Recombinant Photobacterium profundum Methionine--tRNA ligase (metG), partial

What is Methionine--tRNA ligase (metG) and what is its fundamental role in bacterial protein synthesis?

Methionine--tRNA ligase (metG), also known as methionyl-tRNA synthetase (MetRS), is a critical enzyme responsible for attaching methionine to its cognate tRNA (tRNAMet) during protein synthesis. This aminoacylation reaction occurs in two steps:

• Activation of methionine using ATP: Methionine + ATP → Methionyl-AMP + PPi

• Transfer of activated methionine to tRNAMet: Methionyl-AMP + tRNAMet → Methionyl-tRNAMet + AMP

In bacterial systems, metG is essential as methionine serves not only as a standard amino acid within proteins but also as the initiating amino acid for all bacterial protein synthesis. The metG gene typically exists as a single genomic copy in most bacteria, though additional copies can sometimes be found on mobile genetic elements, which has implications for bacterial adaptation and antimicrobial resistance .

What expression systems are most effective for producing recombinant P. profundum metG?

For expression of recombinant P. profundum metG, researchers should consider the following methodological approach:

Recommended Expression System Setup:

• Vector selection: pET series vectors with T7 promoter for high-level expression

• Host strain: E. coli BL21(DE3) or Arctic Express for cold-adapted proteins

• Induction conditions: Lower temperatures (16-20°C) to promote proper folding of psychrophilic proteins

• Expression time: Extended periods (24-48 hours) at lower temperatures

Purification Protocol:

• Cell lysis by sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

• Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

• Ion exchange chromatography for further purification

• Size exclusion chromatography as a final polishing step

This approach is supported by successful strategies used for other recombinant tRNA synthetases, including the engineered MetRS described in research where a single genomic copy was sufficient to support both robust cell growth and efficient protein production .

How do the biochemical properties of P. profundum metG differ from mesophilic homologs?

As a protein from a piezophilic (pressure-loving) and psychrophilic (cold-loving) marine bacterium, P. profundum metG exhibits distinctive biochemical properties compared to mesophilic counterparts:

These adaptations reflect evolutionary strategies for maintaining enzymatic activity under the extreme conditions of the deep sea environment. The enhanced flexibility in the active site typically allows for maintained catalytic efficiency at lower temperatures, while structural adaptations accommodate high hydrostatic pressure.

How can engineered metG be utilized for incorporation of unnatural amino acids in protein synthesis?

Engineered metG variants provide powerful tools for incorporating unnatural amino acids (UAAs) into proteins through site-specific modifications of the enzyme's substrate binding pocket. The methodology involves:

• Structural analysis to identify key residues in the methionine-binding pocket

• Site-directed mutagenesis to alter substrate specificity while maintaining tRNA charging ability

• Genomic integration of the engineered metG gene for stable expression and consistent results

Research has demonstrated that a strain carrying a single genomic copy of an engineered MetRS (metG*) successfully incorporates azidonorleucine (ANL) into proteins with up to 90% replacement efficiency while maintaining robust cell growth and high protein production (>20 mg/L culture) .

Applications include:

• Bioorthogonal protein labeling via click chemistry

• Protein structure-function analysis with novel chemical moieties

• Creation of proteins with enhanced stability or novel functions

• Surface display of modified proteins for cell labeling applications

The study by Yoo and Tirrell showed that ANL-containing OmpC, an outer membrane protein, could be covalently modified using copper-catalyzed azide-alkyne cycloaddition, demonstrating the practical utility of this approach for surface protein engineering .

What role does metG play in bacterial antimicrobial resistance development?

Recent research reveals a surprising connection between metG and antimicrobial resistance (AMR) through mechanisms involving bacterial persistence:

• Mobile genetic element association: Additional copies of metG carried on phage-plasmids (like pWPMR2) were identified in bacterial lineages evolving toward antimicrobial resistance .

• Persister phenotype induction: Expression of additional metG copies, particularly mutated versions, creates subpopulations of persister cells that can survive antibiotic exposure .

• Cross-species mechanism: This phenomenon appears across multiple bacterial phyla, suggesting it represents a widespread strategy for developing antimicrobial resistance .

The mechanism was elucidated through bacterial genome-wide association studies of Shigella sonnei isolates, which identified a strong association between AMR trajectory and the presence of additional metG copies. Functional microbiology and experimental evolution studies confirmed that these additional copies predispose bacteria to evolve resistance to third-generation cephalosporins by creating persister phenotypes .

This discovery has important implications for antimicrobial resistance surveillance and potential intervention strategies.

What experimental approaches are most effective for characterizing metG mutants?

Comprehensive characterization of metG mutants requires a multi-faceted experimental approach:

Enzymatic Activity Assessment:

• Aminoacylation assays: Measure the rate of methionyl-tRNA formation using radiolabeled methionine or HPLC-based detection.

• ATP-PPi exchange assays: Quantify the first step of the aminoacylation reaction.

• Thermal stability assays: Determine temperature sensitivity using differential scanning fluorimetry.

• Substrate specificity profiling: Test activity with methionine analogs using competition assays.

Structural Analysis:

• X-ray crystallography or cryo-EM to determine three-dimensional structures

• Hydrogen-deuterium exchange mass spectrometry to assess conformational dynamics

• Molecular dynamics simulations to predict substrate interactions

Cellular Impact Studies:

• Growth phenotype analysis under various conditions

• Proteome-wide analysis of mistranslation using mass spectrometry

• Ribosome profiling to assess translation fidelity

• Stress response evaluation using reporter systems

When studying engineered metG variants, researchers have successfully integrated a single genomic copy of metG* that maintains robust cell growth while enabling efficient incorporation of unnatural amino acids, demonstrating the feasibility of creating stable, functional metG variants .

How might the unique properties of P. profundum metG be exploited for biotechnological applications?

The unique properties of P. profundum metG as a cold-adapted, piezophilic enzyme offer several biotechnological opportunities:

Cold-Active Protein Expression Systems:

• Development of low-temperature protein expression systems for heat-sensitive proteins

• Creation of cold-active cell-free protein synthesis platforms

• Engineering of hybrid metG variants combining cold-activity with altered substrate specificity

Pressure-Based Applications:

• Design of pressure-regulated expression systems

• Development of high-pressure biocatalysis processes

• Creation of biosensors for pressure detection in deep-sea applications

Enzyme Engineering Platform:

• Using P. profundum metG as a scaffold for evolving enzymes with novel properties

• Development of pressure-resistant protein production pipelines

• Creation of aminoacyl-tRNA synthetases with expanded substrate range stable under extreme conditions

The adaptations that allow P. profundum metG to function at low temperatures and high pressures could be particularly valuable for biotechnology applications requiring these conditions, such as food processing, deep-sea bioprospecting, and cold-environment bioremediation.

What methodological challenges arise when studying metG's role in translation fidelity?

Investigating metG's role in translation fidelity presents several methodological challenges that researchers must address:

Challenge 1: Discriminating direct vs. indirect effects

• Solution: Use ribosome profiling to directly measure translation events at codon resolution

• Implementation: Compare wild-type and metG variant strains under identical conditions to identify specific mistranslation events

• Analysis: Apply computational pipelines to detect codon-specific mistranslation signatures

Challenge 2: Detecting low-frequency mistranslation events

• Solution: Develop reporter systems with selectable or screenable phenotypes

• Implementation: Design constructs where only mistranslation of specific codons activates reporter

• Analysis: Quantify reporter activation using flow cytometry or selection-based approaches

Challenge 3: Connecting mistranslation to physiological outcomes

• Solution: Combine proteomics with phenotypic assays

• Implementation: Correlate proteome-wide mistranslation patterns with stress response activation

• Analysis: Apply network analysis to identify critical nodes affected by mistranslation

Recent research on additional copies of metG has shown that even subtle changes in metG expression or sequence can have significant effects on bacterial phenotypes, including antimicrobial resistance development through persister formation . This suggests that metG plays important roles beyond its canonical function in protein synthesis.

What controls should be included when studying recombinant P. profundum metG activity?

Proper experimental design for studying recombinant P. profundum metG activity requires rigorous controls:

Essential Controls for in vitro Activity Assays:

Validation Approaches:

• Orthogonal activity measurement techniques (radiometric, HPLC, colorimetric)

• Independent protein preparations to ensure reproducibility

• Complementation testing in MetRS-deficient strains

When studying engineered MetRS variants, researchers have successfully validated activity through both in vitro assays and functional complementation, demonstrating that a single genomic copy of an engineered MetRS can support robust growth while enabling efficient incorporation of unnatural amino acids .

How can researchers investigate the potential role of P. profundum metG in cold adaptation mechanisms?

To investigate P. profundum metG's role in cold adaptation, researchers should employ a comprehensive strategy:

Comparative Genomics and Structural Biology Approach:

• Sequence alignment of metG from psychrophilic, mesophilic, and thermophilic organisms

• Identification of cold-adaptation signatures (increased glycine content, reduced proline, fewer salt bridges)

• Structural modeling to identify regions with enhanced flexibility

• Targeted mutagenesis to convert cold-adapted features to mesophilic equivalents

Functional Characterization Strategy:

• Enzyme kinetics at temperature range (0-37°C) to determine temperature coefficient (Q₁₀)

• Thermodynamic parameter analysis (ΔH, ΔS, ΔG) using temperature-dependent kinetics

• Conformational dynamics assessment using hydrogen-deuterium exchange

• Complementation studies in mesophilic hosts at low temperatures

Systems Biology Integration:

• Global translation rate measurement at different temperatures

• Transcriptome and proteome analysis under cold stress

• MetG interaction partner identification using pull-down assays

• Creation of chimeric MetRS enzymes to isolate cold-adaptive domains

This approach would provide insights into how P. profundum metG contributes to cold adaptation and potentially identify features that could be transferred to other systems for biotechnological applications in cold environments.

What strategies can be employed to study the potential involvement of P. profundum metG in antimicrobial resistance?

Based on recent findings linking additional metG copies to antimicrobial resistance through persister formation , researchers can employ several strategies to investigate P. profundum metG's potential involvement:

Genetic Manipulation Approaches:

• Expression of P. profundum metG in heterologous hosts to assess persister formation

• Creation of strains with variable P. profundum metG copy numbers

• Introduction of mutations corresponding to those found in resistance-associated metG variants

• Complementation of metG deletion strains with P. profundum metG

Resistance Development Analysis:

• Experimental evolution under antibiotic pressure with strains expressing P. profundum metG

• Comparison of mutation rates and resistance acquisition timelines

• Assessment of cross-resistance to multiple antibiotics

• Persister formation quantification using time-kill curves

Molecular Mechanism Investigation:

• Ribosome profiling to assess translation patterns

• Metabolomic analysis to identify stress-related metabolite changes

• Protein-protein interaction studies to identify metG interaction partners

• Transcriptome analysis to detect stress response activation

Research has shown that phage-plasmid borne metG copies can create persister phenotypes that predispose bacteria to evolve resistance to third-generation cephalosporins . Investigating whether P. profundum metG confers similar properties would provide valuable insights into the potential role of deep-sea bacterial enzymes in antimicrobial resistance development.

How should researchers interpret differences in substrate specificity between P. profundum metG and mesophilic homologs?

When analyzing substrate specificity differences between P. profundum metG and mesophilic homologs, researchers should consider several factors:

Methodological Approach to Data Interpretation:

• Temperature-dependent effects: Always compare kinetic parameters at both the optimal temperature for each enzyme and at standardized temperatures.

  Parameter	P. profundum metG	E. coli metG
  Km for methionine at 10°C	x mM	y mM
  Km for methionine at 37°C	z mM	w mM
  Kcat/Km ratio (10°C:37°C)	a	b

• Substrate panel analysis: Test a comprehensive panel of methionine analogs to develop a complete specificity profile.

• Structural basis investigation: Correlate specificity differences with structural features using homology modeling and molecular docking.

• Evolutionary context consideration: Analyze selective pressures in the deep-sea environment that might drive specificity differences.

Engineered MetRS variants have demonstrated dramatically altered substrate specificity, enabling incorporation of unnatural amino acids while maintaining protein synthesis capability . When interpreting P. profundum metG specificity data, researchers should consider whether similar engineering approaches might be applied to further modify this cold-adapted enzyme for biotechnological applications.

What statistical approaches are most appropriate for analyzing metG mutation effects on protein function?

Robust statistical analysis of metG mutation effects requires appropriate methodologies:

Recommended Statistical Framework:

• Multiple mutation comparison:

  • ANOVA with post-hoc tests for comparing multiple variants

  • Bonferroni or Tukey's correction for multiple comparisons

  • Mixed-effects models when analyzing data across multiple conditions

• Structure-function correlations:

  • Principal component analysis to identify patterns in mutational effects

  • Hierarchical clustering to group functionally similar mutations

  • Regression analysis to correlate structural parameters with functional outcomes

• Evolutionary analysis:

  • dN/dS ratios to identify selection pressures on specific residues

  • Ancestral sequence reconstruction to infer evolutionary trajectories

  • Coevolutionary analysis to identify functionally linked residues

Sample Size and Power Considerations:

• Minimum triplicates for each experimental condition

• Power analysis to determine sufficient sample size for detecting effect sizes of interest

• Bootstrap resampling for robust parameter estimation with limited sample sizes

When analyzing engineered metG variants, researchers have demonstrated that single amino acid changes can dramatically alter substrate specificity while maintaining essential function . Similar approaches could be applied to analyze the effects of mutations in P. profundum metG on cold adaptation and substrate specificity.

How can researchers reconcile conflicting data on metG function across different experimental systems?

When faced with conflicting data on metG function across experimental systems, researchers should employ a systematic reconciliation approach:

Methodological Reconciliation Framework:

• System-specific variable identification:

  • Compare buffer compositions, pH, salt concentrations

  • Assess temperature and pressure differences between experiments

  • Evaluate protein expression systems and purification methods

  • Consider tag effects on protein function

• Experimental design harmonization:

  • Develop standardized protocols applicable across systems

  • Perform side-by-side comparisons under identical conditions

  • Use multiple orthogonal techniques to measure the same parameter

  • Exchange materials between laboratories for validation

• Integrated data analysis:

  • Apply meta-analysis techniques to combine datasets

  • Develop mathematical models that account for system-specific variables

  • Use Bayesian approaches to update hypotheses based on all available data

  • Identify consistent trends despite quantitative differences

When reconciling data on engineered metG variants, researchers should consider that genomic integration versus plasmid-based expression can significantly affect outcomes . Similarly, when studying metG's role in antimicrobial resistance, both genetic context and environmental conditions can influence results .

What are the potential applications of engineered P. profundum metG variants in synthetic biology?

Engineered P. profundum metG variants offer unique capabilities for synthetic biology applications:

Cold-Adapted Synthetic Biology Systems:

• Development of low-temperature protein expression systems using cold-adapted translation machinery

• Creation of synthetic circuits functional at low temperatures for environmental applications

• Engineering of cold-active cell-free protein synthesis platforms for temperature-sensitive products

Pressure-Responsive Genetic Elements:

• Design of pressure-regulated gene expression systems

• Development of biosensors for deep-sea applications

• Creation of pressure-dependent protein interaction networks

Expanded Genetic Code Applications:

• Integration of P. profundum metG's cold adaptation features with substrate specificity engineering

• Development of translation systems that incorporate unnatural amino acids at low temperatures

• Creation of psychrophilic organisms with expanded genetic codes

Research has demonstrated that engineered MetRS variants can efficiently incorporate unnatural amino acids while supporting robust cell growth . Combining these capabilities with P. profundum metG's cold adaptation features could enable new synthetic biology applications in extreme environments.

How might understanding metG's role in antimicrobial resistance lead to new therapeutic strategies?

The discovery that additional metG copies contribute to antimicrobial resistance opens several potential therapeutic avenues:

Target-Based Approaches:

• Development of inhibitors specifically targeting variant metG proteins

• Design of molecules that prevent interaction between multiple metG copies

• Creation of compounds that selectively disrupt persister formation pathways

Diagnostic Applications:

• Development of rapid tests to detect additional metG copies in clinical isolates

• Creation of biomarkers for predicting resistance development potential

• Design of surveillance tools for tracking metG-mediated resistance spread

Resistance Prevention Strategies:

• Design of antibiotic combination therapies targeting both regular cells and persisters

• Development of persister-targeting adjuvants to conventional antibiotics

• Creation of anti-persister compounds based on metG inhibition

Research has shown that phage-plasmid borne methionine tRNA ligase contributes to antimicrobial resistance by creating persister phenotypes . Targeting this mechanism could provide a novel approach to combating antimicrobial resistance in a wide range of bacterial pathogens.

What technological advancements would facilitate deeper investigation of P. profundum metG structure-function relationships?

Advancing our understanding of P. profundum metG structure-function relationships would benefit from several technological developments:

Structural Biology Advancements:

• High-pressure crystallography or cryo-EM systems to determine structure under native conditions

• Time-resolved structural techniques to capture conformational changes during catalysis

• Advanced computational modeling incorporating pressure effects

• In-cell structural determination methods for native conformation assessment

Functional Analysis Technologies:

• Single-molecule enzymology under pressure and low temperature

• High-throughput mutagenesis and activity screening platforms for cold-adapted enzymes

• Real-time aminoacylation assays with fluorescent readouts

• Microfluidic systems for measuring enzyme kinetics under pressure

Systems Biology Integration:

• Global translation profiling technologies under extreme conditions

• Proteome-wide mistranslation detection methods

• Metabolic flux analysis under cold and high-pressure conditions

• In vivo protein interaction mapping under extreme conditions

Engineering MetRS variants has already demonstrated the potential for altering substrate specificity while maintaining essential function . Advanced technologies would enable similar engineering of P. profundum metG while preserving or enhancing its unique adaptations to extreme environments.