Recombinant Pseudomonas syringae pv. tomato ATP synthase subunit alpha (atpA), partial

What is the role of the C-terminal domain in P. syringae ATP synthase subunit alpha?

The C-terminal domain of ATP synthase subunit alpha in P. syringae, similar to the mycobacterial domain studied in related research, likely influences ATPase activity regulation. Research indicates that species-specific C-terminal extensions can significantly impact ATP hydrolysis capabilities. For instance, in mycobacterial ATP synthase, a 36-amino acid long C-terminal domain in subunit α suppresses ATPase activity . Testing this functionality in P. syringae requires generating deletion mutants of the C-terminal domain using site-directed mutagenesis followed by ATP hydrolysis assays in both membrane-embedded and purified enzyme contexts. Researchers should compare wild-type and C-terminal truncation mutants across different pH and temperature conditions to fully characterize domain function.

How does the ATP synthase in P. syringae differ from other bacterial species?

ATP synthase in P. syringae exhibits distinctive characteristics compared to other bacterial species. While sharing the fundamental F1F0 structure common to F-ATP synthases, P. syringae likely possesses unique residues that affect its catalytic properties. Comparative analyses should include:

To determine these differences experimentally, researchers should perform multiple sequence alignments of atpA genes across bacterial species, followed by structural modeling and functional assays comparing enzymatic properties under standardized conditions.

What are the most effective protocols for expressing recombinant P. syringae atpA?

For optimal expression of recombinant P. syringae atpA, a systematic approach combining appropriate vector selection and expression conditions is essential. Based on successful expression systems for similar proteins, researchers should:

• Clone the atpA gene into a pET vector system with a His-tag or GST-tag for purification

• Transform into E. coli BL21(DE3) or similar expression strains

• Test induction conditions using IPTG concentrations between 0.1-1.0 mM

• Optimize expression temperature (18-30°C) and duration (4-24 hours)

• Evaluate protein solubility using SDS-PAGE analysis of soluble and insoluble fractions

For functional studies, co-expression with other ATP synthase subunits may be necessary to achieve proper folding and activity. Verification of proper assembly can be performed using co-immunoprecipitation with antibodies against other ATP synthase subunits, similar to techniques used in mitochondrial ATP synthase studies .

How can I measure ATP synthase activity in P. syringae membrane preparations?

To effectively measure ATP synthase activity in P. syringae membrane preparations, researchers should employ a multi-faceted approach:

• Membrane vesicle preparation: Isolate bacterial membranes through differential centrifugation following cell lysis by French press or sonication.

• ATP synthesis activity: Measure ATP production using a luciferin-luciferase assay following establishment of a proton gradient using NADH or succinate.

• ATP hydrolysis activity: Quantify inorganic phosphate release using malachite green or similar colorimetric assays.

• Proton pumping: Assess using fluorescent probes such as ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor pH gradient formation.

Control experiments should include specific inhibitors (e.g., DCCD, oligomycin) to confirm ATP synthase-specific activity. For comparative studies with mutant variants, normalize activities to protein content determined by Bradford or BCA assay and ATP synthase content determined by western blotting with anti-atpA antibodies.

What mutations in atpA affect thermal stability of P. syringae ATP synthase?

Mutations in atpA that affect thermal stability of P. syringae ATP synthase should target conserved residues in catalytic domains and interfacial regions. Based on findings from cyanobacterial research, the C252 position (or its equivalent in P. syringae) represents a critical target, as mutations at this position have been shown to improve high temperature tolerance in other organisms .

To systematically identify thermal stability-enhancing mutations:

• Perform computational analysis to identify conserved residues in ATP synthase across thermophilic and mesophilic organisms

• Design a targeted mutagenesis strategy focusing on these residues

• Create a library of atpA mutants using site-directed mutagenesis

• Screen mutants using a three-step procedure involving transformation, initial screening under selective conditions, and rescreening to confirm phenotypes

• Characterize thermal stability by measuring enzyme activity after heat treatment at various temperatures (30-65°C)

The most promising mutants should undergo thorough biochemical characterization to understand the structural basis of improved thermal stability.

How does subunit gamma interaction with atpA affect ATP synthase function in P. syringae?

The interaction between subunit gamma and atpA is crucial for proper ATP synthase function in P. syringae. Research on mycobacterial ATP synthase revealed that the C-terminal domain of subunit α interacts with subunit γ residues 104-109, affecting rotation of the subunit γ and subsequently the enzyme's activity . To investigate this interaction in P. syringae:

• Perform cross-linking studies using chemical cross-linkers that preserve native protein-protein interactions

• Conduct single-molecule rotation assays to measure angular velocity of the power-stroke after ATP binding in wild-type and mutant enzymes

• Employ solution NMR or X-ray scattering to determine the structural interactions between atpA and subunit gamma

• Generate mutants with modified interface residues to assess their impact on coupling efficiency

Studies should focus on measuring changes in both ATP synthesis and hydrolysis activities when interface residues are modified. Decreased angular velocity of subunit gamma rotation would suggest steric hindrance similar to what has been observed in mycobacterial ATP synthase .

How does ATP synthase activity influence P. syringae virulence in tomato plants?

ATP synthase activity significantly impacts P. syringae virulence in tomato plants through multiple mechanisms. Energy production via ATP synthase is essential for various virulence-associated processes. To investigate this relationship:

• Generate conditional atpA mutants with reduced expression or activity

• Assess bacterial growth rates in planta using competitive index assays

• Quantify expression of virulence genes (including Type III secretion system components) in wild-type and ATP synthase-impaired strains

• Measure ATP production in apoplastic conditions that mimic the plant environment

Research on related pathogens suggests that ATP synthase function may be particularly important during the apoplastic colonization phase. P. syringae pv. tomato enters the plant apoplast through natural openings, driven by chemotaxis towards plant-derived compounds . ATP synthase activity likely provides the energy required for this process and subsequent multiplication in the apoplastic space, which is critical for establishing infection .

What plant metabolites affect P. syringae ATP synthase during infection?

Several plant metabolites affect P. syringae ATP synthase during infection, potentially as part of the plant defense response. Key compounds include:

To study these interactions:

• Measure ATP synthase activity in the presence of various plant metabolites at physiologically relevant concentrations

• Assess expression changes in atpA and other ATP synthase genes when exposed to these compounds

• Create reporter constructs linking atpA promoter to luxCDABE reporter genes to monitor real-time expression changes

• Determine if specific plant metabolites directly bind to ATP synthase subunits using isothermal titration calorimetry or surface plasmon resonance

Understanding these interactions may reveal how plants attempt to disrupt bacterial energy metabolism during infection and how P. syringae adapts to overcome these defenses.

What methods can be used to track ATP synthase localization in P. syringae during infection?

Tracking ATP synthase localization in P. syringae during infection requires sophisticated imaging techniques. Based on successful approaches in other systems:

• Generate translational fusions of atpA with fluorescent proteins (GFP, mCherry) using chromosomal integration to maintain native expression levels

• Utilize photoactivatable GFP (paGFP) fusions similar to those used in mitochondrial ATP synthase studies to track movement of specific ATP synthase populations

• Employ super-resolution microscopy techniques (STORM, PALM) to visualize ATP synthase localization with nanometer precision

• For co-localization studies, use MitoTracker or membrane-specific dyes alongside fluorescent ATP synthase fusions

For dynamic studies during infection:

• Develop a plant leaf chamber compatible with confocal microscopy

• Create transparent plant tissue models using cleared leaf techniques

• Use time-lapse imaging to track ATP synthase redistribution during different infection stages

This approach has been successful in tracking mitochondrial ATP synthase trafficking to the plasma membrane in other systems and should be adaptable to study bacterial ATP synthase during plant infection.

How can cryo-EM be optimized for structural determination of P. syringae ATP synthase?

Optimizing cryo-EM for structural determination of P. syringae ATP synthase requires addressing several technical challenges:

• Sample preparation:

  • Purify intact ATP synthase complexes using gentle detergents (DDM, LMNG)

  • Ensure sample homogeneity through size exclusion chromatography

  • Optimize protein concentration (2-5 mg/ml) and grid parameters (blotting time, temperature)

• Data collection strategy:

  • Collect micrographs with dose fractionation (40-60 e-/Ų)

  • Use beam-tilt pair acquisition to improve angular sampling

  • Implement energy filters to enhance contrast

• Image processing considerations:

  • Apply motion correction algorithms optimized for membrane proteins

  • Use 2D classification to identify intact complexes versus dissociated components

  • Implement symmetry-free reconstructions initially, followed by local symmetry refinement

• Validation methods:

  • Cross-validate structures with biochemical crosslinking data

  • Compare with homologous structures from related bacterial species

  • Verify key functional residues align with biochemical data

This approach has successfully yielded high-resolution structures of ATP synthases from other bacterial species and should be adaptable to P. syringae with appropriate optimization.

How can the P. syringae atpA gene be modified to improve stability under stress conditions?

Modifying the P. syringae atpA gene to improve stability under stress conditions requires a systematic protein engineering approach:

• Identify stability-determining regions through comparative analysis with extremophilic organisms

• Apply rational design principles targeting:

  • Increasing salt bridge and hydrophobic interactions

  • Optimizing surface charge distribution

  • Modifying flexible loops that may contribute to unfolding

  • Introducing disulfide bonds at strategic positions

• Implement a high-throughput mutagenesis and screening system:

  • Develop a three-step procedure for efficiently isolating thermotolerant mutants as described for cyanobacterial ATP synthase

  • Test mutants under multiple stress conditions simultaneously (high temperature, pH extremes, oxidative stress)

  • Quantify stability improvements through thermal inactivation assays

• Combine beneficial mutations:

  • Test additive and synergistic effects of multiple mutations

  • Analyze potential structural conflicts using molecular dynamics simulations

  • Optimize combinations for maximal stability without compromising activity

The AtpA-C252F mutation strategy employed in cyanobacteria provides a useful model, where a systematic screening approach successfully identified mutations improving both high light and high temperature tolerance .

What genomic integration strategies are most effective for introducing modified atpA genes in P. syringae?

For effective genomic integration of modified atpA genes in P. syringae, researchers should consider several complementary approaches:

• Homologous recombination-based methods:

  • Implement the sacB-based strategy using suicide vectors like pEX18Tc

  • Design homology arms of 500-1000 bp flanking the atpA gene

  • Include selectable markers (Gmr-lacZ) for positive selection

  • Verify integration through PCR and sequencing

• Site-specific integration systems:

  • Utilize the mini-CTX integration system targeting the attB site

  • Design integration constructs containing the entire gene with upstream promoter region

  • Perform biparental or triparental mating for construct delivery

  • Confirm proper integration and expression levels

• CRISPR-Cas9 genome editing:

  • Design guide RNAs targeting the native atpA locus

  • Provide repair templates containing the modified atpA sequence

  • Select transformants using appropriate antibiotics

  • Screen for successful editing through phenotypic and genotypic analyses

Each method offers distinct advantages depending on the specific modification goals. The mini-CTX system has been successfully used for complementation experiments in Pseudomonas species and may be particularly useful for introducing modified atpA genes while maintaining native expression patterns.

How can I overcome expression toxicity when working with recombinant ATP synthase components?

Overcoming expression toxicity when working with recombinant ATP synthase components requires strategic optimization of expression systems:

• Implement tight expression control:

  • Use strict inducible promoters (T7-lac, araBAD) with minimal basal expression

  • Optimize inducer concentrations to minimize toxicity while maintaining adequate yield

  • Consider glucose repression for tighter control of leaky expression

• Modify host strains:

  • Use C41(DE3) or C43(DE3) E. coli strains specifically designed for toxic membrane proteins

  • Consider using Lemo21(DE3) with tunable T7 lysozyme levels to modulate expression

  • Implement strains with altered membrane compositions that better accommodate membrane proteins

• Adjust expression conditions:

  • Reduce cultivation temperature to 18-20°C during induction

  • Use rich media formulations optimized for membrane protein expression

  • Implement slow induction protocols with gradual inducer addition

• Explore fusion partners:

  • Test solubility-enhancing tags (MBP, SUMO) that may reduce toxicity

  • Consider periplasmic targeting to reduce impact on cellular metabolism

  • Evaluate secretion strategies that may alleviate cytoplasmic accumulation

Successful expression of mycobacterial ATP synthase components has been achieved using similar approaches , suggesting these strategies should be effective for P. syringae proteins.

What are common pitfalls in measuring ATP synthase activity and how can they be avoided?

Common pitfalls in measuring ATP synthase activity and their solutions include:

To ensure reliable results:

• Always include positive controls with known activity levels

• Implement multiple complementary activity assays (ATP synthesis, hydrolysis, proton pumping)

• Normalize activities to ATP synthase content determined by quantitative western blotting

• Consider the impact of detergents and lipid environment on enzyme function

• Verify intact complex formation through native gel electrophoresis or analytical ultracentrifugation

These precautions will significantly improve the reliability and reproducibility of ATP synthase activity measurements in research settings.