Recombinant Streptococcus pneumoniae Thymidylate synthase (thyA)
Description
ThyA as a Selection Marker in Streptococcal Systems
In Streptococcus thermophilus, thyA mutants have been used to develop food-grade host-vector systems, where thymidine auxotrophy serves as a selectable marker. This approach involves generating thymidine-requiring mutants through trimethoprim treatment and using recombinant vectors to restore thymidine autotrophy . While not directly applicable to S. pneumoniae, similar strategies could theoretically be adapted for genetic engineering in pneumococcal systems.
Thymidine Auxotrophy and Host-Pathogen Interactions
In Haemophilus influenzae, inactivation of thyA increases resistance to trimethoprim (TxS) but alters bacterial physiology, including impaired division and self-aggregation . These findings suggest that thymidine auxotrophy modulates bacterial fitness and pathogenicity, though analogous studies in S. pneumoniae are lacking in the provided sources.
Limitations and Future Directions
The absence of targeted research on S. pneumoniae thyA in the provided sources highlights a gap in understanding its role in pneumococcal metabolism and pathogenesis. Future studies could investigate:
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ThyA expression dynamics during infection or under stress conditions.
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Interactions with antimicrobial agents targeting thymidine biosynthesis.
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Structural biology of S. pneumoniae thyA for inhibitor design.
Data Table: ThyA-Related Findings in Streptococcal Species
Product Specs
Target Background
KEGG: snt:SPT_0693
Q&A
What is the enzymatic role of thymidylate synthase (thyA) in Streptococcus pneumoniae, and why is its recombinant form significant?
Thymidylate synthase catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a rate-limiting step in DNA biosynthesis. Recombinant thyA enables precise study of folate metabolism and antibiotic resistance mechanisms. Key applications include:
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Genetic selection systems: thyA-deficient strains require thymidine supplementation, allowing positive/negative selection in recombineering .
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Antibiotic target validation: Trimethoprim resistance studies rely on thyA mutations affecting tetrahydrofolate cycling .
Methodological Insight:
To express recombinant thyA, use E. coli BL21(DE3) with a T7 promoter system. Verify protein functionality via complementation assays in thymidine-free media .
Which expression systems are optimal for producing recombinant thyA, and how do host factors influence yield?
Prokaryotic systems dominate due to cost-effectiveness and post-translational modification simplicity:
| Host System | Advantages | Limitations |
|---|---|---|
| E. coli BL21 | High yield (≥50 mg/L), scalable | Insoluble aggregates require refolding |
| Streptococcus thermophilus | Native folding, food-grade safety | Lower yield (10–15 mg/L), complex media |
Critical Step: Codon-optimize the thyA gene for GC content matching the host. For S. thermophilus, use the pSintA1 vector with a temperature-sensitive origin .
How is catalytic activity measured for recombinant thyA, and what controls are essential?
A standard spectrophotometric assay monitors dTMP production at 340 nm (NADPH oxidation). Key components:
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100 mM Tris-HCl (pH 7.4), 25 mM MgCl₂, 50 µM dUMP, 150 µM methylenetetrahydrofolate
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Include negative controls with heat-inactivated enzyme and thymidine supplementation to confirm thyA-specific activity .
Data Interpretation: Normalize activity to protein concentration (Bradford assay). Activity <5 nmol/min/mg suggests improper folding or cofactor depletion.
What genetic tools utilize thyA as a dual selection marker?
The thyA system enables seamless DNA modification through:
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Positive selection: Transformants grow in thymidine-deficient media.
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Negative selection: Add trimethoprim (50 µg/mL) to eliminate wild-type thyA .
Case Study: In S. thermophilus, pBSt1 vectors with thyA achieved 90% selection efficiency, outperforming erythromycin resistance markers .
How can solubility issues in recombinant thyA purification be resolved?
Problem: Aggregation in E. coli due to hydrophobic N-terminal residues.
Solutions:
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Fusion tags: Thioredoxin (Trx) improves solubility by 3.2-fold .
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Buffer optimization: 20 mM Tris (pH 8.0), 500 mM arginine, 2% CHAPS reduces non-specific interactions.
Validation: Compare circular dichroism spectra to native S. pneumoniae thyA. Deviations >15% indicate misfolding .
How should conflicting kinetic data between recombinant and native thyA be reconciled?
Case Example: Recombinant thyA shows 40% lower V<sub>max</sub> than native enzyme in S. pneumoniae lysates .
Resolution Strategies:
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Post-translational modifications: Check for phosphorylation at Ser12 via mass spectrometry.
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Cofactor stability: Pre-incubate with 10 mM DTT to stabilize tetrahydrofolate.
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Assay interference: Test inhibitors like FdUMP (5-fluoro-dUMP) to confirm target specificity.
What cross-species insights from Staphylococcus aureus thyA studies apply to S. pneumoniae?
Key Findings:
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thyA inactivation reduces virulence factors (e.g., α-hemolysin) by 80% in S. aureus .
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thyA mutants upregulate adhesion genes (fnbA, clfB) by 4.5-fold, altering infectivity dynamics.
Methodological Transfer:
Use allelic replacement vectors (e.g., pMAD) to introduce S. aureus thyA mutations into S. pneumoniae. Monitor biofilm formation via crystal violet assays.
How can molecular dynamics simulations improve thyA mutagenesis experiments?
Protocol:
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Model wild-type thyA (PDB 1TJS) using GROMACS.
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Introduce mutations (e.g., G148W) and simulate for 50 ns.
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Identify destabilized regions (RMSF >2 Å) for experimental targeting.
Validation: Compare simulated Km with enzyme kinetics. A 2014 study achieved 88% correlation between predicted and observed dUMP binding affinities .
What structural insights guide thyA conjugation to polysaccharide vaccines?
Challenge: Carrier protein conformation affects epitope exposure during conjugation .
Approaches:
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Site-specific conjugation: Introduce cysteine residues at surface loops (e.g., Loop 3) via PCR mutagenesis.
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Analytical validation: Use MALDI-TOF to confirm a 1:1 polysaccharide:thyA ratio.
Data: Conjugating thyA to group A meningococcal polysaccharide increased Th1/Th2 ratios by 2.3-fold vs. TT carriers .
