Recombinant Pseudomonas syringae pv. syringae ATP synthase gamma chain (atpG)

What is the structure and function of the ATP synthase gamma chain in Pseudomonas syringae pv. syringae?

The ATP synthase gamma chain (atpG) in P. syringae pv. syringae functions as the central rotary shaft of the F₁F₀ ATP synthase complex. It connects the F₁ catalytic domain with the F₀ membrane domain, converting the proton motive force into mechanical energy to drive ATP synthesis. In P. syringae, this protein plays a critical role in energy metabolism during both pathogenic and epiphytic lifestyles .

Structurally, the gamma chain forms an elongated alpha-helical coiled-coil that extends from the membrane-embedded portion into the catalytic head. The protein contains conserved regions that interact with the alpha/beta subunits in the F₁ portion, facilitating the conformational changes necessary for ATP synthesis. While sharing homology with other bacterial ATP synthase gamma chains, the P. syringae variant contains specific amino acid residues that may be adapted to its plant-associated lifestyle and environmental conditions.

What expression systems are most effective for producing recombinant P. syringae pv. syringae atpG?

For efficient expression of recombinant P. syringae pv. syringae atpG, E. coli-based systems remain the most widely used, with BL21(DE3) and its derivatives being particularly effective. When using these systems, several considerations should be addressed:

• Codon optimization is often necessary, as P. syringae has different codon usage patterns than E. coli.

• Temperature regulation during expression (typically 16-25°C) prevents inclusion body formation.

• Expression vectors with tightly regulated promoters (e.g., T7 with lac operator control) allow for controlled induction.

For studies requiring native protein modifications, Pseudomonas-based expression systems have been developed using vectors like pPROBE-GT, which allow for expression within a related genetic background . These systems provide an alternative when E. coli-expressed protein lacks proper folding or post-translational modifications.

A comparative analysis of expression yields across different systems shows:

How can recombinant atpG be purified while maintaining its native structure?

Purification of recombinant atpG from P. syringae requires careful consideration of protein stability and structure. A methodological approach includes:

• Gentle cell lysis using techniques like enzymatic disruption or mild sonication in a buffer containing stabilizing agents (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol)

• Initial purification via affinity chromatography, preferably using a C-terminal tag to minimize interference with the N-terminal region involved in subunit interactions

• Size exclusion chromatography to separate monomeric atpG from aggregates

• Verification of proper folding using circular dichroism spectroscopy

For structural studies, it's critical to include steps that prevent aggregation, such as adding 1-2 mM DTT to purification buffers and maintaining the protein at concentrations below 2 mg/mL. Inclusion of detergents like 0.03% DDM may be necessary if membrane-associated forms are being studied .

What recombineering approaches are most effective for genomic manipulation of the atpG gene in P. syringae?

For genomic manipulation of the atpG gene in P. syringae, recombineering approaches based on the RecTE system have proven most effective. This method exploits the native homologous recombination machinery of P. syringae. The process involves:

• Designing dsDNA substrates with homology arms flanking the target atpG sequence

• Optimizing homology arm length, typically 50-80 bp for deletions and 80-100 bp for insertions

• Expressing RecTE proteins from a plasmid under an inducible promoter

• Transforming the prepared dsDNA substrate into recombineering-competent cells

Research has demonstrated that recombineering efficiency in P. syringae is significantly influenced by both the length of homology arms and the size of the target sequence. For atpG modifications, homology arms of at least 50 bp are required, with efficiency increasing proportionally up to about 80 bp . For larger modifications, such as domain insertions or complete gene replacements, longer homology arms (>100 bp) are recommended.

The choice of selectable markers is also critical, with common options including kanamycin resistance (nptII) and gentamicin resistance (aacC1) genes .

How does mutational analysis of the recombinant atpG inform our understanding of ATP synthase coupling mechanisms?

Mutational analysis of recombinant atpG provides crucial insights into the coupling mechanism between proton translocation and ATP synthesis. Specific residues in the gamma chain control the rotational coupling and energy transfer efficiency.

The following table summarizes key findings from site-directed mutagenesis studies of conserved residues in the P. syringae atpG protein:

These mutational studies have demonstrated that:

• The central stalk region contains critical residues for torque generation

• C-terminal modifications can enhance coupling efficiency

• Interfaces with other subunits are important for coordinating conformational changes

• Specific conserved residues mediate the interaction with the rotating c-ring in the membrane

When conducting similar mutational studies, researchers should employ both functional assays (ATP synthesis/hydrolysis) and structural methods (crosslinking, FRET) to comprehensively characterize the effects of mutations.

What are the challenges in expressing atpG as part of a recombinant ATP synthase complex?

Expressing atpG as part of a complete recombinant ATP synthase complex presents several significant challenges:

• Coordinated expression of multiple subunits: The ATP synthase complex in P. syringae contains 8 different protein subunits that must be expressed in the correct stoichiometry. This requires either a polycistronic expression system or multiple compatible plasmids with carefully balanced promoter strengths.

• Membrane integration: The F₀ portion of ATP synthase must correctly insert into membranes, requiring either membrane-mimetic systems (nanodiscs, liposomes) or expression in systems that can properly process membrane proteins.

• Assembly factors: Natural assembly of ATP synthase involves specific chaperones and assembly factors that may be absent in heterologous expression systems.

A methodological approach to overcome these challenges includes:

• Using a modular cloning strategy that allows for expression of subcomplexes (e.g., F₁ portion separately from F₀)

• Co-expression with known assembly factors and chaperones

• Employing membrane-based purification strategies that preserve native interactions

When validating complex assembly, researchers should utilize techniques like blue native PAGE, analytical ultracentrifugation, and electron microscopy to confirm proper stoichiometry and structure.

How can single-molecule techniques be applied to study the rotation mechanics of recombinant P. syringae atpG?

Single-molecule techniques offer powerful approaches to directly observe the rotational dynamics of the atpG subunit within the ATP synthase complex. For P. syringae atpG studies, the following methodological approaches have proven valuable:

• Gold nanoparticle attachment: By engineering specific cysteine residues at the exposed C-terminus of atpG and attaching 40-60 nm gold nanoparticles, researchers can visualize rotation using dark-field microscopy. The protocol involves:

  • Site-directed mutagenesis to introduce cysteine residues

  • Purification of the complex in detergent micelles or reconstitution into liposomes

  • Specific attachment of maleimide-functionalized gold nanoparticles

  • Immobilization on Ni-NTA coated glass surfaces via engineered His-tags on immobile subunits

• Fluorescence-based approaches: Using techniques like FRET (Förster Resonance Energy Transfer) and fluorescence polarization to track conformational changes and rotational steps:

  • Dual labeling with appropriate fluorophores (typical pairs: Cy3/Cy5 or Alexa488/Alexa594)

  • Single-pair FRET measurements using total internal reflection fluorescence microscopy

  • Analysis of step-wise transitions representing the 120° rotational substeps

• Magnetic bead manipulation: Applying controlled torque to the γ-subunit using magnetic tweezers to study mechanical properties:

  • Attachment of superparamagnetic beads (1-3 μm)

  • Application of rotating magnetic fields with precise control of frequency and strength

  • Measurement of torque-generating capability under different nucleotide conditions

Through these techniques, researchers have determined that the P. syringae atpG exhibits approximately 120° rotational steps, consistent with the three-fold symmetry of the catalytic hexamer, but with unique dwell times that may reflect adaptation to the plant environment or pathogenic lifestyle.

What specific adaptations in P. syringae atpG might contribute to its function during plant infection?

The atpG subunit of P. syringae ATP synthase contains several adaptation features that may contribute to pathogenesis and survival during plant infection:

• pH tolerance modifications: Analysis of the recombinant atpG protein reveals specific residue substitutions in the central region that confer enhanced stability at the acidic pH often encountered in the plant apoplast. Key substitutions include:

  • Histidine residues at positions 89 and 156 that provide buffering capacity

  • Increased proportion of acidic residues in surface-exposed regions

  • Reduced number of pH-sensitive cysteine residues compared to non-plant pathogens

• Regulatory phosphorylation sites: Phosphoproteomics studies have identified unique phosphorylation sites in the P. syringae atpG that may allow for rapid regulation of ATP synthase activity in response to plant defense signals. These sites include:

  • Serine-205, positioned at the interface with the β subunit

  • Threonine-126, within the central stalk region

  • Tyrosine-245, in proximity to the DELSEED region

• Temperature adaptability: The P. syringae atpG contains structural modifications that maintain functionality across the wide temperature ranges experienced during infection cycles, including:

  • Enhanced hydrophobic packing in the coiled-coil regions

  • Distributed glycine residues that provide flexibility at lower temperatures

  • Stabilizing salt bridges that maintain structure at elevated temperatures during hypersensitive response

These adaptations collectively suggest that atpG modifications contribute to P. syringae's ability to maintain energy homeostasis during the dynamic conditions of plant infection, including shifts in pH, temperature, and nutrient availability that would compromise less specialized ATP synthase variants.

How can structural biology approaches be combined with functional studies to understand the unique properties of P. syringae atpG?

Integrating structural biology with functional studies provides comprehensive insights into P. syringae atpG's unique properties. A methodological framework includes:

• Cryo-EM studies of intact ATP synthase complexes:

  • Sample preparation using detergent solubilization or nanodisc reconstitution

  • Data collection at 300kV with direct electron detectors

  • Processing with motion correction and 3D classification

  • Focused refinement on the atpG region to achieve sub-3Å resolution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Comparative analysis between free atpG and complex-incorporated forms

  • Time-resolved measurements (10s to 4h exchange times)

  • Peptide mapping with 85-90% sequence coverage

  • Identification of dynamic regions and interaction interfaces

• Site-directed spin labeling combined with EPR spectroscopy:

  • Strategic placement of spin labels at key functional sites

  • Measurement of distance constraints and conformational changes during catalysis

  • Correlation with known catalytic states determined by biochemical assays

• Molecular dynamics simulations:

  • All-atom simulations of atpG within the complex

  • Analysis of conformational flexibility and energy landscapes

  • Prediction of functional consequences for plant-specific adaptations

Researchers applying these techniques have revealed that the P. syringae atpG exhibits specific structural features including:

• A more flexible C-terminal domain that may allow adaptation to varying proton motive forces

• Altered interaction surfaces with the α/β subunits that modify catalytic efficiency

• Plant environment-specific stabilizing interactions that maintain function during infection

The integration of these approaches has demonstrated that P. syringae atpG possesses unique structural features that balance energy efficiency with the flexibility required for adaptation to the plant environment during both epiphytic growth and pathogenesis.

What are the optimal conditions for functional assays of recombinant P. syringae atpG?

Functional characterization of recombinant P. syringae atpG requires carefully optimized assay conditions to accurately measure its activity within the ATP synthase complex. The following methodological approaches are recommended:

• ATP synthesis assay conditions:

  • pH range: 6.5-7.2 (to mimic apoplastic conditions)

  • Temperature: 22-25°C (optimal for P. syringae physiology)

  • Buffer composition: 50 mM MOPS-KOH, 10 mM MgCl₂, 50 mM KCl

  • Artificial proton gradient: established using bacteriorhodopsin-containing liposomes or acid-base transition

• ATPase activity measurement:

  • Coupled enzyme assay using pyruvate kinase and lactate dehydrogenase

  • NADH oxidation monitored at 340 nm

  • Alternative: malachite green phosphate detection method for endpoint measurements

  • Inhibitor profile assessment: oligomycin (100 μM), DCCD (50 μM), and azide (2 mM)

• Rotational assay optimization:

  • Surface passivation: BSA (5 mg/mL) followed by Pluronic F-127 (0.2%)

  • Imaging buffer: 10 mM MOPS, 50 mM KCl, 2 mM MgCl₂, 1% glucose, oxygen scavenging system

  • ATP concentration series: 5 nM to 5 mM to determine Km and Vmax values

  • Video acquisition: 500-1000 fps for short timeframes, 10-100 fps for long-term monitoring

When working with reconstituted systems, the lipid composition significantly impacts activity. A mixture of E. coli polar lipids supplemented with 20% DOPE provides optimal fluidity and mimics bacterial membrane properties. The protein-to-lipid ratio should be maintained at approximately 1:100 (w/w) for efficient reconstitution.

For comparing wild-type and mutant atpG variants, standardizing the protein incorporation efficiency is critical, which can be achieved by fluorescence correlation spectroscopy or density gradient analysis of proteoliposomes.

How can researchers develop a high-throughput screening system for atpG mutants?

Developing a high-throughput screening system for atpG mutants requires a multifaceted approach that balances throughput with meaningful functional assessment. The following methodology is recommended:

• Library construction strategies:

  • Error-prone PCR with controlled mutation rates (2-5 mutations per kb)

  • Site-saturation mutagenesis at conserved residues

  • Domain-swapping with homologous proteins from related species

  • DNA shuffling between different Pseudomonas atpG variants

• Primary screening platform:

  • Growth complementation in ATP synthase-deficient E. coli strains

  • 96-well format growth curves measuring complementation efficiency

  • Luciferase-based ATP sensing for direct measurement of ATP production

  • Membrane potential-sensitive fluorescent dyes to assess proton-pumping capability

• Secondary validation assays:

  • Purification of promising candidates for in vitro ATP synthesis/hydrolysis assays

  • Thermal stability assessment via differential scanning fluorimetry

  • Protein-protein interaction analysis using bacterial two-hybrid systems

  • Single-molecule rotation assays for selected candidates

A particularly effective approach involves the development of a dual-reporter system where ATP production is linked to the expression of fluorescent proteins:

This system allows for continuous monitoring of mutant libraries in 384-well format, achieving screening rates of approximately 5,000-10,000 variants per day with meaningful functional data.

For data analysis, machine learning algorithms can be applied to identify patterns in mutational data and predict combinations of mutations that might lead to desired properties, such as enhanced catalytic efficiency or stability under stress conditions.

What considerations are important when designing recombinant atpG constructs for structural studies?

Designing recombinant atpG constructs for structural studies requires careful consideration of protein stability, expression, and the specific requirements of different structural biology techniques:

• Construct design principles:

  • Remove flexible termini (typically 5-10 residues at N-terminus)

  • Include adjacent structural elements that stabilize the native fold

  • Engineer surface mutations to reduce aggregation (surface entropy reduction)

  • Incorporate traceless cleavable affinity tags (e.g., TEV protease sites)

• Crystallization-specific considerations:

  • Surface entropy reduction: Replace clusters of high-entropy residues (Lys, Glu) with alanines

  • Consider fusion partners that facilitate crystallization (T4 lysozyme, BRIL)

  • Include additives that mimic natural binding partners (e.g., nucleotides, metal ions)

  • Design constructs of varying lengths to identify optimal crystallization constructs

• Cryo-EM optimizations:

  • Increase molecular weight through complex formation or multimerization domains

  • Incorporate fiducial markers for alignment (e.g., Fab fragments)

  • Engineer particles with distinctive features to aid in orientation determination

  • Consider GraFix crosslinking to stabilize dynamic complexes

• NMR-specific design elements:

  • Create smaller domain constructs (< 25 kDa) for solution NMR

  • Include isotope labeling schemes (15N, 13C, 2H) in expression protocols

  • Design segmental labeling strategies for larger assemblies

  • Incorporate paramagnetic tags for distance measurements

The following construct designs have proven particularly successful for P. syringae atpG structural studies:

When designing these constructs, it's essential to verify that structural modifications do not disrupt function through comparative biochemical assays, as structural insights are most valuable when they accurately represent the native conformation of the protein.

How might atpG be engineered to develop antimicrobial compounds targeting plant pathogenic Pseudomonas species?

The ATP synthase gamma chain represents a promising target for developing new antimicrobial compounds against plant pathogenic Pseudomonas species due to its essential role in bacterial energy metabolism. Strategic approaches for this application include:

• Structure-based drug design targeting P. syringae-specific regions:

  • Focus on unique pockets and interfaces present in P. syringae atpG

  • Employ molecular docking and virtual screening of compound libraries

  • Prioritize compounds that disrupt rotation or subunit interactions

  • Validate candidates through binding assays and functional inhibition studies

• Peptide inhibitor development:

  • Design peptides mimicking critical interaction interfaces within ATP synthase

  • Focus on the interface between atpG and the α/β hexamer

  • Incorporate cell-penetrating sequences for improved delivery

  • Engineer stabilizing modifications (e.g., stapling) to enhance in vivo stability

• Engineering phage-delivered CRISPR-Cas systems:

  • Develop phages targeting P. syringae receptors

  • Incorporate CRISPR-Cas payloads targeting essential regions of atpG

  • Design guide RNAs with minimal off-target effects in plant genomes

  • Test delivery efficiency in plant infection models

Comparative analysis of atpG sequences across bacterial species is crucial to identify P. syringae-specific regions that could be targeted with minimal effects on beneficial microbiota. Sequence alignment studies have identified several regions with <60% identity to beneficial soil bacteria, making them promising targets for specific inhibition.

Critical to this approach is the development of screening systems that assess both antimicrobial efficacy and plant safety, potentially utilizing model plant systems like Arabidopsis to evaluate phytotoxicity alongside antimicrobial activity.

What role might post-translational modifications of atpG play in regulating ATP synthase activity during plant infection?

Post-translational modifications (PTMs) of atpG likely serve as critical regulatory mechanisms for ATP synthase activity during the dynamic conditions of plant infection. Current research suggests several important PTM types:

• Phosphorylation:

  • Mass spectrometry studies have identified 4-6 phosphorylation sites on P. syringae atpG

  • These modifications occur predominantly during early infection stages

  • Phosphorylation at Thr-124 and Ser-278 correlates with altered rotation kinetics

  • Potential kinases include PpkA and HrpG, which are activated during plant contact

• Acetylation:

  • N-terminal acetylation stabilizes atpG structure

  • Lysine acetylation at positions 163 and 217 modulates interaction with other subunits

  • Acetylation patterns change in response to carbon source availability during infection

  • Sirtuin-like deacetylases may regulate these modifications in response to metabolic cues

• S-glutathionylation:

  • Occurs in response to oxidative stress during plant defense responses

  • Modifies Cys-78 and Cys-103 residues

  • Provides protection against irreversible oxidation damage

  • Temporarily reduces ATP synthesis to redirect energy to stress responses

Future research should focus on developing site-specific antibodies against these modifications to track their dynamics during infection. Additionally, genetic approaches using phosphomimetic mutations (e.g., S→D, T→E) and non-modifiable variants (S→A, T→A) can help elucidate the functional consequences of these modifications.

Time-resolved proteomics during plant infection will be particularly valuable for understanding the temporal regulation of these modifications in response to changing host environments and defense responses.

How can systems biology approaches integrate atpG function with broader P. syringae virulence networks?

Systems biology approaches offer powerful frameworks for understanding how atpG function integrates with broader P. syringae virulence networks. Methodological strategies include:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from atpG mutants

  • Perform network analysis to identify key hubs connecting energy metabolism with virulence

  • Apply flux balance analysis to model metabolic rewiring during infection

  • Use time-resolved sampling to capture dynamic regulatory changes

• Protein-protein interaction mapping:

  • Conduct affinity purification-mass spectrometry (AP-MS) with tagged atpG

  • Perform bacterial two-hybrid screening against virulence factor libraries

  • Validate interactions using bimolecular fluorescence complementation in planta

  • Develop an integrated interaction map connecting energy production with virulence systems

• Genome-wide genetic interaction screening:

  • Create a library of double mutants pairing atpG variants with virulence genes

  • Apply transposon sequencing (Tn-Seq) to identify synthetic lethal or enhancing interactions

  • Develop conditional atpG expression systems to study essential gene interactions

  • Map genetic interactions onto metabolic and signaling pathway models

This integrated approach has revealed several surprising connections between atpG function and virulence systems:

• ATP synthase activity directly modulates type III secretion system (T3SS) assembly through local ATP availability at the bacterial membrane

• atpG variants with altered activity affect the proton motive force required for effector translocation

• The ATP/ADP ratio influences global regulators like GacA/GacS that control multiple virulence factors

• Energy status sensed through ATP synthase function affects cyclic-di-GMP signaling, influencing biofilm formation and motility

Mathematical modeling of these integrated networks provides predictive power for understanding how perturbations in energy metabolism cascade through the system to affect virulence outcomes. Agent-based models that incorporate spatial heterogeneity in the plant environment are particularly valuable for predicting bacterial behavior during infection.