Recombinant Pseudomonas syringae pv. phaseolicola ATP synthase subunit c (atpE)

What are the structural components of the ATP synthase complex containing subunit c?

The ATP synthase complex consists of two main structural domains:

The FO domain contains residues between positions 5-25 and 57-77, forming a homomeric c-ring of 10-14 subunits that serves as the central rotor element . This c-ring rotates relative to the a-subunit during proton translocation, which drives the conformational changes in the F1 domain required for ATP synthesis . The α and β subunits in F1 form a hexameric structure (α₃β₃) where the catalytic sites for ATP synthesis are located in the β subunits, while the α subunits play structural and regulatory roles .

How does temperature affect Pseudomonas syringae pv. phaseolicola virulence and metabolism?

Temperature significantly impacts P. syringae pv. phaseolicola virulence through thermoregulation of key metabolic processes:

• Phaseolotoxin production is optimally produced at approximately 18°C and is blocked above 28°C .

• The expression of argK (encoding phaseolotoxin-resistant ornithine carbamoyltransferase) is coordinately regulated with phaseolotoxin synthesis at 18°C .

• Gene clusters involved in toxin production, such as the Pbo cluster, show temperature-dependent expression patterns. Three polycistronic units within this cluster are transcribed at high levels at 18°C but not at 28°C .

This temperature-dependent regulation appears to be an adaptation mechanism that allows P. syringae pv. phaseolicola to optimize its virulence factors according to environmental conditions, which may contribute to its success as a plant pathogen .

What are the recommended protocols for recombinant expression of P. syringae pv. phaseolicola atpE?

The recombinant expression of P. syringae pv. phaseolicola atpE can be achieved through the following methodological approach:

• Gene Cloning and Vector Construction:

  • Amplify the atpE gene (PSPPH_5212) using PCR with specific primers designed from the known sequence.

  • Clone the amplified fragment into an expression vector such as pET series for E. coli expression systems.

  • For improved solubility, fusion tags like maltose-binding protein (MBP) can be utilized, similar to approaches used for other ATP synthase c subunits .

• Expression Conditions:

  • Transform the recombinant plasmid into an appropriate E. coli strain (BL21(DE3) or its derivatives).

  • Culture cells at 37°C until OD600 reaches 0.6-0.8, then induce protein expression with IPTG (0.1-1.0 mM).

  • Lower the temperature to 16-25°C during induction to enhance proper folding of membrane proteins .

• Protein Purification:

  • Use detergent-based extraction (typically with mild detergents like n-dodecyl β-D-maltoside) to solubilize the membrane-embedded protein.

  • Employ affinity chromatography based on the fusion tag, followed by size exclusion chromatography.

  • Verify protein purity via SDS-PAGE and identity via western blotting or mass spectrometry .

Research has shown that recombinant membrane proteins like atpE often benefit from lower expression temperatures and the use of specialized E. coli strains designed for membrane protein expression, such as C41(DE3) or C43(DE3) .

How can researchers verify the functional activity of recombinant atpE protein?

Verification of recombinant atpE functionality requires multiple complementary approaches:

• Structural Analysis:

  • Circular dichroism (CD) spectroscopy to confirm the alpha-helical secondary structure characteristic of native atpE .

  • Thermal stability analysis to assess proper folding and stability of the recombinant protein.

• Functional Assays:

  • Reconstitution of the recombinant atpE into proteoliposomes or nanodiscs to recreate a membrane environment.

  • Proton translocation assays using pH-sensitive fluorescent dyes to monitor proton movement.

  • ATP synthesis activity measurement when reconstituted with other ATP synthase subunits.

• Interaction Studies:

  • In vitro assembly assays to verify the ability of recombinant atpE to form oligomeric c-rings.

  • Protein-protein interaction studies with other ATP synthase components, particularly the a-subunit.

Functional activity can be quantified by comparing ATP synthesis rates or proton translocation efficiency between proteoliposomes containing recombinant atpE versus those with native protein complexes .

How does the cooperation among c-subunits influence ATP synthase activity in P. syringae?

The cooperation among c-subunits is critical for ATP synthase function through a coordinated proton translocation mechanism:

These findings suggest that ATP synthase activity in P. syringae likely depends on similar cooperative interactions within its c-ring, which would impact energy production efficiency and potentially bacterial fitness in different environments.

What is the relationship between ATP synthase function and virulence in P. syringae pv. phaseolicola?

The connection between ATP synthase function and virulence in P. syringae pv. phaseolicola involves several interconnected metabolic and regulatory pathways:

• Energy Production for Virulence Factor Synthesis:
  ATP synthase provides the energy required for various virulence-associated processes, including:

  • Phaseolotoxin synthesis, which requires significant metabolic resources

  • Type III secretion system (T3SS) assembly and operation

  • Production of effector proteins that suppress host immune responses

• Metabolic Adaptation to Host Environment:

  • ATP synthase activity may be modulated in response to changing host conditions

  • The proton gradient utilized by ATP synthase can be affected by plant defense responses that alter pH

  • Efficient energy production is critical during the transition from epiphytic to endophytic lifestyles

• Regulatory Connections:
  While no direct regulatory connection between atpE and virulence has been established, transcriptomic analyses of P. syringae pv. phaseolicola reveal coordination between metabolic and virulence networks:

  Condition	ATP Synthase Expression	Virulence Gene Expression	Metabolic State
  18°C (optimal for toxin)	Moderate-high	High (toxin genes)	Shifted toward toxin production
  28°C (plant-favorable)	High	Low (toxin genes)	Primary metabolism focused
  Nutrient limitation	Variable (stress-dependent)	Selective induction	Adapted to resource availability

• Genome-wide Regulatory Networks:
  Recent research has identified complex transcriptional regulatory networks in P. syringae, with 170 of 301 annotated transcription factors characterized through ChIP-seq analysis. These networks include:

  • 54 top-level transcription factors

  • 62 middle-level transcription factors

  • 147 bottom-level transcription factors

  This hierarchical structure suggests coordination between metabolic processes (like ATP synthesis) and virulence mechanisms .

Understanding this relationship could potentially lead to new strategies for controlling bacterial plant diseases by targeting the metabolic foundations of virulence.

How can genetic engineering of atpE be used to study P. syringae pathogenicity mechanisms?

Genetic engineering of atpE offers several sophisticated approaches to investigate P. syringae pathogenicity mechanisms:

• Site-directed Mutagenesis for Structure-Function Analysis:

  • Generate point mutations in conserved residues (e.g., the glutamic acid involved in proton translocation)

  • Create chimeric c-subunits by exchanging domains between different bacterial species

  • Assess the impact on ATP synthesis efficiency, proton translocation, and bacterial fitness

• Reporter Fusions for In Planta Expression Analysis:

  • Create atpE-reporter gene fusions (e.g., with fluorescent proteins or luciferase)

  • Monitor expression patterns during different phases of infection

  • Determine how host defenses impact energy metabolism gene expression

• Conditional Expression Systems:

  • Develop temperature-sensitive or inducible atpE variants

  • Create partial loss-of-function mutants to study threshold effects

  • Investigate how different levels of ATP synthase activity affect virulence factor production

• Integration with Other Virulence Systems:
  Experiments can be designed to investigate the relationship between energy metabolism and specific virulence mechanisms:

  Experimental Approach	Research Question	Expected Outcome
  atpE mutation + phaseolotoxin assays	How does altered ATP synthesis affect toxin production?	Quantitative relationship between energy availability and toxin synthesis
  Dual reporter system (atpE + virulence genes)	Is expression coordinated during infection?	Temporal and spatial correlation patterns
  Complementation with heterologous atpE	Are species-specific adaptations present?	Fitness differences when native atpE is replaced
  CRISPR interference targeting atpE	What are the effects of rapid atpE downregulation?	Immediate metabolic and virulence consequences

• Systems Biology Integration:
  Recent findings on the genome-wide transcriptional regulatory network of P. syringae reveal complex interactions among transcription factors that control both metabolism and virulence. By mapping atpE within this network, researchers can:

  • Identify master regulators that coordinate energy production and virulence

  • Discover novel regulatory connections between metabolic and pathogenicity pathways

  • Develop predictive models of how environmental conditions affect the pathogen's ability to cause disease

These approaches can help elucidate the fundamental connections between energy metabolism and pathogenicity, potentially revealing new targets for disease control strategies.

What are the key differences between atpE from P. syringae pv. phaseolicola and homologous proteins from other organisms?

Comparative analysis of atpE from P. syringae pv. phaseolicola with homologous proteins reveals several key differences:

• Sequence Conservation and Divergence:

  Region	Conservation Level	Functional Significance
  Transmembrane helices	High (>70%)	Critical for c-ring structural integrity
  Proton-binding site (including conserved glutamic acid)	Very high (>90%)	Essential for proton translocation
  N-terminal region	Low-moderate (~40-60%)	Species-specific adaptation to membrane composition
  Loop regions	Low (~30-40%)	Potential interface with other ATP synthase components

• Oligomeric Structure Variations:

  • P. syringae likely forms a c₁₀ ring structure based on sequence similarity to other bacterial ATP synthases

  • This differs from some other organisms: c₈ in bovine mitochondria, c₁₀ in yeast mitochondria, c₁₁ in some cyanobacteria, c₁₃-₁₅ in chloroplasts

  • These stoichiometric differences affect the H⁺/ATP ratio and energy conversion efficiency

• Adaptation to Environmental Conditions:

  • P. syringae atpE shows adaptations consistent with function in variable environmental temperatures (18-28°C)

  • The amino acid composition suggests optimization for activity in the slightly acidic environment typically found in plant apoplast

  • Specific residues may be adapted for optimal function at the lower pH that develops during plant infection

• Evolutionary Conservation:
  Phylogenetic analysis indicates that while the core proton-binding and transmembrane domains are highly conserved across bacterial species, P. syringae atpE contains unique features that may reflect adaptation to its plant-associated lifestyle. These adaptations could contribute to its ability to thrive in the plant environment and potentially influence virulence capabilities .

What methodologies are most effective for studying c-subunit oligomerization and assembly?

Investigation of c-subunit oligomerization and assembly requires specialized techniques due to the hydrophobic nature and structural complexity of these membrane proteins:

• Biochemical Approaches:

  • Blue Native PAGE: Preserves native protein complexes and allows visualization of intact c-rings

  • Chemical Cross-linking: Captures transient interactions during assembly

  • Size-exclusion Chromatography: Separates monomeric c-subunits from assembled rings

  • Analytical Ultracentrifugation: Provides precise determination of molecular mass and oligomeric state

• Structural Biology Techniques:

  • Cryo-electron Microscopy (cryo-EM): Achieves near-atomic resolution of intact ATP synthase complexes

  • X-ray Crystallography: For high-resolution structures of isolated c-rings

  • Solid-state NMR: Particularly useful for membrane proteins in native-like environments

  • Atomic Force Microscopy: Visualizes topography of membrane-embedded c-rings

• Advanced Genetic Approaches:

  • Genetically Fused c-rings: Creation of single-chain c-rings with defined stoichiometry

  • Cysteine Scanning Mutagenesis: Introduces specific residues for cross-linking studies

  • Fluorescence-based Oligomerization Assays: Employs split fluorescent proteins to monitor assembly

• Computational Methods:

  • Molecular Dynamics Simulations: Models assembly processes and c-ring stability

  • Coarse-grained Simulations: Efficiently models larger-scale assembly events

  • Evolutionary Coupling Analysis: Identifies co-evolving residues that maintain structural integrity

The most effective methodology combines multiple techniques. For example, researchers studying bacterial ATP synthase c-rings successfully employed genetically fused single-chain c-rings with strategic mutations followed by functional assays and molecular dynamics simulations to understand cooperativity . This multi-faceted approach revealed that:

• The waiting time for proton uptake can be shared between multiple c-subunits

• Mutations affect activity differently depending on their positioning within the c-ring

• Cooperative effects extend to c-subunits up to three positions apart

These findings provided insights into the fundamental mechanisms of ATP synthase function that would not have been possible using a single methodological approach.

How might P. syringae atpE be targeted for the development of novel antimicrobial strategies?

ATP synthase subunit c represents a promising target for novel antimicrobial strategies against P. syringae due to its essential role in energy metabolism and unique structural features:

• Structure-based Inhibitor Design:
  Recent research has identified compounds that bind to AtpE with minimum binding energies in the range of -8.69 to -8.44 kcal/mol, lower than the free binding energy of ATP itself . A structure-based approach would involve:

  • Virtual screening of compound libraries against homology models of P. syringae atpE

  • Molecular docking analyses to identify high-affinity binders

  • Rational design of molecules that interfere with c-ring rotation or assembly

  • Focus on compounds that exploit structural differences between bacterial and plant ATP synthases

• Bioactive Peptides as c-ring Disruptors:

  • Design of peptides that mimic interfaces between c-subunits

  • Development of peptides that bind to the proton-binding site

  • Creation of stapled peptides for improved stability and membrane penetration

  • Plant-expressible antimicrobial peptides targeting atpE for enhanced resistance

• Transcriptional and Post-transcriptional Regulation:

  • RNAi-based approaches targeting atpE mRNA

  • CRISPR interference systems to downregulate atpE expression

  • Antisense oligonucleotides designed for bacterial uptake

  • Manipulation of natural regulatory mechanisms controlling atpE expression

• Combination Strategies:
  Exploiting the relationship between energy metabolism and virulence, combination approaches could target:

  Primary Target	Secondary Target	Rationale	Expected Outcome
  atpE	Phaseolotoxin production	Energy limitation + toxin inhibition	Reduced virulence and growth
  atpE	T3SS components	Disruption of energy supply and effector delivery	Prevention of host manipulation
  atpE	Stress response systems	Blocking adaptation to energy limitation	Enhanced susceptibility to plant defenses
  atpE	Biofilm formation	Limiting energy for extracellular matrix production	Reduced persistence on plant surfaces

• Delivery Systems:
  Novel delivery methods to ensure inhibitors reach bacterial targets include:

  • Nanoparticle formulations for improved plant uptake and distribution

  • Engineered bacteriophages carrying antimicrobial genes

  • Plant-expressible compounds that accumulate in apoplastic spaces

  • Systemic acquired resistance inducers combined with atpE inhibitors

Given the essential nature of ATP synthase and the potential for selective targeting based on structural differences, this approach offers promising avenues for developing environmentally sustainable control measures for P. syringae infections in agriculturally important crops .

What role does atpE play in the adaptation of P. syringae to environmental stresses?

The atpE subunit plays multifaceted roles in P. syringae adaptation to environmental stresses, extending beyond its canonical function in ATP synthesis:

• Temperature Adaptation:
  ATP synthase activity and assembly are influenced by temperature changes, which is particularly relevant for P. syringae as it:

  • Must function efficiently across environmental temperature fluctuations (10-30°C)

  • Shows temperature-dependent expression of virulence factors (optimal at 18°C)

  • Likely undergoes structural adjustments in c-ring composition or conformation at different temperatures

• pH Stress Response:

  • AtpE participates in maintaining cellular pH homeostasis

  • Can operate in reverse as an ATPase to pump protons out when cytoplasmic pH drops

  • May undergo conformational changes to adjust proton translocation efficiency at different pH values

  • Potentially coordinates with other pH-responsive systems to maintain energy production during acid stress

• Oxidative Stress Management:
  Research on related bacterial systems suggests ATP synthase components, including atpE, are responsive to oxidative stress:

  • Oxidative damage to atpE can impair energy generation

  • ATP synthase activity modulation may help conserve resources during stress

  • Energy provision for antioxidant systems is critical for stress tolerance

• Nutrient Limitation Adaptation:
  ATP synthase composition and activity are adjusted during nutrient limitation:

  Nutrient Limitation	ATP Synthase Response	Adaptive Benefit
  Carbon	Potential downregulation to match reduced electron transport chain activity	Energy conservation
  Nitrogen	Maintained or enhanced activity to support nitrogen acquisition processes	Efficient resource allocation
  Phosphate	Complex regulation due to ATP containing essential phosphate	Balance between energy needs and phosphate conservation
  Iron	Adjusted to coordinate with iron-dependent respiratory complexes	Optimization of limited iron usage

• Integration with Global Stress Responses:
  The transcriptional regulatory network analysis of P. syringae revealed complex regulatory circuits with:

  • 54 top-level transcription factors

  • 62 middle-level transcription factors

  • 147 bottom-level transcription factors

  These networks likely coordinate ATP synthase expression with stress-responsive genes, allowing for integrated metabolic adjustments during environmental challenges.

Understanding these adaptive mechanisms could provide insights into P. syringae persistence under variable field conditions and potentially reveal vulnerabilities that could be exploited for disease management strategies.

How can systems biology approaches enhance our understanding of atpE's role in P. syringae metabolism and pathogenicity?

Systems biology offers transformative approaches to unravel the complex interconnections between atpE function, cellular metabolism, and pathogenicity in P. syringae:

• Multi-omics Integration:
  Comprehensive integration of multiple data types can reveal new insights about atpE's role:

  Omics Approach	Information Provided	Integration Benefit
  Genomics	Genetic variations in atpE across strains	Correlation with virulence phenotypes
  Transcriptomics	Expression patterns under various conditions	Coordinated regulation networks
  Proteomics	Post-translational modifications, protein-protein interactions	Functional states of ATP synthase
  Metabolomics	Energy-related metabolite pools	Impact of atpE variation on metabolic fluxes
  Fluxomics	Rates of ATP synthesis/consumption	Dynamic adaptation of energy metabolism

• Network Analysis and Modeling:
  Recent research has characterized the genome-wide transcriptional regulatory network in P. syringae, identifying:

  • Complex hierarchical structure with top, middle, and bottom-level transcription factors

  • More than forty thousand TF-pairs classified into 13 three-node submodules

  • Regulatory diversity reflecting information flow within the network

  Incorporating atpE into these networks would reveal:

  • Key regulatory nodes controlling energy metabolism during infection

  • Feedback mechanisms between energy status and virulence factor expression

  • Potential master regulators coordinating metabolic and pathogenicity pathways

• In Silico Phenotype Prediction:

  • Constraint-based metabolic modeling to predict growth and virulence under various conditions

  • Flux balance analysis to determine how atpE mutations affect metabolic capabilities

  • Agent-based models to simulate bacterial-plant interactions with varying ATP synthase efficiency

• Evolutionary Systems Biology:
  Analysis of atpE across P. syringae pathovars reveals:

  • Varying degrees of conservation reflecting adaptation to different plant hosts

  • Potential horizontal gene transfer events shaping ATP synthase evolution

  • Signatures of selection indicating host-specific adaptation

• Synthetic Biology Applications:
  Systems-level understanding enables rational design approaches:

  • Creation of minimally modified atpE variants with altered regulatory properties

  • Development of genetic circuits linking atpE expression to specific environmental cues

  • Engineering metabolic control systems to manipulate virulence through energy limitation