Recombinant Pseudomonas syringae pv. tomato Probable tRNA threonylcarbamoyladenosine biosynthesis protein Gcp (gcp)

What is the threonylcarbamoyladenosine biosynthesis protein Gcp in Pseudomonas syringae pv. tomato?

The threonylcarbamoyladenosine biosynthesis protein Gcp in Pseudomonas syringae pv. tomato is a critical enzyme involved in the synthesis of threonylcarbamoyladenosine (t6A), a universally conserved tRNA modification found in all three kingdoms of life. This protein likely functions similarly to the TsaD/Kae1/Qri7 protein family characterized in other organisms. In bacteria, the t6A modification plays an essential role in translation fidelity by serving as a determinant for aminoacylation of tRNA by bacterial-type isoleucyl-tRNA synthetases. Unlike in eukaryotes, where t6A modification may be dispensable in some contexts, this modification pathway appears to be essential in most prokaryotes, including Pseudomonas species .

Why is studying Gcp in P. syringae pv. tomato particularly valuable for researchers?

Studying Gcp in P. syringae pv. tomato is particularly valuable because this pathogen serves as an ideal model system at the intersection of molecular microbiology and plant pathology. P. syringae pv. tomato DC3000 has emerged as a premier bacterial model for studying plant-pathogen interactions due to its ability to infect both tomato and the model plant Arabidopsis thaliana. This dual-host capability enables researchers to leverage the genetic and genomic tools available for Arabidopsis while studying a pathogen of agricultural importance. The essential nature of tRNA modifications in bacteria makes Gcp a potential target for antimicrobial development, while its role in bacterial physiology may influence pathogenicity mechanisms that are extensively studied in this particular pathovar .

What recombineering approaches are most effective for creating targeted mutations in the gcp gene of P. syringae pv. tomato?

For creating targeted mutations in the gcp gene of P. syringae pv. tomato, a recombineering approach based on the RecTE system from P. syringae pv. syringae B728a has proven highly effective. This method exploits homologous recombination between genomic loci and linear DNA substrates introduced directly into P. syringae cells via electroporation.

The methodology involves:

• Expressing the RecT and RecE homologs from P. syringae pv. syringae B728a in the target strain (P. syringae pv. tomato DC3000)

• Designing linear DNA substrates with homology arms flanking the target region of the gcp gene

• Introducing these substrates via electroporation

• Selecting for recombinants using appropriate markers

For single-stranded DNA oligonucleotides, the P. syringae RecT homolog alone is sufficient to promote recombination, while efficient recombination of double-stranded DNA requires expression of both RecT and RecE homologs. This system can be delivered using vectors like pUCP24/47, which contains the Bacillus subtilis sacB gene as a counterselectable marker to expedite plasmid elimination after recombination .

Given the likely essential nature of gcp, constructing conditional mutants rather than complete knockouts may be necessary unless creating specific point mutations that maintain function while altering specific properties of interest.

What are the optimal conditions for expressing and purifying recombinant Gcp protein from P. syringae pv. tomato for in vitro studies?

For optimal expression and purification of recombinant Gcp protein from P. syringae pv. tomato, the following methodological approach is recommended:

Expression System Selection:

• E. coli BL21(DE3) strain provides high-level expression while minimizing proteolytic degradation

• Vector considerations: pET28a(+) with an N-terminal His-tag facilitates purification while minimizing interference with protein function

Expression Conditions:

• Culture medium: LB supplemented with appropriate antibiotics

• Induction parameters: 0.5 mM IPTG at OD600 of 0.6-0.8

• Post-induction incubation: 16-18 hours at 18°C (lower temperature improves protein solubility)

Lysis and Purification Protocol:

• Harvest cells by centrifugation (6,000 × g, 15 min, 4°C)

• Resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol)

• Lyse cells using sonication or French press

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Purify using Ni-NTA affinity chromatography with stepwise imidazole elution

• Further purify via size exclusion chromatography using a Superdex 200 column

Storage Conditions:

• Store purified protein in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

• Flash-freeze aliquots in liquid nitrogen and store at -80°C

The purification protocol may need adjustment based on the specific properties of P. syringae Gcp, which shares functional characteristics with t6A synthesis proteins from other organisms .

How can researchers design experiments to study the role of Gcp in tRNA modification within P. syringae pv. tomato?

To study the role of Gcp in tRNA modification within P. syringae pv. tomato, researchers should employ a multi-faceted experimental approach:

1. Genetic Approaches:

• Construct conditional mutants using inducible promoters if gcp is essential

• Create point mutations in conserved domains to generate hypomorphic alleles

• Complement mutations with wild-type or variant gcp genes to confirm specificity

• Use the P. syringae RecTE recombineering system for precise genomic modifications

2. Biochemical Characterization:

• Isolate tRNA from wild-type and mutant strains

• Analyze tRNA modifications using liquid chromatography-mass spectrometry (LC-MS/MS)

• Develop an in vitro t6A synthesis assay using purified components

• Assess tRNA aminoacylation efficiency using purified isoleucyl-tRNA synthetase

3. Physiological Impact Assessment:

• Monitor growth rates under various conditions (temperature, pH, osmotic stress)

• Analyze proteome changes using quantitative mass spectrometry

• Examine translation fidelity using reporter constructs

• Test bacterial fitness in planta using tomato and Arabidopsis infection models

4. Structural Studies:

• Determine protein structure using X-ray crystallography or cryo-EM

• Identify binding partners through pull-down assays coupled with mass spectrometry

• Perform molecular docking to understand substrate interactions

This comprehensive approach would provide insights into both the molecular function of Gcp in tRNA modification and its broader physiological significance in P. syringae pv. tomato biology and pathogenicity.

How can researchers resolve data inconsistencies when analyzing the effects of Gcp mutations on P. syringae phenotypes?

When confronting data inconsistencies in Gcp mutation studies, researchers should implement a systematic approach to identify and resolve contradictions:

Step 1: Data Validation and Quality Control

• Apply statistical tests to identify outliers in phenotypic measurements

• Implement the R-based validation approach using dplyr to group data by experimental conditions and identify inconsistent values within groups

• Verify experimental controls performed as expected across all replicates

Step 3: Investigate Potential Sources of Inconsistency

• Examine genetic stability of mutants (secondary mutations)

• Evaluate environmental variation between experiments

• Consider strain-specific effects if using different P. syringae isolates

• Assess the impact of growth phase on phenotype expression

Step 4: Resolution Strategies

• Increase biological replicates focusing on conditions with inconsistencies

• Implement more stringent controls for environmental variables

• Use complementation studies to confirm phenotype causality

• Apply dose-dependent or time-course approaches to capture phenotypic transitions

This structured approach transforms data inconsistencies from experimental liabilities into opportunities for deeper biological insights about Gcp function in P. syringae .

What statistical approaches are most appropriate for analyzing the impact of Gcp on bacterial growth and virulence?

For analyzing the impact of Gcp on bacterial growth and virulence, several statistical approaches are particularly appropriate:

1. Growth Curve Analysis:

• Model: Fit growth data to a modified Gompertz model or logistic growth equation

• Parameters: Compare lag phase duration, maximum growth rate, and carrying capacity

• Analysis of Variance (ANOVA): Use repeated measures ANOVA with post-hoc tests to compare growth parameters between wild-type and mutant strains

• Non-linear Mixed Effects Models: Account for both fixed effects (strain, treatment) and random effects (experimental replicates)

2. Virulence Assays (in planta):

• Survival Analysis: Use Kaplan-Meier curves and log-rank tests to analyze time-to-symptom data

• Disease Severity Indices: Apply ordinal logistic regression for categorical disease ratings

• Bacterial Population Studies: Employ non-parametric tests (Mann-Whitney U) for CFU data that typically shows non-normal distribution

• Area Under Disease Progress Curve (AUDPC): Calculate and compare using parametric tests after confirming normality

3. High-dimensional Data Analysis:

• Principal Component Analysis (PCA): Reduce dimensionality when examining multiple phenotypic variables

• Hierarchical Clustering: Group similar phenotypes to identify patterns

• Linear Discriminant Analysis (LDA): Maximize separation between wild-type and mutant phenotypic profiles

4. Advanced Statistical Considerations:

• Multiple Testing Correction: Apply Benjamini-Hochberg procedure to control false discovery rate

• Power Analysis: Determine appropriate sample sizes to detect biologically meaningful differences

• Bayesian Approaches: Incorporate prior knowledge about tRNA modification systems when available

How can researchers effectively distinguish between direct effects of Gcp mutation and secondary consequences in omics data?

Distinguishing between direct effects of Gcp mutation and secondary consequences in omics data requires a sophisticated experimental design and analytical framework:

Experimental Design Strategies:

• Time-resolved Sampling: Collect samples at multiple time points after Gcp depletion/inactivation to capture primary versus secondary effects

• Conditional Expression Systems: Use tunable promoters to create graded reductions in Gcp activity rather than binary comparisons

• Parallel Multi-omics: Simultaneously analyze transcriptome, proteome, and metabolome to build comprehensive response networks

• Targeted Mutations: Compare effects of catalytic site mutations versus complete gene deletion

Analytical Framework:

• Network Analysis Pipeline:

  • Construct protein-protein interaction networks centered on Gcp

  • Apply pathway enrichment analysis to identify overrepresented functional categories

  • Use directed acyclic graphs to infer causal relationships between observed changes

• Temporal Sequence Mapping:

  • Plot the emergence of changes in a temporal sequence

  • Earlier changes are more likely direct effects; later changes represent downstream consequences

  • Apply mathematical modeling to predict the propagation of effects through cellular networks

• Integration with Known tRNA Modification Networks:

  • Compare observed changes with effects documented for other tRNA modification defects

  • Identify signature patterns specific to t6A deficiency versus general translational stress

• Specific Analysis for tRNA Modification Effects:

  • Codon usage bias analysis to identify transcripts vulnerable to t6A deficiency

  • Ribosome profiling to detect translation efficiency changes at specific codons

  • Proteomics focusing on mistranslation products

This integrated approach enables researchers to construct a causality map distinguishing direct molecular consequences of Gcp dysfunction from adaptive responses and systemic disruptions, providing deeper insights into the functional role of t6A modifications in P. syringae .

How might understanding Gcp function in P. syringae pv. tomato contribute to developing novel antimicrobial strategies?

Understanding Gcp function in P. syringae pv. tomato could contribute significantly to novel antimicrobial development through multiple strategic pathways:

Exploiting Essential Nature for Targeted Antimicrobials:
The essentiality of Gcp and t6A biosynthesis in most prokaryotes but not in eukaryotes presents an attractive target for narrow-spectrum antimicrobials. This differential essentiality provides a selectivity window for targeting bacterial pathogens while minimizing host toxicity. Structure-based drug design using solved Gcp structures could facilitate the development of small-molecule inhibitors that disrupt t6A biosynthesis specifically in prokaryotic systems .

Targeting Translation Fidelity Mechanisms:
Gcp's role in ensuring translational fidelity through t6A modification represents a novel vulnerability. Compounds that selectively interfere with bacterial t6A-dependent tRNA recognition by isoleucyl-tRNA synthetases could induce proteotoxic stress by increasing mistranslation rates. This strategy differs from traditional antibiotics by exploiting quality control mechanisms rather than directly inhibiting essential processes, potentially circumventing established resistance mechanisms .

Bacteriophage-Based Biocontrol Applications:
The discovery of novel bacteriophages associated with P. syringae strains presents opportunities for phage therapy approaches. The phage described in research with P. syringae from ornamental pear trees demonstrated efficacy in symptom mitigation regardless of the Pseudomonas strain tested. Engineering phages to specifically target virulence factors or essential genes like gcp could enhance their efficacy as biocontrol agents against plant pathogens .

Phage-Antibiotic Synergy Exploration:
Combining Gcp inhibitors with bacteriophages could yield synergistic effects through complementary mechanisms of action. This combined approach might reduce the emergence of resistance while enhancing efficacy against established infections. The unique nature of the P. syringae RecTE recombineering system could facilitate rapid testing of such combination approaches in relevant model systems .

These approaches leverage fundamental understanding of Gcp function to address the growing challenge of antimicrobial resistance while potentially providing new tools for agricultural disease management .

What are the challenges and potential solutions for studying the impact of Gcp on the P. syringae effectorome and type III secretion system?

Studying the impact of Gcp on the P. syringae effectorome and type III secretion system presents significant challenges along with potential methodological solutions:

Challenges:

• Essentiality Constraint: The likely essential nature of Gcp makes traditional knockout approaches problematic for studying its influence on virulence systems.

• Indirect Effects vs. Direct Regulation: Distinguishing between direct regulatory roles of Gcp and indirect effects caused by general translation perturbation is conceptually and technically challenging.

• Temporal Dynamics: Type III secretion system (T3SS) expression and effector deployment occur in precise temporal sequences that may be difficult to capture when studying tRNA modification effects.

• Host-Dependent Expression: P. syringae effectorome expression is highly responsive to host factors, requiring complex in planta experimental systems.

Methodological Solutions:

• Conditional Expression Systems:

  • Develop tightly regulated inducible/repressible promoter systems for gcp

  • Create partial loss-of-function alleles through targeted mutagenesis

  • Apply CRISPR interference (CRISPRi) for tunable repression without complete gene deletion

• High-Resolution Temporal Analysis:

  • Implement time-course RNA-seq and proteomics after Gcp depletion

  • Use reporter fusions to key T3SS genes to monitor expression dynamics

  • Apply single-cell approaches to capture population heterogeneity in T3SS expression

• Codon-Based Analysis:

  • Examine codon usage patterns in effector genes that might depend on t6A modification

  • Create synonymous variants of effector genes with altered codon bias

  • Analyze translation efficiency and accuracy of effector proteins using ribosome profiling

• Advanced In Planta Systems:

  • Develop leaf microfluidic devices for real-time imaging of bacterial behavior

  • Apply FRET-based sensors to monitor effector translocation efficiency

  • Use plant tissue-specific induction of Gcp repression in bacteria

These approaches would enable researchers to dissect the complex interplay between tRNA modification by Gcp and the sophisticated virulence mechanisms that make P. syringae pv. tomato an effective plant pathogen and important model organism .

How can comparative genomics and phylogenetics enhance our understanding of Gcp evolution and function across Pseudomonas species?

Comparative genomics and phylogenetics provide powerful frameworks for understanding Gcp evolution and function across Pseudomonas species through several integrated approaches:

Phylogenomic Framework Construction:
Developing a robust phylogenetic framework of Pseudomonas species serves as the foundation for evolutionary analysis of Gcp. This requires whole-genome sequencing of diverse isolates, multilocus sequence analysis, and core genome alignment methodologies similar to those used in characterizing P. syringae isolates from Callery pears. Such analyses have already revealed the complex evolutionary history of the P. syringae species complex and identified distinct phylogroups with different host specificities .

Gcp Sequence-Structure-Function Analysis:
A comprehensive comparison of Gcp protein sequences across Pseudomonas species can identify:

• Universally conserved residues essential for catalytic function

• Lineage-specific variations that might reflect adaptation to different niches

• Selection pressures acting on different domains of the protein

• Co-evolution patterns with interacting partners in the t6A modification pathway

Genomic Context Conservation:
Analysis of the genomic neighborhood of gcp across Pseudomonas genomes can reveal:

• Operon structures and potential co-regulated genes

• Evidence of horizontal gene transfer events

• Regulatory elements that might influence expression patterns

• Functional associations through conserved genomic proximity

Correlation with Host Range and Virulence Profiles:
By mapping Gcp sequence variations onto established phylogenies and correlating with host range data:

• Researchers can identify Gcp variants associated with specific host preferences

• Potential connections between tRNA modification efficiency and virulence can be explored

• Adaptive evolution of translation quality control mechanisms in different ecological niches can be assessed

Table 1: Comparative Analysis of Gcp Conservation Across Pseudomonas Phylogroups

This integrated comparative approach can reveal how Gcp function has evolved in conjunction with the diversification of Pseudomonas species into various ecological niches and pathogenic lifestyles, providing insights into both fundamental mechanisms of translation quality control and the evolution of bacterial pathogenicity .

What is the recommended protocol for monitoring t6A modification levels in tRNAs from P. syringae strains with altered Gcp expression?

Protocol for Monitoring t6A Modification Levels in P. syringae tRNAs

Materials Required:

• P. syringae cultures (wild-type and Gcp-modified strains)

• RNase-free reagents and equipment

• Nuclease P1, Bacterial Alkaline Phosphatase

• HPLC system with C18 column

• LC-MS/MS system with electrospray ionization

Procedure:

1. tRNA Isolation and Purification:

• Harvest cells in mid-log phase (OD600 0.5-0.7)

• Extract total RNA using TRIzol reagent followed by DNase treatment

• Enrich tRNA fraction using size exclusion chromatography

• Verify tRNA quality by Bioanalyzer or urea-PAGE analysis

2. tRNA Hydrolysis to Nucleosides:

• Denature 10-20 μg purified tRNA at 95°C for 5 minutes

• Rapidly cool on ice for 5 minutes

• Add nuclease P1 (1U/10 μg RNA) in 10 mM ammonium acetate (pH 5.3)

• Incubate at 45°C for 2 hours

• Add bacterial alkaline phosphatase (1U/10 μg RNA) in dephosphorylation buffer

• Incubate at 37°C for 1 hour

• Remove enzymes by filtration (10 kDa MWCO)

3. HPLC Separation and Quantification:

• Inject hydrolyzed sample onto C18 reverse-phase column

• Use gradient elution: Buffer A (5 mM ammonium acetate, pH 5.3) to Buffer B (40% acetonitrile)

• Monitor absorbance at 254 nm

• Collect relevant fractions for further LC-MS/MS analysis

4. LC-MS/MS Analysis:

• Configure MS/MS for multiple reaction monitoring (MRM)

• Target parent ion m/z: 413.1 (t6A)

• Monitor diagnostic fragment ions: m/z 281.1 and 136.1

• Quantify t6A relative to canonical nucleosides using calibration curves

• Calculate modification index as (t6A/A) × 1000

5. Data Analysis and Interpretation:

• Compare t6A levels between wild-type and Gcp-modified strains

• Normalize to total tRNA concentration

• Analyze specific tRNA species separately if needed using tRNA-specific RT-PCR

Troubleshooting Guide:

This protocol enables precise quantification of t6A levels, allowing researchers to correlate Gcp function with tRNA modification status in P. syringae strains .

How can researchers optimize recombineering efficiency when working with the gcp gene in P. syringae pv. tomato?

Optimizing Recombineering Efficiency for gcp Manipulation in P. syringae pv. tomato

The efficient genetic manipulation of the essential gcp gene requires maximizing recombineering efficiency while accommodating the constraints of targeting an essential gene. Based on established RecTE systems from P. syringae, researchers can optimize the following parameters:

Vector System Optimization:

• Use the pUCP24/47 vector system expressing the P. syringae RecTE homologs

• Ensure the vector contains the B. subtilis sacB gene for counterselection

• Consider using temperature-sensitive plasmid variants for transient RecTE expression

• Optimize promoter strength to balance RecTE expression with potential toxicity

DNA Substrate Design Principles:

• For point mutations: Use 60-100 nucleotide ssDNA oligonucleotides with the mutation centered

• For gene replacements: Include 50-80 bp homology arms flanking the target region

• Target the lagging strand of DNA replication for ssDNA recombineering

• Minimize secondary structures in oligonucleotide design

• When targeting essential genes like gcp, design substrates that maintain function (silent mutations or conservative amino acid changes)

Electroporation Parameter Optimization:

• Cell preparation: Harvest cells at mid-log phase (OD600 0.4-0.6)

• Wash cells 3× in ice-cold 300 mM sucrose

• DNA concentration: 100-500 ng for dsDNA, 0.5-5 μM for ssDNA

• Pulse settings: 2.5 kV, 25 μF, 200 Ω (optimize for P. syringae pv. tomato)

• Recovery: 3 hours in SOC medium at 28°C before selection

Selection Strategy for Essential Gene Modification:

• Two-step replacement strategy:

  • First introduce a merodiploid state (second copy of gcp)

  • Then modify or delete the original copy

  • Finally remove the supplementary copy if viable

• Conditional approach:

  • Introduce an inducible promoter upstream of gcp

  • Maintain expression during recombineering

  • Test viability under repressing conditions

Efficiency Enhancement Techniques:

• Transient inactivation of the mismatch repair system

• Optimization of RecTE expression timing relative to substrate introduction

• Use of single-stranded DNA binding proteins to protect oligonucleotides

• Implementation of CRISPR-Cas9 to increase selection pressure for desired recombinants

Verification Protocols:

• Colony PCR with primers flanking the modification site

• Restriction digestion if the modification creates/removes a site

• Sanger sequencing to confirm precise modifications

• Whole genome sequencing to verify absence of off-target modifications

This optimized protocol leverages the natural recombination machinery of P. syringae while accommodating the challenges of manipulating an essential gene like gcp .

What are the key considerations for designing in vivo assays to assess the impact of Gcp mutations on P. syringae virulence in tomato and Arabidopsis?

Key Considerations for Designing In Vivo Virulence Assays for Gcp Mutants

When assessing how Gcp mutations affect P. syringae virulence in plant models, researchers must carefully design experiments that account for both the essential nature of Gcp and the complex plant-pathogen interaction dynamics:

Plant System Selection and Preparation:

• Host Selection Strategy:

  • Test both tomato (natural host) and Arabidopsis (model system)

  • Include resistant and susceptible cultivars/ecotypes

  • Consider age-dependent susceptibility differences

• Growth Conditions Standardization:

  • Maintain consistent light intensity (150-200 μmol·m⁻²·s⁻¹)

  • Control temperature (21-23°C) and humidity (60-70%)

  • Synchronize plant development stage (4-5 week old plants)

  • Minimize abiotic stress factors that could confound results

Bacterial Strain Construction Considerations:

• Mutation Design for Essential Genes:

  • Create conditional or partial loss-of-function alleles

  • Use site-directed mutagenesis targeting conserved residues

  • Implement tunable expression systems (tetracycline-responsive)

  • Always include complemented strains to confirm phenotype specificity

• Strain Validation Requirements:

  • Verify growth characteristics in vitro before plant inoculation

  • Confirm stability of mutations during passage

  • Evaluate t6A modification levels in each strain

  • Assess Type III secretion system functionality in inducing conditions

Inoculation Methods and Parameters:

• For Arabidopsis Assays:

  • Syringe infiltration: Use 1×10⁵ CFU/ml for symptom development

  • Spray inoculation: Use 1×10⁸ CFU/ml with 0.02% Silwet L-77

  • Dip inoculation: 1×10⁶ CFU/ml with 0.02% Silwet L-77

• For Tomato Assays:

  • Syringe infiltration: 1×10⁴-10⁵ CFU/ml for symptom development

  • Dip inoculation for seedlings: 1×10⁶ CFU/ml

  • Use multiple inoculation techniques to assess different aspects of pathogenicity

Data Collection and Analysis Framework:

• Quantitative Measurements:

  • Bacterial population dynamics (in planta growth curves over 0-7 days)

  • Lesion area measurement (digital image analysis)

  • Chlorophyll fluorescence (Fv/Fm) to assess photosystem II efficiency

  • Electrolyte leakage as indicator of membrane damage

• Molecular Responses:

  • Expression of plant defense genes (PR1, PDF1.2)

  • Reactive oxygen species accumulation (DAB staining)

  • Callose deposition (aniline blue staining)

  • Phytohormone accumulation (SA, JA, ethylene)

• Experimental Design Considerations:

  • Minimum 10 plants per treatment

  • Three biological replicates

  • Randomized complete block design

  • Include appropriate positive and negative controls

  • Blind scoring of disease symptoms where possible

This comprehensive approach ensures that virulence phenotypes can be reliably attributed to specific aspects of Gcp function while accounting for the complexities of plant-pathogen interactions with P. syringae pv. tomato .