Recombinant Pseudomonas syringae pv. tomato tRNA modification GTPase MnmE (mnmE)

What is the molecular mechanism of GTP hydrolysis by MnmE in Pseudomonas syringae pv. tomato?

MnmE employs a distinct GTPase mechanism that differs from classical small GTP-binding proteins. Unlike many GTPases that require auxiliary GTPase-activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs), MnmE's catalytic activity is driven by potassium-dependent homodimerization of its G domains . This dimerization creates a unique catalytic site where potassium ions serve as a critical cofactor for GTP hydrolysis.

The GTPase cycle involves several sequential steps:

• GTP binding to each monomer of MnmE

• Potassium-dependent dimerization of the G domains

• GTP hydrolysis at the dimer interface

• Release of phosphate and subsequent conformational changes

• GDP release and cycle reset

This cycle triggers large-scale conformational changes throughout the protein structure that are essential for its tRNA modification function . These structural rearrangements likely position the tRNA substrate and catalytic residues optimally for the modification reaction to proceed.

How does the MnmE-MnmG complex form and function in tRNA modification?

The MnmE-MnmG complex represents a sophisticated molecular machine responsible for the carboxymethylaminomethyl modification of wobble uridine in tRNA (cmnm5U) . Formation of this complex involves:

• Initial binding of MnmE and MnmG independently to tRNA

• GTP-dependent dimerization of MnmE's G domains

• Conformational changes that promote stable association with MnmG

• Formation of a catalytically active heterotetrameric complex (MnmE2-MnmG2)

The resulting complex creates a reaction chamber where the tRNA wobble uridine is positioned for modification. During the reaction cycle, GTP hydrolysis by MnmE drives conformational changes throughout the complex that are transmitted to MnmG, coordinating the chemistry of the modification process. This includes activation of the C5 position of uridine, transfer of the carboxymethylaminomethyl group, and release of the modified tRNA .

What is the evolutionary conservation of MnmE across bacterial species compared to Pseudomonas syringae?

MnmE represents a highly conserved GTPase family found across bacterial species and even into eukaryotes (where orthologs are targeted to mitochondria) . In Pseudomonas syringae pv. tomato, MnmE maintains the core structural and functional characteristics seen in other bacterial species, including:

• Multi-domain architecture with a conserved G domain for GTP binding and hydrolysis

• Potassium-dependent activation mechanism

• Functional association with MnmG protein

• Role in tRNA modification at the wobble position

Comparative analysis reveals that while the G domain shows high sequence conservation across species (>70% identity in most bacteria), the peripheral domains may exhibit greater variability, reflecting adaptation to specific cellular environments or subtle differences in substrate recognition. The conservation of MnmE across diverse bacterial pathogens like Pseudomonas syringae underscores its fundamental importance in bacterial physiology and potentially in pathogenicity .

What are the optimal protocols for expressing and purifying recombinant MnmE from Pseudomonas syringae pv. tomato?

Successful expression and purification of recombinant MnmE from Pseudomonas syringae pv. tomato requires careful optimization at several steps:

Expression System Selection:

• E. coli BL21(DE3) typically provides high expression levels

• Use of pET-based vectors with T7 promoter offers tight regulation

• Consider codon optimization if rare codons are present in P. syringae sequence

Expression Conditions:

• Induction at OD600 = 0.6-0.8 with 0.5 mM IPTG

• Lower temperature induction (16-18°C overnight) often improves folding

• Supplementation with 5-10 mM KCl can enhance stability due to potassium-dependent activity

Purification Strategy:

• Initial capture via affinity chromatography (His-tag or GST-tag)

• Ion exchange chromatography (typically anion exchange at pH 8.0)

• Size exclusion chromatography for final polishing and buffer exchange

Stabilization Considerations:

• Include 5 mM MgCl2 in all buffers to stabilize nucleotide binding

• Maintain 50-100 mM KCl throughout purification

• Addition of 5-10% glycerol prevents aggregation

• Consider flash-freezing aliquots in liquid nitrogen for long-term storage

This approach typically yields protein with >95% purity suitable for enzymatic, structural, and interaction studies .

How can small-angle X-ray scattering (SAXS) be effectively used to study the MnmE-MnmG complex structure?

SAXS represents a powerful technique for investigating the solution structure and conformational changes of the MnmE-MnmG complex. The methodological approach includes:

Sample Preparation:

• Purify individual MnmE and MnmG proteins to >95% homogeneity

• Form complex by mixing equimolar ratios with gentle overnight incubation at 4°C

• Perform final purification via size exclusion chromatography

• Concentrate to 2-10 mg/ml in low-scattering buffer (typically HEPES or Tris)

Data Collection Parameters:

• Multiple concentrations (2, 5, and 10 mg/ml) to account for concentration-dependent effects

• Temperature control at 10°C to minimize radiation damage

• Include GTP, GDP, and nucleotide-free states to capture conformational cycle

• Collect matched buffer blanks before and after each sample

Analysis Workflow:

• Buffer subtraction and data processing using standard SAXS software (ATSAS)

• Guinier analysis to determine radius of gyration (Rg) and assess sample quality

• Pair-distance distribution function calculation to establish maximum dimension (Dmax)

• Ab initio modeling to generate low-resolution envelopes

• Rigid body modeling using available crystal structures of individual components

• Molecular dynamics flexible fitting to refine models against SAXS data

Conformational Analysis:

• Compare Rg values in different nucleotide states to detect GTP-dependent conformational changes

• Assess complex formation through changes in Dmax

• Develop structural models of the complex in different functional states

This approach provides valuable insights into the structural dynamics of the complex that complement crystallographic and cryo-EM studies.

What approaches are most effective for measuring the GTPase activity of MnmE in vitro?

Multiple complementary assays can be employed to measure the GTPase activity of recombinant MnmE from Pseudomonas syringae pv. tomato:

Colorimetric Phosphate Detection:

• Malachite green assay - Measures released inorganic phosphate through color change

  • Sensitivity: 0.1-10 nmol phosphate

  • Linear range: 0.1-10 μM phosphate

  • Add 100 μl malachite green reagent to 100 μl reaction, measure absorbance at 630 nm

Coupled Enzyme Assays:

• NADH-coupled assay

  • Principle: GTP hydrolysis → GDP → regeneration of GTP via pyruvate kinase and lactate dehydrogenase with oxidation of NADH

  • Monitor decrease in absorbance at 340 nm

  • Real-time continuous measurement possible

Direct Nucleotide Analysis:

• HPLC separation of GTP and GDP

  • Use C18 reverse phase column with ion-pairing reagent

  • UV detection at 254 nm

  • Quantify GDP/GTP ratio

Radioactive Assays:

• [γ-32P]GTP hydrolysis

  • Highly sensitive for low activity measurements

  • Separate [32P]Pi from [γ-32P]GTP by thin-layer chromatography

  • Quantify by phosphorimaging

Optimization Parameters:

• Test multiple K+ concentrations (0-200 mM) to determine optimal activation

• Evaluate effects of Mg2+ (typically 5 mM optimal)

• Assess temperature dependence (usually 25-37°C optimal)

• Determine pH optimum (typically pH 7.5-8.0)

For most research applications, the malachite green assay provides sufficient sensitivity while being technically accessible and cost-effective .

How does recombination affect the evolution of the MnmE gene in Pseudomonas syringae strains?

Recombination plays a crucial role in the evolution of genes in Pseudomonas syringae, including those involved in basic cellular processes like tRNA modification. Analysis of Pseudomonas syringae population structures reveals:

• Recombination contributes more significantly than mutation to genetic variation between isolates, contrary to previous assumptions about clonality in bacterial plant pathogens .

• For genes like MnmE, recombination can lead to:

  • Introduction of novel functional variants from related species

  • Repair of deleterious mutations

  • Optimization of gene function for specific host environments

• Multiple recombination breakpoints are detectable within sequenced gene fragments across the P. syringae genome, suggesting active genetic exchange .

• Population genetic tests applied to housekeeping genes (which could include MnmE) indicate a higher recombination rate than previously appreciated in Pseudomonas species .

Methodological approaches to detect recombination in MnmE include:

• Sequence-based methods like RDP4 or GARD for breakpoint detection

• Population genetic tests such as the phi test or Tajima's D

• Phylogenetic incongruence analyses comparing MnmE trees with other housekeeping genes

Understanding recombination patterns in MnmE can provide insights into how fundamental cellular processes are maintained or adapted during the evolution of plant pathogenicity in different P. syringae strains .

What role might tRNA modifications by MnmE play in host adaptation of Pseudomonas syringae pv. tomato?

The tRNA modification activities of MnmE may contribute significantly to host adaptation in Pseudomonas syringae pv. tomato through several mechanisms:

• Translational Efficiency Regulation

  • MnmE-mediated tRNA modifications at the wobble position enhance translational efficiency and accuracy, particularly for specific codons

  • Different host environments may exert distinct translational demands, requiring adaptation of the tRNA modification profile

  • Host adaptation may involve optimization of translation for host-specific virulence factors

• Stress Response Modulation

  • tRNA modifications can regulate bacterial stress responses through selective translation of stress-response proteins

  • Plant defense responses create distinct stress environments (oxidative, pH, antimicrobial compounds)

  • Modulation of MnmE activity could allow rapid adaptation to host-induced stress

• Virulence Factor Expression Control

  • Type III secretion system (T3S) effectors, critical for pathogenicity, may have codon usage patterns that depend on MnmE-modified tRNAs

  • The unusual host range of PtoDC3000 (including both Solanaceae and Brassicaceae plants) may be partially enabled by optimized translation of specific effector proteins

• Host Range Determination

  • P. syringae pv. tomato strain DC3000 exhibits an unusually wide host range compared to other tomato isolates

  • This could potentially relate to optimized translation through tRNA modifications

  • Efficient expression of a broader range of effector proteins might enable infection of diverse plant species

Methodological approaches to investigate this connection include:

• Comparative tRNA modification profiling across strains with different host specificities

• Mutational analysis of MnmE and assessment of host range impacts

• Ribosome profiling to identify differentially translated genes in MnmE mutants

• Correlation of codon usage patterns in virulence factors with tRNA modification activities

These investigations could reveal how fundamental cellular processes like tRNA modification contribute to pathogenicity and host adaptation in this economically important plant pathogen .

How do potassium levels affect MnmE activity in planta during Pseudomonas syringae infection?

The potassium-dependent activation of MnmE GTPase activity presents an intriguing connection between bacterial physiology and the plant infection environment:

Potassium Dynamics During Infection:

• Plant Defense Responses

  • Potassium efflux is a known early response to pathogen attack in plants

  • K+ concentrations in the apoplast fluctuate during infection (typically 10-100 mM range)

  • Guard cell K+ fluxes control stomatal closure, a key defense against bacterial entry

• Bacterial Response Mechanisms

  • MnmE activity is highly sensitive to K+ concentrations, with half-maximal activation typically at 20-40 mM K+

  • Fluctuations in local K+ concentrations could modulate MnmE-dependent tRNA modification

  • This may serve as a regulatory mechanism linking environmental sensing to translation control

Experimental Approaches to Study This Relationship:

• In Planta K+ Measurement During Infection

  • Use K+-selective microelectrodes to measure apoplastic K+ concentrations during infection

  • Apply non-invasive ion flux measurement techniques (MIFE) to detect dynamic changes

  • Correlate with bacterial population growth and disease progression

• MnmE Activity Profiling

  • Develop reporters for MnmE activity based on:

    • Expression of tRNA modification-dependent genes

    • Direct measurement of modified tRNA nucleosides by LC-MS/MS

  • Compare activity in different plant tissues and infection stages

• Genetic Manipulation Approaches

  • Engineer MnmE variants with altered K+ sensitivity

  • Create plant lines with manipulated K+ homeostasis

  • Assess impacts on bacterial virulence and tRNA modification patterns

This research direction could reveal novel connections between environmental sensing and the regulation of basic cellular processes during plant-pathogen interactions .

What conformational changes occur in MnmE during its GTPase cycle and how do they affect tRNA modification?

The GTPase cycle of MnmE involves dramatic conformational changes that are essential for its tRNA modification function:

Conformational States During GTPase Cycle:

• Nucleotide-Free State

  • G domains remain separated and flexible

  • N-terminal domains maintain distance from each other

  • Relatively open conformation with accessible nucleotide binding site

• GTP-Bound State (Before Hydrolysis)

  • Initial GTP binding causes local conformational changes

  • K+-dependent dimerization of G domains brings monomers into close proximity

  • Formation of complete active site at the dimer interface

• Transition State (During GTP Hydrolysis)

  • Maximum compaction of the structure

  • Close association of G domains

  • Catalytic residues optimally positioned for GTP hydrolysis

• GDP-Bound State (After Hydrolysis)

  • Partial relaxation of the compact structure

  • Maintained dimerization with altered interface geometry

  • Changed positions of C-terminal domains

Functional Implications for tRNA Modification:

The conformational cycle directly coordinates the tRNA modification process:

• In the open conformation, the MnmE-MnmG complex can recruit the tRNA substrate

• GTP binding and K+-dependent dimerization create a catalytically active complex

• The conformational changes position the tRNA wobble uridine optimally in the active site

• The transition state conformation activates the modification chemistry

• GTP hydrolysis may provide energy for the chemistry or conformational work

• Return to open state after GDP release allows product release and cycle reset

Methodological Approaches to Study Conformational Changes:

• Small-angle X-ray scattering (SAXS) to capture solution conformations

• Fluorescence resonance energy transfer (FRET) between labeled domains

• Hydrogen-deuterium exchange mass spectrometry to map flexible regions

• Electron paramagnetic resonance (EPR) with site-specific spin labels

• Single-molecule FRET to observe conformational dynamics in real-time

These sophisticated biophysical approaches reveal how MnmE uses the energy of GTP hydrolysis to drive the complex tRNA modification reaction through precisely coordinated structural rearrangements .

How can cryo-electron microscopy be optimized for structural analysis of the MnmE-MnmG-tRNA complex?

Cryo-electron microscopy (cryo-EM) represents a powerful approach for visualizing the complete MnmE-MnmG-tRNA complex in different functional states, but requires careful optimization:

Sample Preparation Optimization:

• Complex Formation

  • Purify individual components (MnmE, MnmG, tRNA) to high homogeneity (>95%)

  • Form stable complex by mixing in optimal ratios (typically 2:2:1 MnmE:MnmG:tRNA)

  • Include appropriate nucleotides (GTP, non-hydrolyzable GTP analogs, or GDP)

  • Stabilize with chemical crosslinking if necessary (test GraFix method)

• Grid Preparation

  • Test multiple grid types (Quantifoil R1.2/1.3, UltrAuFoil)

  • Optimize blotting parameters (time, force, humidity)

  • Consider glow discharge or plasma cleaning settings

  • Evaluate additives to optimize particle distribution (detergents like OG at 0.01-0.05%)

Data Collection Strategies:

• Collection Parameters

  • Use energy filters to improve signal-to-noise ratio

  • Collect at 300kV with direct electron detector

  • Frame rate: 40 frames per exposure, total dose ~40-60 e-/Ų

  • Defocus range: -0.8 to -2.5 μm

• Conformational States

  • Collect datasets in different nucleotide states:

    • Apo (nucleotide-free)

    • GTP-bound (use GTPγS or GppNHp)

    • GDP-bound (post-hydrolysis state)

  • Consider time-resolved approaches if conformational changes are rapid

Data Processing Workflow:

• Initial Processing

  • Motion correction with dose weighting

  • CTF estimation with CTFFIND4 or Gctf

  • Particle picking using reference-free approaches initially

• Classification Strategy

  • Extensive 2D classification to identify heterogeneity

  • Ab initio model generation without symmetry constraints

  • 3D classification to separate conformational states

  • Consider multi-body refinement for domains with independent movement

• Final Refinement

  • High-resolution refinement with CTF refinement and particle polishing

  • Local resolution estimation

  • Map sharpening and filtering

Challenges and Solutions:

With this optimized approach, resolutions of 3-4 Å should be achievable for the complete functional complex, providing unprecedented insights into the structural basis of tRNA modification .

What are the molecular determinants of tRNA recognition by the MnmE-MnmG complex in Pseudomonas syringae?

Understanding how the MnmE-MnmG complex specifically recognizes and modifies tRNA substrates involves delineating multiple molecular interaction determinants:

tRNA Structural Elements Critical for Recognition:

Protein Domains Involved in tRNA Binding:

• MnmE Contributions

  • N-terminal domain likely provides tRNA body contacts

  • G domain dimerization creates part of the tRNA binding pocket

  • C-terminal domain may interact with anticodon stem

• MnmG Contributions

  • FAD-binding domain provides structural framework

  • NADH-binding domain may interact with anticodon loop

  • C-terminal domain forms part of the active site around U34

Methodological Approaches to Define Recognition Determinants:

• Mutational Analysis

  • Systematic mutagenesis of potential RNA-binding residues

  • tRNA variant testing to identify critical recognition elements

  • Charge-reversal mutations to test electrostatic interactions

• Structural Studies

  • High-resolution cryo-EM of the complete complex

  • X-ray crystallography of complex with tRNA anticodon stem-loop

  • NMR studies of specific domain-RNA interactions

• Biophysical Interaction Analysis

  • Microscale thermophoresis to measure binding affinities

  • Hydrogen-deuterium exchange to map interaction surfaces

  • RNA footprinting to identify protected nucleotides

• Computational Approaches

  • Molecular dynamics simulations of complex formation

  • Electrostatic potential mapping to identify interaction hotspots

  • RNA-protein docking with experimental constraints

Understanding these recognition determinants will provide crucial insights into the molecular basis of tRNA modification specificity and may reveal potential targets for antimicrobial development against Pseudomonas syringae .

How can understanding MnmE function in Pseudomonas syringae contribute to developing novel plant disease control strategies?

The essential role of MnmE in tRNA modification presents several potential avenues for developing innovative plant disease control strategies against Pseudomonas syringae:

Potential Intervention Approaches:

• Small Molecule Inhibitor Development

  • Target the unique potassium-dependent GTPase mechanism

  • Design compounds that interfere with MnmE-MnmG complex formation

  • Exploit structural differences between bacterial and plant homologs

  • Focus on allosteric inhibitors that disrupt conformational cycling

• Genetic Resistance Strategies

  • Engineer plant decoy proteins that mimic tRNA structures

  • Develop transgenic plants expressing inhibitory RNA aptamers

  • Create plant lines with altered potassium homeostasis in the apoplast

• Biological Control Applications

  • Develop competing bacterial strains with modified MnmE that interfere with pathogen colonization

  • Design phage-based delivery of inhibitory proteins targeting MnmE function

Developmental Pathway for MnmE-Targeting Interventions:

• Target Validation

  • Confirm essentiality of MnmE for virulence in greenhouse and field conditions

  • Demonstrate lack of off-target effects on beneficial microbiota

  • Establish relationship between tRNA modification and pathogenicity

• Screening Approaches

  • Develop high-throughput GTPase activity assays

  • Design phenotypic screens in planta using reporter systems

  • Implement fragment-based drug discovery against the MnmE active site

• Efficacy Testing

  • Evaluate impact on bacterial growth, colonization, and symptoms

  • Test in multiple plant species under varied environmental conditions

  • Compare with conventional bactericides for effectiveness and resistance development

Potential Advantages of MnmE-Based Strategies:

While technical challenges remain in delivery and stability of potential interventions, the fundamental role of MnmE in bacterial physiology makes it a promising target for next-generation disease control approaches that could reduce reliance on conventional bactericides in agriculture .

What biotechnological applications could utilize recombinant MnmE from Pseudomonas syringae pv. tomato?

Recombinant MnmE from Pseudomonas syringae pv. tomato offers several unique properties that could be exploited for biotechnological applications:

Potential Biotechnological Applications:

• Synthetic Biology Tools

  • Use as an inducible molecular switch responding to potassium levels

  • Develop as a component of synthetic gene circuits using GTP hydrolysis as a signal

  • Create MnmE-based biosensors for potassium in agricultural or environmental monitoring

• RNA Modification Technology

  • Engineer MnmE-MnmG systems to install specific modifications on designer tRNAs

  • Develop tools for studying translation regulation through controlled tRNA modification

  • Create systems for site-specific RNA labeling using modified nucleotides

• Structural Biology Applications

  • Utilize as a model system for studying GTPase mechanisms

  • Develop as a crystallization chaperone for difficult-to-crystallize proteins

  • Create potassium-responsive biomaterials with MnmE as the sensing component

Technical Implementation Approaches:

• Protein Engineering Strategies

  • Create chimeric proteins with altered specificity or activity

  • Develop split-MnmE systems for protein complementation assays

  • Engineer variants with enhanced stability or altered nucleotide specificity

• Expression and Production Systems

  • Optimize high-yield expression in heterologous hosts

  • Develop purification strategies for large-scale production

  • Create immobilization techniques for enzyme reuse

• Formulation and Delivery

  • Develop stabilization methods for long-term storage

  • Create encapsulation systems for controlled release

  • Design immobilization matrices for continuous-flow applications

Proof-of-Concept Projects:

• MnmE-Based Potassium Biosensor

• Synthetic tRNA Modification System

These applications leverage the unique structural and enzymatic properties of MnmE while addressing important needs in biotechnology and synthetic biology fields .