Recombinant Mycoplasma gallisepticum Threonine--tRNA ligase (thrS), partial

What is Mycoplasma gallisepticum and why is it significant for research?

Mycoplasma gallisepticum (Mgal) is a common bacterial pathogen affecting poultry worldwide that has also spread to wild birds, particularly North American house finches, following a host shift in 1994. The significance of this organism lies in both its economic impact on the poultry industry and its unique minimal genome, making it valuable for studying host-pathogen interactions and bacterial evolution. The molecular determinants of Mgal virulence and host specificity remain largely unknown, creating important research opportunities for exploring fundamental aspects of bacterial pathogenesis . Research with Mgal contributes to our understanding of bacterial adaptation to new hosts and the genetic basis of virulence in minimal bacteria, providing insights that extend beyond this specific pathogen to broader principles of microbial evolution.

What is the role of threonine-tRNA ligase (thrS) in bacterial systems?

Threonine-tRNA ligase (thrS) is an essential aminoacyl-tRNA synthetase responsible for catalyzing the attachment of the amino acid threonine to its cognate tRNA molecule during protein synthesis. This enzyme plays a critical role in translation accuracy by ensuring that threonine is correctly incorporated into growing polypeptide chains. In minimal genome bacteria like Mycoplasma gallisepticum, the proper functioning of each aminoacyl-tRNA synthetase is particularly crucial due to the reduced genetic redundancy. The thrS gene product maintains the fidelity of the genetic code by preventing misincorporation of incorrect amino acids, making it an essential component of cellular machinery. Disruptions in thrS function can lead to proteome-wide mistranslation and potentially affect bacterial fitness, virulence, and antibiotic resistance.

How does the genome engineering of Mycoplasma gallisepticum differ from other bacterial systems?

Genome engineering in Mycoplasma gallisepticum presents unique challenges compared to model bacterial systems due to several factors. Unlike well-established systems such as E. coli or B. subtilis, Mgal lacks many of the native recombination machinery components that facilitate genetic manipulation. Successful genetic engineering in Mgal has been achieved using heterologous recombination systems, particularly the RecET-like system from Bacillus subtilis, which has proven effective for gene inactivation and targeted replacement . The efficiency of this system contrasts with the unsuccessful application of the recombination system from the more closely related Spiroplasma phoeniceum.

The implementation of genetic tools in Mgal follows these methodological steps:

• Introduction of exogenous recombination systems into Mgal cells

• Transformation with recombination templates (20 μg of DNA)

• Selection of transformants on chloramphenicol-containing media

• PCR screening to identify successful recombination events

• Application of the Cre-lox system for antibiotic marker removal

This approach differs from systems with established genetic tools and represents a significant advancement in the molecular biology of minimal bacteria.

What are the optimal conditions for transformation of Mycoplasma gallisepticum with recombinant DNA?

The transformation of Mycoplasma gallisepticum requires specific conditions to achieve successful genetic modification. Based on recent research, the optimal approach involves:

• DNA Template Preparation: Using approximately 20 μg of recombination template DNA, which can be either double-stranded (ds) or single-stranded (ss), and in linear or circular format. Research indicates that single-stranded circular templates may yield more transformants .

• Expression System: Implementing a heterologous RecET-like recombination system from Bacillus subtilis rather than systems from more closely related species. This counterintuitive approach has proven more successful, with 18 transformants obtained using the B. subtilis RecET-like system compared to zero with the Spiroplasma phoeniceum system .

• Selection Method: Employing chloramphenicol resistance as a selective marker and cultivating transformants on chloramphenicol-containing plates.

• Verification Protocol: Confirming successful transformations through PCR screening, looking for specific recombination profiles that indicate proper integration at the target locus.

• Clone Purification: Performing multiple filter-cloning steps (0.45 μm filter) to ensure clonal populations and eliminate mixed colonies.

The success rate varies by template type, with transformation efficiency data showing:

• Single-stranded circular: 2 successful clones from 12 transformants

• Single-stranded linear: 1 successful clone from 1 transformant

• Double-stranded circular: 1 successful clone from 4 transformants

• Double-stranded linear: 0 successful clones from 1 transformant

These parameters provide a methodological framework for researchers aiming to genetically modify M. gallisepticum.

How can fractional factorial designs be applied to optimize expression of recombinant thrS in Mycoplasma gallisepticum?

Fractional factorial experimental designs offer an efficient approach to optimize recombinant thrS expression in Mycoplasma gallisepticum by identifying significant factors while minimizing experimental runs. When working with complex biological systems, researchers face numerous variables that can influence recombinant protein expression, including temperature, media composition, induction timing, and genetic construct design.

The application of this methodology follows these steps:

• Factor Identification: Identify key variables potentially affecting thrS expression (e.g., promoter strength, codon optimization, culture temperature, media composition, induction parameters).

• Design Selection: Implement a two-level fractional factorial design based on the sparsity of effects principle, which states that most responses are affected by a small number of main effects and lower-order interactions, while higher-order interactions are relatively unimportant .

• Experimental Execution: For a system with 7 potential factors affecting thrS expression, a 2^(7-3) design would require only 16 experimental runs instead of 128 for a full factorial design, while still capturing main effects and important two-factor interactions.

• Analysis Methods: Since unreplicated fractional factorial designs don't provide degrees of freedom for calculating error sum of squares, employ techniques like normal probability plots of effects or Lenth's method to identify significant factors .

• Follow-up Experimentation: Use the identified significant factors from the screening experiment for more detailed investigation in subsequent experiments.

This approach allows researchers to efficiently identify the most important factors affecting thrS expression without conducting exhaustive experiments testing all possible combinations of variables. For example, if temperature, codon optimization, and media composition emerge as the three most significant factors affecting expression, these can be examined more thoroughly in subsequent experiments.

What methods are most effective for purifying recombinant thrS protein from Mycoplasma gallisepticum?

Purification of recombinant threonine-tRNA ligase from Mycoplasma gallisepticum requires a tailored approach that accounts for the unique characteristics of mycoplasma proteins. A comprehensive purification strategy involves:

• Expression System Selection: Consider using either native M. gallisepticum with the RecET-like system from B. subtilis for homologous expression , or a heterologous expression system such as E. coli with appropriate codon optimization for the AT-rich M. gallisepticum genome.

• Affinity Tag Design: Incorporate a suitable affinity tag (His6, FLAG, or Strep-tag) at either the N- or C-terminus of the thrS protein, carefully positioned to minimize interference with enzymatic activity.

• Cell Lysis Protocol:

  • For M. gallisepticum: Use gentle osmotic lysis with 10-15% glycerol in hypotonic buffer

  • For heterologous systems: Apply standard mechanical or chemical lysis methods appropriate for the host organism

• Purification Sequence:
  a. Initial capture by affinity chromatography using the incorporated tag
  b. Intermediate purification by ion exchange chromatography
  c. Polishing step using size exclusion chromatography to remove aggregates and ensure homogeneity

• Activity Preservation: Maintain buffers containing 5-10% glycerol and 1-2 mM DTT throughout purification to preserve enzymatic activity, and consider adding specific stabilizing agents like threonine or ATP.

• Quality Assessment: Verify purity using SDS-PAGE (≥95%), confirm identity by mass spectrometry, and assess activity through aminoacylation assays measuring the attachment of threonine to cognate tRNA.

This methodological approach provides a systematic framework for obtaining pure, active threonine-tRNA ligase from M. gallisepticum for structural and functional studies, while addressing the specific challenges associated with proteins from this minimal bacterium.

How can CRISPR-Cas systems be adapted for gene editing in Mycoplasma gallisepticum, particularly for thrS modifications?

While CRISPR-Cas systems have revolutionized genome editing in many organisms, their application in Mycoplasma gallisepticum presents unique challenges that require specific adaptations. For researchers interested in modifying the thrS gene, a specialized approach combining CRISPR technology with the proven RecET-like recombination system from B. subtilis offers the most promising strategy.

The methodological framework involves:

• CRISPR-Cas9 Component Optimization:

  • Codon-optimize Cas9 for expression in the AT-rich M. gallisepticum genome

  • Engineer small guide RNAs (sgRNAs) with specificity for the thrS locus

  • Develop promoters compatible with M. gallisepticum transcriptional machinery

• Delivery System Development:

  • Package CRISPR components and homology-directed repair templates in a single vector

  • Utilize transformation protocols established for the RecET system, requiring approximately 20 μg of DNA

  • Consider transient expression systems to minimize off-target effects

• Integration with RecET Recombination:

  • Employ CRISPR-Cas9 to create targeted double-strand breaks at the thrS locus

  • Simultaneously provide the RecET system from B. subtilis to enhance homologous recombination efficiency

  • Design repair templates with appropriate homology arms (>500 bp recommended)

• Selection Strategy:

  • Incorporate chloramphenicol resistance markers flanked by loxP sites

  • Apply the proven Cre-lox system for subsequent marker removal

  • Implement PCR screening to identify successful recombinants

• Validation Protocol:

  • Sequence the modified thrS region to confirm precise editing

  • Assess aminoacylation activity of the modified thrS gene product

  • Evaluate whole-cell phenotypes resulting from thrS modification

This integrated approach leverages the demonstrated effectiveness of the B. subtilis RecET-like system in M. gallisepticum while incorporating the precision of CRISPR-based targeting, providing a powerful methodology for thrS gene editing in this challenging minimal bacterium.

What are the implications of thrS mutations on antibiotic resistance in Mycoplasma gallisepticum?

The relationship between threonine-tRNA ligase (thrS) mutations and antibiotic resistance in Mycoplasma gallisepticum represents a complex area of research with significant implications for both basic science and clinical applications. Recent studies indicate that aminoacyl-tRNA synthetases, including thrS, may play unexpected roles in antibiotic susceptibility through several mechanisms:

• Direct Interactions with Antibiotics:

  • Certain thrS mutations may alter binding sites that interact with antibiotics targeting protein synthesis, particularly those affecting the aminoacylation process

  • Structural changes in thrS can impact the effectiveness of antibiotics like tylosin, which has shown variable susceptibility patterns in M. gallisepticum isolates

• Mistranslation-Mediated Resistance:

  • Specific mutations in thrS may lead to controlled mistranslation, generating protein diversity that contributes to stress response and antibiotic tolerance

  • This "adaptive mistranslation" mechanism can produce heterogeneous populations with varying antibiotic susceptibility profiles

• Metabolic Consequences:

  • Alterations in thrS function can affect the cellular pool of charged tRNAs, influencing bacterial growth rate and potentially modifying antibiotic susceptibility

  • Changes in translation efficiency may trigger stress responses that activate efflux pumps or other resistance mechanisms

• Experimental Evidence:

  • M. gallisepticum isolates have demonstrated changing sensitivity to tylosin, necessitating routine testing to optimize treatment protocols

  • The relationship between specific thrS mutations and resistance profiles represents a critical area for further investigation

• Research Methodology:

  • Minimum inhibitory concentration (MIC) determination for various antibiotics against M. gallisepticum strains with defined thrS mutations

  • Whole genome sequencing to correlate spontaneous resistance with thrS polymorphisms

  • Site-directed mutagenesis of thrS using the RecET-like system from B. subtilis to confirm causative relationships

This research area highlights the importance of understanding the multifaceted roles of essential genes like thrS beyond their primary functions, particularly in minimal genome organisms where individual gene products may serve multiple purposes.

What data tables are required for NIH grant applications focusing on recombinant Mycoplasma gallisepticum thrS research?

When preparing NIH grant applications for research on recombinant Mycoplasma gallisepticum threonine-tRNA ligase, researchers must include specific data tables that comprehensively document the project's scope, participating personnel, and expected outcomes. The appropriate tables vary based on whether the application is new or a renewal, and the type of training grant being requested.

For a typical research project focused on M. gallisepticum thrS, the following tables would be required:

For new predoctoral training projects, researchers should additionally include:

• Table 5A: Publications of Trainees Supported by this Program

• Table 6A: Training Program Candidates, Entrants, and Their Characteristics for the Past Five Years

• Table 8A (Part II only): Program Outcomes

For renewal applications, all the above tables plus Table 7 (Appointments to the Training Grant for Each Year of the Current Project Period) would be required .

These tables serve several critical functions:

• Documenting the training program's structure, including participating departments and faculty

• Demonstrating the ability to recruit and retain qualified trainees

• Showcasing faculty research support and mentoring capabilities

• Providing evidence of program effectiveness through trainee publications and outcomes

Researchers can prepare these tables either using MS Word fillable templates or the NIH xTRACT system, which helps pre-populate information from existing NIH databases . The xTRACT system offers significant advantages for data management, including automatic calculation of values for Tables 4 and 6, and simplified tracking of trainee outcomes for Table 8.

How should researchers design experiments to investigate the structure-function relationship of Mycoplasma gallisepticum thrS?

Investigating the structure-function relationship of Mycoplasma gallisepticum threonine-tRNA ligase requires a multidisciplinary approach combining genetic manipulation, biochemical characterization, and structural biology. A comprehensive experimental design should incorporate the following methodological elements:

• Genetic Engineering Strategy:

  • Generate a series of thrS variants using the successful RecET-like system from B. subtilis

  • Create targeted mutations in functional domains (ATP-binding, threonine-binding, tRNA-binding, editing)

  • Develop a complementation system to study essential residues by expressing the variant thrS before inactivating the chromosomal copy

• Biochemical Characterization:

  • Express and purify wild-type and variant thrS proteins with appropriate affinity tags

  • Assess aminoacylation kinetics using purified M. gallisepticum tRNA^Thr

  • Measure substrate binding affinities (threonine, ATP, tRNA) through isothermal titration calorimetry

  • Evaluate editing activity using mischarged tRNA substrates

• Structural Analysis Pipeline:

  • Determine three-dimensional structure through X-ray crystallography or cryo-electron microscopy

  • Perform molecular dynamics simulations to analyze conformational changes

  • Use hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Fractional Factorial Experimental Design:

  • Apply two-level fractional factorial design principles to efficiently explore multiple variables

  • Focus on main effects and lower-order interactions while minimizing experimental runs

  • Example design for investigating 5 factors affecting thrS activity:

• Integration and Analysis:

  • Correlate structural features with biochemical properties

  • Map mutations with altered function onto the three-dimensional structure

  • Apply computational methods to predict effects of additional mutations

This comprehensive approach enables researchers to systematically investigate structure-function relationships in M. gallisepticum thrS while minimizing the number of experiments required through efficient experimental design.

What are the best practices for documenting and analyzing data from thrS expression experiments in Mycoplasma gallisepticum?

Effective documentation and analysis of data from threonine-tRNA ligase expression experiments in Mycoplasma gallisepticum require rigorous scientific methodology and comprehensive record-keeping. The following best practices ensure reproducibility, facilitate collaboration, and maximize the value of experimental results:

• Experimental Design Documentation:

  • Implement detailed protocols that specify all relevant parameters, including:

    • Genetic constructs and sequence verification

    • Expression conditions (temperature, media composition, induction parameters)

    • Cell harvest and processing methods

    • Purification protocols with buffer compositions

  • Apply fractional factorial designs where appropriate to efficiently explore multiple variables

  • Include positive and negative controls in all experimental runs

• Data Collection Standards:

  • Record raw data in machine-readable formats when possible

  • Establish consistent units and normalization methods

  • Document all instrument settings and calibration status

  • Maintain complete records of all experimental attempts, including failed experiments

• Structured Laboratory Notebook System:

  • Implement electronic laboratory notebooks with standardized templates

  • Include cross-references to stored samples, sequence data, and raw files

  • Create an indexing system for experimental series

  • Record observations, deviations from protocols, and real-time interpretations

• Data Analysis Framework:

  • Apply appropriate statistical methods based on experimental design

  • For fractional factorial designs, use techniques such as:

    • Normal probability plots of effects

    • Pareto charts of standardized effects

    • Main effects and interaction plots

  • Validate analytical methods with simulated data sets

  • Use scripting languages (R, Python) to ensure reproducible analysis

• Data Visualization Standards:

  • Create standardized visualization formats for common experiment types

  • Include error bars representing standard deviation or standard error

  • Use consistent color schemes and symbols across related experiments

  • Generate both detailed technical visualizations and simplified versions for presentations

• Data Storage and Sharing Practice:

  • Implement a tiered storage system with regular backups

  • Structure data repositories to facilitate future meta-analyses

  • Prepare datasets in formats compatible with public repositories

  • Document metadata following community standards (minimum information requirements)

• Quality Control Metrics:

  • Establish acceptance criteria for each experiment type

  • Include internal standards where applicable

  • Implement regular testing of critical reagents

  • Document lot numbers and sources of all materials

These comprehensive best practices ensure that thrS expression experiments in M. gallisepticum generate reliable, reproducible data that can be effectively communicated to the scientific community and integrated with other research efforts in the field.

What are common problems encountered when expressing recombinant thrS in Mycoplasma gallisepticum and how can they be resolved?

Researchers working with recombinant threonine-tRNA ligase expression in Mycoplasma gallisepticum frequently encounter several challenges that can compromise experimental outcomes. These issues, along with their systematic resolution strategies, include:

• Low Transformation Efficiency

  • Problem: Failed transformation or few transformants when introducing recombinant thrS constructs.

  • Resolution Strategy:

    • Use the B. subtilis RecET-like recombination system instead of systems from more closely related species, as this has proven more effective in M. gallisepticum

    • Optimize DNA template quantity (approximately 20 μg recommended)

    • Consider single-stranded circular DNA templates, which have shown higher success rates

    • Ensure high DNA purity and appropriate concentration of competent cells

• Protein Misfolding and Insolubility

  • Problem: Expressed thrS protein forms inclusion bodies or shows poor solubility.

  • Resolution Strategy:

    • Reduce expression temperature to slow translation and facilitate proper folding

    • Co-express molecular chaperones to assist protein folding

    • Design constructs with solubility-enhancing fusion partners

    • Optimize induction parameters through fractional factorial experimental design

• Poor Enzymatic Activity

  • Problem: Purified recombinant thrS shows reduced or absent aminoacylation activity.

  • Resolution Strategy:

    • Verify protein integrity through mass spectrometry

    • Ensure cofactors (Mg²⁺, ATP) are present in activity assays

    • Check for inhibitory contaminants in purification

    • Test activity under various buffer conditions (pH, ionic strength)

    • Consider the addition of stabilizing agents like glycerol or reducing agents

• Genomic Integration Issues

  • Problem: Difficulty achieving stable genomic integration of modified thrS constructs.

  • Resolution Strategy:

    • Extend homology arms in recombination templates to increase integration efficiency

    • Screen more transformants by PCR to identify successful recombinants

    • Perform multiple 0.45 μm filter-cloning steps to ensure clonal populations

    • Consider alternative selection markers if chloramphenicol resistance is problematic

• Phenotypic Instability

  • Problem: Loss of expression or reversion to wild-type phenotype during cultivation.

  • Resolution Strategy:

    • Maintain selection pressure throughout cultivation

    • Regularly verify genotype by PCR or sequencing

    • Prepare frozen stocks from verified clones at early passages

    • Monitor growth curves to detect fitness defects that might select for revertants

• Unexpected Metabolic Effects

  • Problem: thrS expression causes growth defects or unexpected physiological changes.

  • Resolution Strategy:

    • Implement inducible or titratable expression systems

    • Conduct metabolomic analysis to identify disrupted pathways

    • Test complementation with wild-type thrS to confirm specificity of effects

    • Consider dual expression systems with controlled ratios of wild-type and recombinant thrS

By systematically addressing these common challenges through the proposed resolution strategies, researchers can significantly improve the success rate of recombinant thrS expression experiments in M. gallisepticum, facilitating more productive investigations of this essential enzyme's structure and function.

How can researchers address contradictory data when analyzing thrS function in Mycoplasma gallisepticum?

When researchers encounter contradictory data in studies of threonine-tRNA ligase function in Mycoplasma gallisepticum, a structured approach to data reconciliation is essential for maintaining scientific integrity and extracting valuable insights from seemingly inconsistent results. The following methodological framework guides researchers through this complex process:

• Systematic Data Validation:

  • Re-examine raw data for potential artifacts or technical errors

  • Verify instrument calibration and experimental conditions

  • Repeat critical experiments with additional controls

  • Consider blinded analysis to minimize confirmation bias

• Experimental Design Assessment:

  • Analyze potential confounding variables using factorial design principles

  • Identify interactions between factors that might explain context-dependent results

  • Evaluate whether sample sizes provide adequate statistical power

  • Consider whether different methodologies might systematically bias results

• Biological Hypothesis Development:

  • Generate alternative hypotheses that could reconcile seemingly contradictory observations

  • Consider post-translational modifications that might affect thrS activity

  • Evaluate potential strain-specific differences in M. gallisepticum

  • Assess whether environmental conditions (like those affecting tylosin sensitivity ) might explain variable results

• Resolution Through Targeted Experimentation:

  • Design experiments specifically to test alternative hypotheses

  • Employ orthogonal techniques to measure the same parameters

  • Use recombination systems to create defined genetic backgrounds

  • Develop in vitro systems to isolate specific aspects of thrS function

• Computational Analysis Integration:

  • Apply machine learning approaches to identify patterns in complex datasets

  • Use Bayesian statistical methods to update confidence in hypotheses as new data emerges

  • Develop predictive models that accommodate apparent contradictions

  • Simulate molecular dynamics to explore condition-dependent protein behavior

• Collaborative Resolution Strategy:

  • Engage collaborators with complementary expertise

  • Implement standardized protocols across laboratories

  • Conduct blind inter-laboratory validation studies

  • Host focused research discussions addressing specific contradictions

• Transparent Reporting Framework:

  • Document both supporting and contradictory evidence

  • Report all experimental attempts, including failed experiments

  • Present alternative interpretations of complex datasets

  • Clearly distinguish between established facts and speculative explanations

This structured approach transforms contradictory data from a frustration into an opportunity for deeper understanding, recognizing that apparent contradictions often reflect biological complexity rather than experimental failure. In the study of thrS in M. gallisepticum, where work is still emerging and methods are being refined, this framework enables researchers to advance knowledge despite the challenges of working with this minimal bacterial system.

What bioinformatic tools and resources are most useful for analyzing recombinant thrS sequences and structures in Mycoplasma species?

Researchers working with threonine-tRNA ligase from Mycoplasma gallisepticum benefit from a specialized bioinformatic toolkit that addresses the unique challenges of analyzing sequences and structures in this minimal genome bacterium. The following comprehensive set of computational resources and methodological approaches facilitates thorough analysis of recombinant thrS:

• Sequence Analysis Tools:

  • Primary Sequence Analysis:

    • MUSCLE or MAFFT for multiple sequence alignment of thrS across Mycoplasma species

    • MEGA X for phylogenetic analysis to understand evolutionary relationships

    • PROVEAN or SIFT for predicting functional impacts of amino acid substitutions

  • Domain Architecture Investigation:

    • InterProScan for identifying conserved domains and functional motifs

    • SMART for visualizing domain organization and detecting class-specific features

    • ConSurf for mapping evolutionary conservation onto sequence regions

• Structure Prediction and Analysis:

  • Structural Modeling:

    • AlphaFold2 for generating highly accurate structural models of thrS

    • SWISS-MODEL for template-based homology modeling

    • I-TASSER for integrative modeling using multiple templates

  • Structure Validation and Analysis:

    • MolProbity for evaluating structural model quality

    • PyMOL or UCSF Chimera for visualization and structural comparison

    • MDWeb for molecular dynamics simulation setup

    • CASTp for identifying binding pockets and catalytic sites

• tRNA-Protein Interaction Analysis:

  • Docking Tools:

    • HADDOCK for modeling thrS-tRNA^Thr complexes

    • NPDock for nucleic acid-protein docking

    • 3dRPC for RNA-protein complex structure prediction

  • Interaction Analysis:

    • NPIDB for analyzing known nucleic acid-protein interfaces

    • PISA for calculating interface areas and binding energies

    • RNABindRPlus for predicting RNA binding residues

• Mycoplasma-Specific Resources:

  • Genomic Databases:

    • Mollicutes DB for Mycoplasma genome browsing

    • MycoDB for comparative genomics of Mycoplasma species

    • MolliGen for genomic and proteomic data integration

  • Codon Usage Tools:

    • OPTIMIZER for codon optimization in the AT-rich Mycoplasma genome

    • GCUA for graphical codon usage analysis

    • Rare Codon Calculator for identifying potential translation bottlenecks

• Integrated Analysis Workflows:

  • Functional Prediction Pipeline:

    • Step 1: Multiple sequence alignment of thrS across Mycoplasma species

    • Step 2: Identification of conserved catalytic and binding residues

    • Step 3: Structural modeling and validation

    • Step 4: Mapping of sequence conservation onto structural models

    • Step 5: Identification of species-specific variations

    • Step 6: Prediction of functional consequences of variations

  • Recombinant Design Workflow:

    • Step 1: Codon optimization for M. gallisepticum expression

    • Step 2: Identification of optimal sites for affinity tags

    • Step 3: Design of mutations for functional studies

    • Step 4: In silico validation of construct stability

These bioinformatic tools and methodological workflows provide researchers with a comprehensive framework for analyzing thrS sequences and structures in Mycoplasma species, facilitating both basic research into thrS function and applied work on recombinant protein expression and engineering.

What are the potential applications of recombinant thrS from Mycoplasma gallisepticum in synthetic biology?

Recombinant threonine-tRNA ligase (thrS) from Mycoplasma gallisepticum offers unique properties that can be leveraged in synthetic biology applications, expanding the toolkit available for designing novel biological systems. The potential applications span from fundamental research to practical biotechnology implementations:

• Minimal Genome Toolkit Development:

  • ThrS from M. gallisepticum can serve as a component in minimal synthetic cells

  • Its natural adaptation to functioning in a minimal genome context makes it valuable for streamlined synthetic systems

  • Integration with other M. gallisepticum components could help create simplified protein synthesis modules

  • The established genetic engineering tools for M. gallisepticum provide a foundation for further toolkit development

• Orthogonal Translation Systems:

  • Modified M. gallisepticum thrS variants could be engineered to recognize non-canonical amino acids

  • Development of orthogonal pairs with specialized tRNAs to expand the genetic code

  • Creation of synthetic circuits that respond to specific amino acid availability

  • Implementation of translation-level regulatory systems based on aminoacyl-tRNA synthetase activity

• Biosensor Development:

  • Engineered thrS variants as sensing components for detecting:

    • Threonine levels in biological samples

    • Antibiotics that target aminoacyl-tRNA synthetases

    • Environmental toxins that affect protein synthesis

  • Coupling thrS activity to reporter systems for quantitative detection

  • Development of whole-cell biosensors using M. gallisepticum as a chassis

• Protein Evolution Platforms:

  • Controlled mistranslation systems using modified thrS to generate protein diversity

  • Directed evolution platforms based on thrS variants with altered fidelity

  • Synthesis of protein libraries with controlled amino acid substitution patterns

  • Exploration of protein sequence space through programmed mistranslation

• Therapeutic Applications:

  • Development of attenuated M. gallisepticum strains through thrS modification

  • Design of thrS-based antimicrobials targeting pathogenic Mycoplasma species

  • Exploration of aminoacyl-tRNA synthetases as vaccine candidates

  • Investigation of thrS inhibitors as species-specific antibiotics

• Methodological Approaches:

  • Application of the RecET-like system from B. subtilis for precise genetic modifications

  • Implementation of Cre-lox recombination for marker removal in engineered strains

  • Use of fractional factorial designs to optimize synthetic biology components

  • Integration of bioinformatic tools for rational design of thrS variants

These diverse applications demonstrate how recombinant thrS from M. gallisepticum can contribute to the expanding field of synthetic biology, offering unique advantages due to its origin in a minimal genome organism and the established genetic engineering tools for its manipulation.

How might comparative studies of thrS across Mycoplasma species inform our understanding of bacterial evolution?

Comparative studies of threonine-tRNA ligase across Mycoplasma species offer a unique window into fundamental aspects of bacterial evolution, particularly in organisms with highly reduced genomes. These studies can illuminate evolutionary processes and constraints that shape essential cellular machinery in minimal bacterial systems.

• Evolutionary Trajectories in Minimal Genomes:

  • ThrS sequence comparison across Mycoplasma species reveals conservation patterns in essential cellular machinery

  • Identification of core functional domains resistant to evolutionary change

  • Analysis of species-specific adaptations in non-catalytic regions

  • Tracking evolutionary rates in aminoacyl-tRNA synthetases compared to other essential genes

• Host Adaptation Signatures:

  • Comparison of thrS from M. gallisepticum strains isolated from different hosts (poultry versus wild birds) following the documented 1994 host shift

  • Identification of potential adaptive mutations associated with host-specific environments

  • Correlation between thrS sequence variations and host range

  • Investigation of selection pressures driving divergence in different ecological niches

• Methodological Framework for Comparative Analysis:

  • Whole-genome sequencing of multiple Mycoplasma strains from diverse hosts

  • Phylogenetic analysis using thrS as a molecular marker

  • Experimental validation of functional differences using recombination systems

  • Application of selection analysis tools to identify sites under positive selection

• Coevolution with tRNA Substrates:

  • Analysis of thrS-tRNA^Thr coevolution patterns across Mycoplasma species

  • Investigation of recognition element changes and corresponding enzyme adaptations

  • Experimental verification of species-specific thrS-tRNA interactions

  • Modeling of evolutionary constraints in the aminoacylation system

• Genome Reduction Consequences:

  • Examination of thrS functional adaptations in response to genome minimization

  • Comparison with orthologs from bacteria with larger genomes

  • Investigation of potential moonlighting functions acquired during genome reduction

  • Analysis of structural simplification versus functional conservation

• Horizontal Gene Transfer Assessment:

  • Evaluation of potential horizontal gene transfer events involving thrS

  • Analysis of incongruent phylogenetic patterns

  • Investigation of recombination signatures within thrS sequences

  • Experimental testing of functional compatibility between thrS from different species

• Antibiotic Resistance Evolution:

  • Correlation between thrS sequence variations and altered antibiotic sensitivity profiles

  • Identification of naturally occurring mutations conferring resistance

  • Experimental validation using genetic engineering approaches

  • Tracking the emergence of resistance-associated polymorphisms across species

This comprehensive approach to comparative thrS analysis across Mycoplasma species provides insights into fundamental evolutionary processes operating in minimal genome bacteria, contributing to our broader understanding of bacterial evolution, host adaptation, and the functional constraints on essential cellular machinery.

What emerging technologies might enhance the study of recombinant thrS function in Mycoplasma gallisepticum?

The study of recombinant threonine-tRNA ligase in Mycoplasma gallisepticum stands to benefit significantly from several emerging technologies that promise to overcome current limitations and open new research avenues. These cutting-edge approaches span multiple disciplines and offer powerful new capabilities for investigating this essential enzyme in minimal bacterial systems.

• Advanced Genome Engineering Technologies:

  • CRISPR-Cas Systems Adapted for Mycoplasma:

    • Development of optimized Cas proteins for efficient genome editing in M. gallisepticum

    • Base editing technologies allowing precise nucleotide changes without double-strand breaks

    • Prime editing systems enabling targeted insertions and deletions without donor templates

    • Integration with established RecET-like recombination systems for enhanced efficiency

  • Genome-wide Engineering Tools:

    • Multiplex automated genome engineering (MAGE) adapted for M. gallisepticum

    • Inducible CRISPR interference (CRISPRi) for titratable gene repression

    • CRISPR activation (CRISPRa) systems for controlled overexpression

    • Recombineering with single-stranded DNA for markerless modifications

• Single-Cell and Single-Molecule Technologies:

  • Single-Cell Genomics and Transcriptomics:

    • Single-cell RNA sequencing to capture heterogeneity in thrS expression

    • Spatial transcriptomics to examine cellular localization patterns

    • Single-cell proteomics to correlate thrS levels with protein synthesis capacity

    • Microfluidic systems for high-throughput single-cell analysis

  • Advanced Microscopy Approaches:

    • Super-resolution microscopy to visualize thrS localization within M. gallisepticum cells

    • Single-molecule fluorescence resonance energy transfer (smFRET) to study thrS conformational dynamics

    • Expansion microscopy to overcome size limitations of these minimal bacteria

    • Live-cell imaging with genetically encoded sensors for aminoacylation activity

• Structural Biology Innovations:

  • Cryo-Electron Microscopy Advances:

    • Time-resolved cryo-EM to capture thrS in different functional states

    • Cryo-electron tomography to visualize thrS in its native cellular context

    • Microcrystal electron diffraction for structural analysis of challenging protein variants

    • Correlative light and electron microscopy to connect function with structure

  • Integrated Structural Biology:

    • AlphaFold2 and RoseTTAFold deep learning for accurate structural prediction

    • Integrative modeling combining multiple experimental data sources

    • Mass photometry for analyzing protein complex assembly in solution

    • Hydrogen-deuterium exchange mass spectrometry to probe protein dynamics

• High-Throughput Functional Analysis:

  • Deep Mutational Scanning:

    • Comprehensive analysis of thousands of thrS variants in parallel

    • Selection systems based on aminoacylation activity or cell fitness

    • Coupling with next-generation sequencing for quantitative assessment

    • Machine learning models to predict functional consequences of mutations

  • Microfluidic Platforms:

    • Droplet-based assays for enzyme kinetics with minimal material requirements

    • Digital PCR approaches for absolute quantification of thrS expression

    • Microfluidic cultivation systems for growth phenotype analysis

    • Lab-on-a-chip systems for integrated analysis of multiple parameters