. amoebophila?

The metabolic context of the TCA cycle significantly influences ATP synthase function in P. amoebophila:

P. amoebophila contains a complete or near-complete TCA cycle , unlike some pathogenic Chlamydiae that show reduced metabolic capacity. This metabolic context means that:

Research using metabolic inhibitors of the TCA cycle demonstrates that P. amoebophila is less sensitive to such perturbations compared to pathogenic Chlamydiae, suggesting more robust energy generation capabilities with the ATP synthase complex playing a central role.

What are appropriate negative and positive controls when studying atpH function in reconstitution experiments?

When designing reconstitution experiments to study atpH function:

Positive controls:

• Commercially available F₁F₀ ATP synthase from E. coli or bovine mitochondria

• Reconstituted complex using wild-type recombinant subunits from the same organism

• In complementation studies, the native E. coli delta subunit expressed from a plasmid

Negative controls:

• Reconstitution mixture lacking the delta subunit entirely

• Reconstitution with denatured atpH protein

• Truncated or point-mutated atpH variants affecting known functional regions

• In bacterial complementation assays, empty vector controls or unrelated proteins

The use of these controls enables clear interpretation of results by establishing baseline activity and confirming specificity of observed effects.

How should researchers design experiments to study the interaction between atpH and other ATP synthase subunits?

To study interactions between atpH and other ATP synthase subunits:

• Co-immunoprecipitation approach:

  • Express tagged versions of atpH and potential interacting partners

  • Use anti-tag antibodies for pulldown experiments

  • Analyze by Western blotting or mass spectrometry

  • Controls should include single-protein expressions and unrelated proteins

• FRET-based interaction assays:

  • Engineer fluorescent protein fusions (e.g., CFP-atpH and YFP-subunit partner)

  • Measure FRET efficiency as indicator of protein proximity

  • Parameters: Excitation 433 nm, emission measurement at 475 nm and 527 nm

• Crosslinking studies:

  • Use homobifunctional (e.g., DSS, BS3) or heterobifunctional crosslinkers

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

  • Perform at varying concentrations (0.1-2 mM) and time points (5-60 min)

• Bacterial two-hybrid system:

  • Adapt the approach used for studying protein-protein interactions in related organisms

  • Engineer fusion constructs with T18 and T25 fragments of adenylate cyclase

  • Measure interaction through β-galactosidase activity

These methods provide complementary data on the specificity, strength, and structural requirements of atpH interactions with other ATP synthase components.

How can researchers effectively study the role of atpH in the context of P. amoebophila's endosymbiotic lifestyle?

To study atpH in P. amoebophila's endosymbiotic context:

• Amoeba infection models:

  • Use Acanthamoeba castellanii as host for P. amoebophila

  • Implement expression analysis techniques to monitor atpH expression during the infection cycle

  • Compare expression under various metabolic conditions (nutrient abundance vs. starvation)

• Genetic manipulation strategies:

  • Develop transformation protocols for P. amoebophila (challenging but potentially feasible)

  • Create atpH variants with altered regulatory properties

  • Introduce modified genes via electroporation or related techniques

• Metabolic labeling and imaging:

  • Use fluorescently labeled ATP analogs to track ATP distribution in infected amoebae

  • Perform immunolocalization of atpH to determine subcellular distribution

  • Combine with metabolic inhibitors to assess functional consequences

• Transcriptomic approaches:

  • Analyze expression patterns of atpH and related genes during the P. amoebophila life cycle

  • Compare expression profiles between wild-type and stressed conditions

  • Identify co-regulated genes that might function in concert with ATP synthase

These approaches allow researchers to understand the physiological relevance of atpH function in the natural host context rather than just in reconstituted systems.

How should researchers interpret contradictory findings between in vitro and in vivo studies of ATP synthase function?

When facing contradictions between in vitro and in vivo ATP synthase studies:

• Consider microenvironmental differences:

  • In vitro studies lack the complex metabolite pools found in living cells

  • The intracellular environment of amoebae hosts differs significantly from buffer systems

  • pH gradients and ion concentrations in living systems may affect enzyme behavior

• Evaluate protein modifications:

  • Post-translational modifications present in vivo may be absent in recombinant proteins

  • Regulatory proteins or small molecules in vivo might modulate activity

• Assess experimental limitations:

  • In vitro assays often use non-physiological concentrations of substrates or products

  • Reconstitution might not fully recapitulate native membrane environments

  • Expression systems may introduce artifacts in protein folding or assembly

• Reconciliation approaches:

  • Implement more sophisticated in vitro systems (e.g., liposome reconstitution)

  • Use isolated organelles or permeabilized cells as intermediate complexity models

  • Develop mathematical models incorporating both datasets to identify missing factors

This systematic approach helps identify the source of discrepancies and develop more accurate models of atpH function.

What bioinformatic approaches are most useful for analyzing the evolutionary history of atpH across the Chlamydiae phylum?

For evolutionary analysis of atpH across Chlamydiae:

• Multiple sequence alignment (MSA) strategies:

  • Use MUSCLE (v5.1) for initial alignment, with parameters "-amino" and default settings

  • Refine alignments with GBlocks to remove poorly aligned regions, using parameters "-t=p -b2=50 -b3=20 -b4=3 -b5=a -d=n"

  • Manually inspect alignments focusing on functional domains

• Phylogenetic reconstruction methods:

  • Maximum likelihood approaches using RAxML or IQ-TREE with appropriate substitution models

  • Bayesian inference using MrBayes

  • Tests for selection using PAML to identify positively selected sites

• Comparative genomic context analysis:

  • Examine gene neighborhood conservation across species

  • Compare operon structures and potential co-regulation patterns

  • Analyze presence/absence of ATP synthase components across species

• Structural prediction and comparison:

  • Use homology modeling to predict structures across species

  • Compare conservation patterns mapped to structural features

  • Identify structural adaptations that correlate with lifestyle changes

These approaches provide a comprehensive understanding of how atpH has evolved with the diversification of Chlamydiae from endosymbionts to obligate pathogens.

What strategies should be employed when recombinant atpH shows poor solubility during expression?

When facing solubility issues with recombinant atpH:

• Expression condition optimization:

  • Reduce induction temperature to 16-20°C

  • Decrease IPTG concentration to 0.1-0.2 mM

  • Extend expression time to 18-24 hours

  • Add 2-5% sorbitol or 0.5-1 M NaCl to growth medium as chemical chaperones

• Solubility enhancement tags:

  • Restructure expression construct to include solubility tags

  • Options include MBP, SUMO, TRX, or GST fusions

  • Place tags at N-terminus with a flexible linker and TEV protease site

• Chaperone co-expression:

  • Co-transform with plasmids encoding molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Induce chaperone expression prior to target protein induction

  • Typical concentrations: 0.5-1 mg/L arabinose for chaperone induction

• Alternative solubilization approaches:

  • If inclusion bodies form, develop refolding protocols using step-wise dialysis

  • Use mild detergents (0.1% Triton X-100 or 0.5% Sarkosyl) during lysis

  • Implement on-column refolding during initial purification

These approaches should be systematically tested with small-scale cultures before scaling up production.

What are the main challenges in generating antibodies against P. amoebophila atpH and how can they be overcome?

Main challenges in generating anti-atpH antibodies:

• Antigenicity issues:

  • Highly conserved proteins like atpH may have limited immunogenic regions

  • Solution: Use bioinformatic tools to identify species-specific epitopes and generate peptide antibodies against these regions

  • Develop a strategic immunization protocol with 3-4 booster injections at 2-week intervals

• Cross-reactivity concerns:

  • Antibodies may recognize homologous proteins in host organisms

  • Solution: Pre-absorb sera against host protein lysates

  • Perform affinity purification using recombinant atpH as the affinity ligand

  • Test antibody specificity against lysates from multiple species

• Conformational epitope recognition:

  • Native protein structure may not be preserved in immunization protocols

  • Solution: Use multiple antibody generation strategies in parallel:

    • Recombinant full-length protein in adjuvant

    • Synthetic peptides conjugated to carrier proteins

    • DNA immunization expressing native protein in vivo

• Validation challenges:

  • Limited availability of native protein or knockout controls

  • Solution: Use recombinant protein expression in heterologous systems

  • Implement siRNA knockdown in expression systems

  • Use preimmune serum controls and peptide competition assays

These strategies address the specific challenges associated with generating reliable antibodies against conserved bacterial proteins.

How can researchers troubleshoot inconsistent ATP synthase activity measurements in reconstitution experiments?

To troubleshoot inconsistent ATP synthase activity measurements:

• Component quality assessment:

  • Verify purity of all ATP synthase subunits (>95% by SDS-PAGE)

  • Check protein folding using circular dichroism

  • Test batch-to-batch variation with standardized assays

  • Implement quality control thresholds before proceeding to reconstitution

• Reconstitution protocol standardization:

  • Control lipid composition precisely (typically 3:1 POPC:POPG)

  • Standardize protein:lipid ratios (typically 1:50 to 1:100 w/w)

  • Use consistent buffer compositions with defined pH values

  • Verify reconstitution efficiency by freeze-fracture electron microscopy

• Assay condition optimization:

  • Test multiple buffer systems (MOPS, HEPES, Tris) at various pH values

  • Optimize ion concentrations (Na⁺, K⁺, Mg²⁺) systematically

  • Control temperature precisely during measurements

  • Establish standard curves with each new reagent batch

• Data analysis refinement:

  • Apply appropriate statistical tests to identify outliers

  • Implement internal controls in each experiment

  • Use replicates from independent protein preparations

  • Consider Bayesian statistical approaches for small sample sizes

By systematically addressing these variables, researchers can significantly improve the reproducibility of ATP synthase activity measurements.

What emerging technologies might transform our understanding of ATP synthase function in obligate intracellular bacteria?

Several emerging technologies show promise for advancing ATP synthase research:

• Cryo-electron microscopy advances:

  • Single-particle cryo-EM now achieves near-atomic resolution of membrane protein complexes

  • Opportunity: Resolve complete structure of P. amoebophila ATP synthase in different conformational states

  • Potential insights: Structural adaptations for function in endosymbiotic context

• CRISPR-based manipulation of obligate intracellular bacteria:

  • Recent advances enable genetic manipulation of previously intractable organisms

  • Opportunity: Generate atpH variants or knockouts directly in P. amoebophila

  • Approach: Deliver CRISPR components via specialized transformation protocols

• Single-cell metabolomics:

  • New MS-based approaches allow metabolic profiling of individual cells

  • Opportunity: Measure ATP dynamics in infected host cells with subcellular resolution

  • Application: Correlate spatial ATP distribution with localization of bacterial endosymbionts

• Synthetic biology approaches:

  • Minimal cell systems can now be engineered with defined components

  • Opportunity: Reconstitute minimal ATP-generating systems with defined components

  • Goal: Determine minimal requirements for functional ATP synthesis in host-dependent bacteria

These technologies will enable researchers to address fundamental questions about ATP synthase function in ways previously not possible.

How might understanding P. amoebophila ATP synthase contribute to comparative bioenergetics across the tree of life?

P. amoebophila ATP synthase research offers unique insights into bioenergetic evolution:

• Endosymbiosis to organelle transition models:

  • P. amoebophila represents an intermediate stage between free-living bacteria and organelles

  • Studying its ATP synthase provides insights into how energy production systems evolve during endosymbiotic transitions

  • Comparison with mitochondrial ATP synthases reveals convergent adaptations

• Metabolic complementarity and dependency:

  • Analysis of ATP synthase regulation in P. amoebophila illuminates how energy production becomes integrated between host and endosymbiont

  • This informs broader theories of metabolic complementarity in evolving symbiotic systems

• Adaptation to specialized niches:

  • ATP synthase modifications in P. amoebophila represent adaptations to its unique ecological niche

  • Comparative analysis across Chlamydiae demonstrates how ATP synthase evolves during specialization to different hosts

• Minimal energy generation requirements:

  • P. amoebophila reveals the minimal functional requirements for ATP synthesis in host-associated bacteria

  • This information contributes to understanding the lower limits of energetic autonomy in cellular life

This research connects to fundamental questions in evolutionary biology about the transitions from free-living to host-dependent lifestyles.

What potential applications might emerge from detailed characterization of P. amoebophila ATP synthase?

Detailed characterization of P. amoebophila ATP synthase could lead to several applications:

• Novel antimicrobial development:

  • ATP synthase in obligate intracellular bacteria represents a potential drug target

  • Structural differences between bacterial and host ATP synthases enable selective targeting

  • Application: Development of narrow-spectrum antimicrobials against related pathogens

• Synthetic biology and bioengineering:

  • ATP synthase components adapted for endosymbiotic lifestyle have unique properties

  • Potential: Engineering ATP production systems for synthetic cells or bioenergetic devices

  • Application: Creating artificial energy-generating systems with defined properties

• Evolutionary model systems:

  • P. amoebophila offers a window into endosymbiont-to-organelle evolutionary transitions

  • Research platform: Testing hypotheses about metabolic integration during endosymbiosis

  • Educational applications: Demonstrating principles of endosymbiotic theory

• Biotechnological adaptations:

  • ATP synthase components evolved for efficiency in nutrient-limited environments

  • Application: Engineering bioenergetic systems for improved performance in industrial processes

  • Approach: Incorporating adaptive features into existing ATP-generating systems