Recombinant Pseudomonas syringae pv. tomato Methionine import ATP-binding protein MetN 2 (metN2)

What is the role of MetN2 in Pseudomonas syringae pv. tomato?

MetN2 functions as an essential component of the methionine ABC transporter system in P. syringae pv. tomato. As an ATP-binding protein, it provides the energy required for the active transport of methionine across the bacterial membrane through ATP hydrolysis. The methionine import system is critical for bacterial metabolism and virulence, as methionine is an essential amino acid for protein synthesis and serves as a precursor for S-adenosylmethionine (SAM), which is involved in numerous cellular processes including methylation reactions.

Similar to characterized MetNI systems in other bacteria, the P. syringae MetN2 likely functions within a complex that includes additional components such as the transmembrane domain protein MetI and the substrate-binding protein MetQ . The entire system facilitates the controlled uptake of methionine from the environment, which is particularly important during bacterial colonization of plant tissues where nutrient availability may fluctuate.

How does MetN2 differ from other ATP-binding proteins in P. syringae pv. tomato?

MetN2 belongs to the ABC transporter family but is specifically tailored for methionine transport. Unlike other ATP-binding proteins in P. syringae, MetN2 contains specific sequence motifs and structural elements that enable it to interact with the methionine transport machinery. It features the characteristic Walker A and Walker B motifs common to ATP-binding proteins, but its substrate specificity is determined by unique regions that facilitate interaction with MetI and potentially MetQ components.

The specificity of MetN2 for the methionine transport system distinguishes it from other ATP-binding proteins involved in the transport of different substrates such as sugars, ions, or other amino acids. This specificity is likely achieved through specialized protein-protein interactions and conformational changes that occur during the transport cycle. By comparison, the MetNI system in other bacteria such as Neisseria meningitides has been shown to have specific binding affinities and conformational states that regulate methionine transport .

What is the genomic context of the metN2 gene in P. syringae pv. tomato DC3000?

The metN2 gene in P. syringae pv. tomato DC3000 is typically located within an operon containing other genes involved in methionine transport and metabolism. Based on comparative genomics with related bacteria, this gene is likely situated in proximity to metI (encoding the transmembrane domain) and metQ (encoding the periplasmic binding protein). This genomic organization facilitates coordinated expression of all components necessary for a functional methionine transport system.

P. syringae pv. tomato DC3000 has been extensively studied as a model plant pathogen, and its genome has been thoroughly characterized . The metN2 gene's genomic context may provide insights into its evolutionary history and functional importance. Multilocus sequence typing (MLST) studies of P. syringae pv. tomato have revealed that recombination has played a significant role in the evolution of this bacterium, contributing more to variation between isolates than mutation . This suggests that genes like metN2 may have been subject to horizontal gene transfer or recombination events that influenced their current genomic location and sequence characteristics.

What are the optimal conditions for expressing recombinant P. syringae pv. tomato MetN2 in E. coli?

For optimal expression of recombinant P. syringae pv. tomato MetN2 in E. coli, a methodological approach involving careful selection of expression vectors, host strains, and growth conditions is essential. The following protocol represents a starting point based on successful expression of similar ATP-binding proteins:

Expression System:

• Vector: pET-28a(+) with an N-terminal His6-tag for purification

• Host strain: E. coli BL21(DE3) for T7 promoter-based expression

• Alternative strain for problematic expression: E. coli B834(DE3), which can also facilitate selenomethionine incorporation for crystallographic studies

Expression Protocol:

• Transform expression plasmid into the appropriate E. coli strain

• Inoculate starter culture in LB medium with appropriate antibiotic

• Grow main culture at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5 mM IPTG

• Reduce temperature to 18-20°C and continue expression for 16-18 hours

• Harvest cells by centrifugation at 4,000g for 20 minutes at 4°C

• Store cell pellet at -80°C until purification

For selenomethionine-labeled protein (useful for crystallographic studies), use a minimal medium like PASM autoinduction media containing 125 μg/mL selenomethionine and allow expression to proceed for 3-5 days at room temperature .

What purification strategy is most effective for obtaining high-purity MetN2 for structural studies?

A multi-step purification strategy is recommended for obtaining high-purity MetN2 suitable for structural studies:

Purification Protocol:

• Cell Lysis: Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM PMSF, and protease inhibitor cocktail). Lyse cells using sonication or high-pressure homogenization.

• Initial Purification: Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin:

  • Equilibrate column with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol)

  • Apply clarified lysate

  • Wash with binding buffer containing 20 mM imidazole

  • Elute with an imidazole gradient (50-300 mM)

• Intermediate Purification: Apply pooled IMAC fractions to ion exchange chromatography:

  • Use Q Sepharose column for anion exchange

  • Equilibrate with 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol

  • Elute with NaCl gradient (50-500 mM)

• Final Purification: Perform size exclusion chromatography:

  • Use Superdex 200 column

  • Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

  • Collect fractions containing pure MetN2

• Concentration: Concentrate protein using centrifugal filters with appropriate molecular weight cut-off (typically 30 kDa)

• Quality Control: Assess purity by SDS-PAGE (>95% purity required for structural studies) and verify identity by mass spectrometry

This purification approach is similar to methods successfully used for other ATP-binding proteins, including the MetN component of the MetNI transporter from Neisseria meningitides .

What methods are available for assessing the ATP hydrolysis activity of purified MetN2?

Multiple complementary approaches can be used to assess the ATP hydrolysis activity of purified MetN2:

1. Colorimetric Phosphate Detection Assay:

• Principle: Measures inorganic phosphate (Pi) released during ATP hydrolysis

• Protocol:

  • Prepare reaction mixture containing purified MetN2 (0.1-1 μM), ATP (0.1-5 mM), and reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2)

  • Incubate at 37°C for specified time intervals

  • Stop reaction with malachite green or molybdate reagent

  • Measure absorbance at appropriate wavelength (typically 620-640 nm)

  • Calculate Pi concentration using a standard curve

2. Coupled Enzyme Assay:

• Principle: Links ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase

• Protocol:

  • Prepare reaction mixture containing MetN2, ATP, phosphoenolpyruvate, NADH, pyruvate kinase, and lactate dehydrogenase

  • Monitor decrease in NADH absorbance at 340 nm

  • Calculate ATP hydrolysis rate from the rate of NADH oxidation

3. Isothermal Titration Calorimetry (ITC):

• Measures heat released during ATP binding and hydrolysis

• Provides thermodynamic parameters of ATP binding

Data Analysis and Interpretation:

The ATP hydrolysis activity of MetN2 should be assayed both independently and in the context of the full MetNIQ transport complex to understand its functional characteristics in different states .

How does recombination influence the evolution of MetN2 in different P. syringae pv. tomato strains?

Recombination plays a critical role in the evolution of P. syringae pv. tomato, including genes encoding transport systems like MetN2. MLST (Multilocus Sequence Typing) studies have demonstrated that recombination contributes more significantly than mutation to genetic variation between P. syringae isolates . For MetN2, this evolutionary mechanism has several important implications:

Recombination Hotspots and MetN2 Evolution:
Population genetic analysis of P. syringae pv. tomato has revealed several recombination breakpoints within sequenced gene fragments , suggesting that recombination may contribute to the diversity observed in functional genes like metN2. The ATP-binding domains of ABC transporters are generally more conserved than other components, but recombination events may introduce subtle variations that affect:

• ATP binding efficiency

• Interaction with membrane components

• Regulatory mechanisms

• Substrate specificity

Comparative Analysis of MetN2 Across Strains:
Examining MetN2 sequences from different P. syringae pv. tomato isolates reveals patterns consistent with recombination-driven evolution. PtoDC3000, an unusual tomato isolate that clusters phylogenetically with isolates from Brassicaceae and Solanaceae species, may contain a version of MetN2 that reflects its broader host range . This suggests that recombination events affecting nutrient acquisition genes like metN2 may contribute to the adaptive capacity of P. syringae across different plant hosts.

Experimental Approaches to Study Recombination Effects:

• Whole-genome sequencing of multiple strains to identify recombination events affecting the metN2 locus

• Construction of chimeric MetN2 proteins to test functional consequences of recombination

• Analysis of selection pressures (dN/dS ratios) on different domains of MetN2

• Population genetics approaches to quantify recombination rates at the metN2 locus

The recombination dynamics affecting metN2 may parallel those observed for type III secreted effectors like AvrPto1, where recombination contributes to the reassortment of effector repertoires between strains . This suggests that recombination may be a general mechanism by which P. syringae pv. tomato adapts its nutrient acquisition systems to different host environments.

What structural mechanisms govern the interaction between MetN2 and other components of the methionine import system?

The structural mechanisms governing interactions between MetN2 and other components of the methionine import system (MetI, MetQ) involve complex conformational changes that facilitate methionine transport. While specific structural data for P. syringae MetN2 is limited, insights can be drawn from related systems:

Structural States and Conformational Changes:
The methionine import system likely undergoes a "Venus-flytrap" mechanism similar to that observed in Neisseria meningitides . This involves:

• Resting State: ATP-bound MetN2 dimers associate with MetI transmembrane domains

• Substrate Binding: Methionine binds to MetQ, inducing conformational changes

• Complex Formation: MetQ associates with MetNI complex, but with different affinities depending on whether it's bound to methionine

• Transport Mechanism: ATP hydrolysis drives conformational changes that facilitate methionine translocation

• Reset Phase: ADP release and ATP binding reset the system

A critical finding from studies of related systems is that ligand-free MetQ associates with the ATP-bound form of MetNI approximately 40 times more tightly than liganded MetQ . This thermodynamic preference is essential for transport to occur and suggests that methionine must dissociate from MetQ for effective transport.

Key Interaction Domains:

• C-loops in MetN2 that coordinate ATP binding

• Q-loops that transmit conformational changes to the transmembrane domains

• Interface regions between MetN2 dimers

• Contact surfaces between MetN2 and MetI

• Binding sites for MetQ docking

Experimental Approaches to Study These Interactions:

• X-ray crystallography of individual components and complexes

• Cryo-EM to capture different conformational states

• FRET analysis to monitor conformational changes in real-time

• Cross-linking studies to identify interaction interfaces

• Mutational analysis of predicted interaction sites

These structural mechanisms are likely conserved across bacterial species, but specific adaptations in P. syringae pv. tomato may reflect its plant-associated lifestyle and particular nutrient requirements.

How does the substrate specificity of MetN2-containing transporters differ between P. syringae pathovars with varying host ranges?

The substrate specificity of MetN2-containing transporters may vary significantly between P. syringae pathovars with different host ranges, potentially contributing to their adaptation to specific plant environments:

Host Range and Transporter Adaptation:
P. syringae pv. tomato DC3000 exhibits an unusually broad host range compared to other tomato isolates, being pathogenic to tomato, Arabidopsis thaliana, and cauliflower . This broader host range may be reflected in adaptations of its nutrient transport systems, including the MetN2-containing methionine transporter. Different plant hosts provide varying nutrient environments, potentially selecting for transporters with modified substrate recognition profiles.

Comparative Analysis of MetN2 Across Pathovars:

Factors Influencing Substrate Specificity:

• Amino acid variations in the MetN2 ATP-binding pocket that affect energy coupling

• Alterations in MetI transmembrane domains that form the translocation pathway

• Modifications in MetQ that affect initial substrate recognition

• Regulatory elements that control expression under different nutrient conditions

Experimental Approaches to Compare Specificities:

• Heterologous expression of MetN2 variants from different pathovars

• Transport assays using radioactively labeled methionine and derivatives

• Growth complementation studies in methionine auxotrophs

• Competitive inhibition assays to determine relative affinities for different substrates

• Structural studies comparing binding sites across pathovars

The taxonomic division of P. syringae pv. tomato into two pathovars proposed based on phylogenetic analysis may correlate with functional differences in methionine transport systems, including substrate specificity of MetN2-containing complexes.

What strategies can resolve expression and solubility issues with recombinant MetN2?

Recombinant expression of membrane-associated proteins like MetN2 frequently encounters solubility challenges. The following strategies can help overcome these issues:

Optimizing Expression Conditions:

• Temperature Modulation: Reduce expression temperature to 16-20°C to slow protein production and allow proper folding

• Induction Optimization: Test different IPTG concentrations (0.1-1.0 mM) and induction times

• Media Formulation: Supplement with glycerol (0.5-2%) and specific ions (Mg²⁺, K⁺) that may stabilize the protein

• Co-expression Strategies: Express with chaperones (GroEL/GroES, DnaK/DnaJ) to assist folding

Enhancing Protein Solubility:

• Fusion Tags:

  • Solubility-enhancing tags (MBP, SUMO, TrxA)

  • Position optimization (N-terminal vs. C-terminal)

  • Linker optimization between tag and target protein

• Buffer Optimization:

  • Screen various pH conditions (6.5-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Add stabilizing agents (glycerol 5-15%, reducing agents like DTT or β-mercaptoethanol)

• Truncation Strategies:

  • Express stable domains identified through bioinformatic analysis

  • Remove flexible or hydrophobic regions that may cause aggregation

Case Study Example:
When expressing selenomethionine-substituted proteins for crystallographic studies, researchers successfully used B834(DE3) E. coli cells grown in PASM autoinduction media containing 125 μg/mL selenomethionine . This specialized approach allowed protein expression for 3-5 days at room temperature, resulting in well-diffracting crystals.

How can researchers address inconsistent ATP hydrolysis activity in purified MetN2 preparations?

Inconsistent ATP hydrolysis activity in purified MetN2 preparations can significantly impact experimental reproducibility. Several methodological approaches can help address this issue:

Identifying Sources of Variability:

• Protein Quality Assessment:

  • Size exclusion chromatography to confirm monodispersity

  • Circular dichroism to verify proper folding

  • Mass spectrometry to confirm integrity and detect modifications

  • DLS (Dynamic Light Scattering) to assess aggregation state

• Activity Assay Optimization:

  • Standardize enzyme concentration and reaction conditions

  • Include appropriate controls in each assay

  • Test multiple substrate concentrations to generate complete kinetic profiles

  • Verify linearity of activity over time

Stabilization Strategies:

• Buffer Optimization:

  • Screen additives (glycerol, detergents, specific ions)

  • Test the effect of different pH values on stability

  • Include metal cofactors (Mg²⁺, Mn²⁺) at optimal concentrations

• Storage Conditions:

  • Aliquot protein to avoid freeze-thaw cycles

  • Test stability at different storage temperatures (-80°C, -20°C, 4°C)

  • Evaluate cryoprotectant addition (glycerol, sucrose)

  • Consider flash-freezing in liquid nitrogen

Reconstitution Approaches:

• Complex Formation:

  • Co-purify or reconstitute with MetI to form the minimal functional unit

  • Add purified MetQ to reconstruct the complete transport system

  • Test activity in the presence of lipids or nanodiscs to mimic membrane environment

• Activity Correlation Table:

These approaches should be implemented systematically, with careful documentation of conditions that yield consistent activity, to establish a robust experimental protocol.

What experimental controls are essential when studying MetN2 interactions with potential inhibitors or regulators?

When investigating MetN2 interactions with potential inhibitors or regulators, rigorous experimental controls are essential to ensure reliable and interpretable results:

Essential Controls for Binding Studies:

• Positive Controls:

  • Known ATP analogs that bind to the nucleotide-binding domain

  • Well-characterized ABC transporter inhibitors

  • Vanadate compounds that trap the transition state

• Negative Controls:

  • Structurally similar compounds without inhibitory activity

  • Denatured MetN2 to control for non-specific binding

  • Buffer-only conditions to establish baseline measurements

• Specificity Controls:

  • Testing against related ATP-binding proteins to determine selectivity

  • Evaluating activity against other components of the methionine transport system

  • Assessing effects on unrelated ATPases to confirm specificity

Methodological Controls for Different Techniques:

• For Thermal Shift Assays:

  • Include DMSO-only controls at equivalent concentrations

  • Run ATP as reference stabilizing ligand

  • Perform concentration-response curves for potential interactors

• For ATPase Activity Assays:

  • Implement internal standards for phosphate detection

  • Include time-course measurements to ensure linearity

  • Verify enzyme concentration dependence

• For Structural Studies:

  • Crystallize protein both with and without potential binding partners

  • Prepare multiple concentrations of ligands to identify partial occupancy

  • Collect diffraction data at multiple wavelengths for anomalous difference maps

Statistical Validation:

• Perform experiments with at least three independent protein preparations

• Include technical replicates (minimum n=3) for each condition

• Apply appropriate statistical tests to determine significance

• Calculate Z-factor values for high-throughput screening assays

Validation in Complex Systems:

• Test effects in reconstituted proteoliposomes

• Validate findings in whole-cell transport assays

• Perform genetic validation through site-directed mutagenesis of predicted interaction sites

By implementing these comprehensive controls, researchers can confidently identify genuine MetN2 interactors and distinguish them from experimental artifacts, enabling robust characterization of potential inhibitors or regulators of this important transport protein.

How might cryo-EM studies advance our understanding of the complete P. syringae MetNIQ transport cycle?

Cryo-electron microscopy (cryo-EM) offers transformative potential for elucidating the complete transport cycle of the P. syringae MetNIQ system, addressing several key knowledge gaps:

Advantages of Cryo-EM for MetNIQ Studies:

• Capturing Multiple Conformational States: Unlike crystallography which often captures single states, cryo-EM can potentially visualize multiple conformations present in a sample simultaneously, providing snapshots of the transport cycle

• Reduced Crystallization Constraints: Membrane protein complexes like MetNIQ are notoriously difficult to crystallize, but cryo-EM bypasses this requirement

• Near-Native Environment: The complex can be studied in nanodiscs or detergent micelles that better mimic the membrane environment

Critical Questions Addressable Through Cryo-EM:

• Conformational Coupling: How ATP binding and hydrolysis by MetN2 drive the conformational changes in MetI transmembrane domains

• MetQ Docking Mechanism: The structural basis for the observation that ligand-free MetQ binds ATP-bound MetNI ~40 times more tightly than liganded MetQ

• Substrate Translocation Pathway: Visualization of the methionine translocation channel and how it opens and closes during transport

• Species-Specific Adaptations: Structural features unique to P. syringae compared to well-characterized systems like E. coli or N. meningitides

Experimental Approach:

• Sample Preparation:

  • Express and purify the complete MetNIQ complex

  • Reconstitute in nanodiscs using MSP scaffold proteins and E. coli lipids

  • Prepare samples with various nucleotides (ATP, ADP, non-hydrolyzable analogs) and with/without methionine

• Data Collection Strategy:

  • Collect multiple datasets representing different states:

    • ATP-bound, no substrate

    • ATP-bound with MetQ (no methionine)

    • ATP-bound with MetQ (methionine-loaded)

    • Transition state (with vanadate)

    • Post-hydrolysis state

• Analysis Framework:

  • Apply 3D classification to identify distinct conformational states

  • Perform focused refinement on dynamic regions

  • Integrate with molecular dynamics simulations to model transitions

Expected Outcomes and Significance:
A comprehensive cryo-EM study would likely reveal a series of coordinated conformational changes that constitute the complete transport cycle, providing a structural framework for understanding how ATP hydrolysis by MetN2 is coupled to substrate translocation. This would advance our understanding beyond the current knowledge based on related systems and potentially reveal unique features of the P. syringae transporter that could be exploited for agricultural applications.

What genetic approaches could reveal the contribution of MetN2 to P. syringae virulence and host adaptation?

Sophisticated genetic approaches can elucidate the precise contributions of MetN2 to P. syringae pv. tomato virulence and host adaptation, revealing potential intervention targets:

Targeted Genetic Manipulation Strategies:

• Gene Deletion and Complementation:

  • Create clean metN2 deletion mutants using allelic exchange

  • Complement with wild-type and mutated variants (Walker A/B motifs, regulatory domains)

  • Evaluate effects on growth in methionine-limited media and during plant infection

• Domain Swapping Experiments:

  • Exchange domains between MetN2 proteins from pathovars with different host ranges

  • Create chimeric proteins between MetN2 and related ATP-binding proteins

  • Test how these modifications affect substrate specificity and transport efficiency

• Targeted Mutagenesis:

  • Introduce point mutations at conserved residues based on structural predictions

  • Create regulatory mutations affecting ATP binding/hydrolysis

  • Develop substrate specificity mutations based on binding pocket analysis

In Planta Functional Analysis:

• Infection Assays:

  • Quantify bacterial growth in different plant hosts (tomato, A. thaliana)

  • Measure disease symptom development over time

  • Compare metN2 mutant performance in resistant vs. susceptible plant varieties

• Competition Assays:

  • Co-inoculate wild-type and metN2 mutants to assess fitness in planta

  • Determine competitive index in different plant tissues and growth conditions

  • Evaluate adaptation over multiple infection cycles

• Metabolomic Analysis:

  • Profile methionine and derivatives in plant tissues during infection

  • Compare utilization patterns between wild-type and metN2 mutants

  • Identify plant metabolites that may specifically interact with the transport system

Population Genetics Approaches:

• Comparative Genomics:

  • Analyze metN2 sequence variation across P. syringae isolates with different host specificities

  • Identify signatures of selection and recombination affecting the metN2 locus

  • Correlate sequence variations with host range and virulence phenotypes

• Experimental Evolution:

  • Subject P. syringae populations to methionine limitation stress

  • Sequence evolved populations to identify adaptive mutations in metN2 and related genes

  • Test evolved strains for altered virulence and host range

Expected Outcomes:
This multi-faceted genetic approach would reveal whether MetN2 functions primarily as a basic housekeeping gene for bacterial nutrition or plays a more specialized role in virulence and host adaptation. The results could identify specific domains or residues critical for function in planta, potentially serving as targets for future intervention strategies aimed at disrupting bacterial infection.

How might synthetic biology approaches enable engineering of MetN2 for biotechnological applications?

Synthetic biology approaches offer exciting opportunities to engineer P. syringae MetN2 for diverse biotechnological applications, building on fundamental understanding of its structure and function:

Engineering Strategies for Modified MetN2:

• Substrate Specificity Engineering:

  • Rational design of the substrate interaction regions based on structural models

  • Directed evolution using random mutagenesis coupled with selection systems

  • Computational design of binding pockets for non-native substrates

• Regulatory Circuit Modifications:

  • Engineering ATP hydrolysis rates through mutations in Walker A/B motifs

  • Creating MetN2 variants responsive to alternative energy sources

  • Developing allosterically regulated versions with novel control mechanisms

• Fusion Protein Development:

  • MetN2-reporter fusions for monitoring transport activity

  • Creation of bifunctional proteins combining transport with catalytic functions

  • Development of stimulus-responsive transport systems by integrating sensory domains

Potential Biotechnological Applications:

Experimental Platform Development:

• Cell-Free Systems:

  • Reconstitution of MetN2 function in liposomes or nanodiscs

  • Development of high-throughput screening platforms for engineered variants

  • Integration with other transport and metabolic components for synthetic cellular systems

• Chassis Organism Engineering:

  • Introduction of engineered MetN2 variants into non-pathogenic Pseudomonas strains

  • Development of minimal bacterial chassis optimized for specific transport functions

  • Creation of orthogonal transport systems that operate independently of native cellular machinery

• Biocontainment Strategies:

  • Engineering MetN2-dependent auxotrophs to enable biological containment

  • Development of synthetic dependency relationships for controlled growth

  • Creating transport systems dependent on non-natural substrates for biocontainment

Challenges and Considerations:
While these applications hold promise, researchers must address challenges including protein stability, integration with native systems, and potential unintended consequences of engineered transport systems. Additionally, engineered systems derived from plant pathogens require careful biosafety assessment to prevent environmental impacts.

The engineering of MetN2 represents a promising frontier where fundamental research on bacterial transport systems can translate into innovative applications across multiple biotechnological sectors.