Recombinant Mycoplasma gallisepticum Tryptophan--tRNA ligase (trpS)

What expression systems are most suitable for recombinant M. gallisepticum Tryptophan--tRNA ligase (trpS)?

E. coli remains the preferred expression system for recombinant M. gallisepticum proteins due to its simplicity, scalability, and cost-effectiveness. For trpS expression, the T7 promoter system in pET vectors is particularly effective, capable of yielding protein levels reaching up to 50% of total cellular protein under optimal conditions . The choice of expression system should consider the following methodological approaches:

• T7 RNAP-based expression: The T7 promoter system with IPTG induction provides tight control of expression, critical for potentially toxic proteins like aminoacyl-tRNA synthetases .

• Tunable promoter systems: For optimization studies, rhamnose-inducible promoters offer superior dose-dependent control compared to IPTG-based systems .

• Codon optimization: M. gallisepticum has a low G+C content (~31%), necessitating codon optimization for efficient expression in E. coli.
  Alternative systems include wheat germ cell-free expression for proteins that may be toxic to bacterial hosts, and yeast-based systems which may provide better folding for eukaryotic applications .

What purification strategies work best for maintaining trpS enzymatic activity?

Maintaining enzymatic activity of trpS requires careful consideration of buffer conditions and purification methodology:

• Affinity tags selection: 6His-tagged recombinant trpS (N-terminal) shows good activity retention after purification .

• Buffer composition: Phosphate buffers (pH 7.5-8.0) containing 5-10% glycerol, 1-5 mM DTT or 2-mercaptoethanol, and 0.1-0.5 mM EDTA help maintain stability.

• Temperature control: All purification steps should be performed at 4°C to prevent activity loss.

• Imidazole gradient: For His-tagged proteins, a gradual imidazole elution (10-250 mM) yields cleaner preparations than single-step elution.
  Activity assays measuring the aminoacylation of tRNA^Trp with [³H]-tryptophan or pyrophosphate exchange assays should be performed immediately after purification to confirm functionality.

How can I verify the purity and identity of recombinant M. gallisepticum trpS?

Multiple complementary approaches should be used to verify both purity and identity:

What are the typical yields of functional recombinant trpS from different expression systems?

Expression yields vary significantly across systems:

How does the GAA trinucleotide repeat region affect expression of recombinant M. gallisepticum genes, and would this impact trpS expression?

The GAA trinucleotide repeat region plays a critical role in regulating gene expression in M. gallisepticum, functioning as a binary on/off switch. This mechanism has significant implications for recombinant expression :

• Exact repeat number matters: Studies using lacZ reporter systems demonstrate that exactly 12 GAA repeats result in gene expression, while any other number (more or fewer) results in gene silencing .

• Phase variation mechanism: The number of GAA repeats can change between generations, causing phase-variable gene expression that alternates between on and off states .

• DNA structure considerations: GAA repeats potentially form triple-helix structures at physiological pH, with the TTC strand folding onto the GAA strand. This may create a single-stranded region that interacts with transcriptional regulators .
  For recombinant trpS expression, researchers should examine whether the native trpS promoter contains GAA repeats. If present, cloning strategies should either:

• Preserve exactly 12 GAA repeats for expression

• Bypass the native regulatory region entirely by using heterologous promoters
  The data from experiments with M9-lacZ fusion genes demonstrated that colonies expressing lacZ had exactly 12 tandem copies of the GAA repeat, while non-expressing colonies had either more (14-16) or fewer (5-11) repeats .

What is the relationship between M. gallisepticum nucleoid structure and trpS expression during different growth phases?

M. gallisepticum undergoes significant nucleoid restructuring between growth phases, affecting global gene expression patterns :

• Exponential phase nucleoid structure: Forms structures with protein-enriched cores and extending DNA loops (200-600 nm length, approximately 1-2 kilobases), comparable to average gene sizes in M. gallisepticum .

• Stationary phase changes:

  • Nucleoid undergoes condensation with shorter DNA loops (100-200 nm)

  • Formation of bead-chain structures (16-20 nm beads along 200-400 nm DNA fragments)

  • Altered protein composition of the nucleoid

• Impact on transcription: Global transcriptional landscape changes dramatically during the stationary phase transition, with most genes significantly repressed .
  This nucleoid restructuring likely affects trpS expression, as aminoacyl-tRNA synthetases are typically downregulated during stationary phase. For optimal recombinant expression, harvesting cells during mid-logarithmic phase would provide highest native trpS expression levels.
  Interestingly, the glycolytic enzyme enolase was identified as a nucleoid structural protein in M. gallisepticum, capable of non-specific DNA binding and forming fibril-like complexes with DNA . This unusual dual functionality may represent adaptation to the minimal genome of M. gallisepticum.

What gene editing approaches can be used to study or modify trpS in M. gallisepticum?

Several gene editing approaches have been developed for mycoplasmas, with varying efficiency in M. gallisepticum:

How can recombinant trpS be used to study M. gallisepticum pathogenesis mechanisms?

Recombinant trpS can serve as a valuable tool for investigating several aspects of M. gallisepticum pathogenesis:

• Cytadherence studies: While not a primary adhesin like GapA or CrmA, aminoacyl-tRNA synthetases in other bacteria have been shown to moonlight as adhesion factors. Testing recombinant trpS for binding to chicken respiratory epithelial cells could reveal secondary adhesion functions .

• Immunomodulation assessment: M. gallisepticum infection modulates host cytokine responses. Purified recombinant trpS could be tested for its ability to stimulate cytokine production (IL1B, IL6, IL10, CXCLi2) in chicken immune cells, similar to studies performed with other M. gallisepticum proteins .

• Vaccine development:

  • As a conserved bacterial protein, trpS could be evaluated as a subunit vaccine component alongside established antigens (GapA, CrmA, VlhAs)

  • Comparative studies using different adjuvants (particularly CpG oligodeoxynucleotide which has shown efficacy with other M. gallisepticum proteins)

• Inhibitor development: Functional recombinant trpS enables screening of potential inhibitors as antimicrobial candidates.
  Recent vaccine development work demonstrated that a rationally designed subunit vaccine containing recombinantly produced M. gallisepticum proteins with CpG oligodeoxynucleotide as adjuvant significantly reduced both bacterial recovery and tracheal pathology .

What structural and functional differences exist between M. gallisepticum trpS and tryptophanyl-tRNA synthetases from other organisms?

Comparing M. gallisepticum trpS with those from other organisms reveals important differences:

• Domain architecture: The minimal M. gallisepticum genome (~1 Mb) typically results in more compact protein structures with fewer auxiliary domains than found in other bacteria.

• Codon usage and adaptation: M. gallisepticum has:

  • Low G+C content (~31%)

  • Distinct codon usage patterns

  • Limited tRNA repertoire

• Catalytic efficiency: M. gallisepticum enzymes often show adaptations to resource-limited environments:

  • Increased kcat/KM ratios for limited substrates

  • Broader substrate specificity

  • Potentially moonlighting functions

• Regulation: Unlike most bacteria, M. gallisepticum lacks the stringent response system for amino acid starvation, suggesting potentially unique regulation of aminoacyl-tRNA synthetases.
  When expressing recombinant M. gallisepticum trpS, these differences may necessitate codon optimization, careful buffer formulation, and structural analysis to understand functional properties.

How might the M. gallisepticum trpS aminoacylation mechanism differ from better-characterized systems?

The aminoacylation mechanism of M. gallisepticum trpS likely exhibits several distinctive features:

• Substrate binding pocket adaptations: The minimal genome of M. gallisepticum suggests potential evolutionary pressure for efficient tryptophan utilization, possibly resulting in a higher affinity binding pocket compared to other bacterial TrpRS enzymes.

• tRNA recognition elements: The coevolution of trpS with M. gallisepticum tRNA^Trp may have led to recognition of distinct identity elements in the tRNA.

• Reaction kinetics: Experimental comparison of aminoacylation kinetics would involve:

  • Measuring aminoacylation of both cognate M. gallisepticum tRNA^Trp and heterologous tRNAs

  • Determining KM values for tryptophan, ATP, and tRNA substrates

  • Assessing temperature optima (expected to align with avian host temperature of 39-42°C)

• Editing mechanisms: M. gallisepticum may have streamlined or eliminated editing domains present in other TrpRS enzymes due to genome reduction pressures. For experimental validation, recombinant expression followed by aminoacylation assays using both radioactive ([³H]-tryptophan) and non-radioactive (pyrophosphate release) methods would provide comparative kinetic parameters.