Recombinant Natranaerobius thermophilus Methionyl-tRNA formyltransferase (fmt)
Description
Overview of Methionyl-tRNA Formyltransferase (FMT)
Methionyl-tRNA formyltransferase (FMT; EC 2.1.2.9) is a critical enzyme in prokaryotic translation initiation. It catalyzes the transfer of a formyl group from 10-formyltetrahydrofolate to methionyl-tRNA, producing formylmethionyl-tRNA (fMet-tRNA), which is essential for initiating protein synthesis in bacteria and organelles .
2.1. Escherichia coli FMT
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Activity: Knockout strains exhibit severe growth defects, underscoring its necessity .
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Regulation: Expression is independent of intracellular FMT concentration, suggesting tight constitutive control .
2.2. Wolbachia pipientis Recombinant FMT (Reference Example)
While not from N. thermophilus, the recombinant FMT from Wolbachia pipientis (UniProt Q73GR6) provides a template for hypothetical properties:
| Parameter | Specification |
|---|---|
| Source | Yeast expression system |
| Purity | >85% (SDS-PAGE) |
| Storage | 12 months (lyophilized, -20°C/-80°C) |
| Reconstitution | 0.1–1.0 mg/mL in sterile water + 50% glycerol |
| Tag | Determined during manufacturing |
| Sequence Coverage | Full-length (1–299 aa) |
3.1. Adaptation to Polyextremophilic Conditions
N. thermophilus thrives at 3.3–3.9 M Na+, pH 9.5, and 53°C . Its FMT would likely exhibit:
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Thermostability: Enhanced structural rigidity for high-temperature function.
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Salt Tolerance: Surface charge modifications to resist ionic interference .
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Alkaline pH Activity: Optimal catalytic efficiency near pH 9.5, aligning with cytoplasmic pH homeostasis mechanisms .
3.2. Genomic and Proteomic Context
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Genomic linkage: fmt may co-occur with Na+/H+ antiporter genes critical for ion homeostasis .
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Expression under stress: Upregulated during osmotic stress, similar to glycine betaine transporters .
Research Gaps and Future Directions
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Recombinant Production: No studies describe cloning, expression, or purification of N. thermophilus FMT. Suggested systems:
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Biochemical Characterization:
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Kinetic assays under high Na+, alkaline pH, and elevated temperature.
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Structural studies (e.g., X-ray crystallography) to identify extremophilic adaptations.
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Essentiality Testing: Gene knockout experiments to assess fmt necessity in N. thermophilus .
Product Specs
Target Background
KEGG: nth:Nther_1334
STRING: 457570.Nther_1334
Q&A
Experimental Design for Studying Recombinant fmt in Natranaerobius thermophilus
Q: How would you design an experiment to study the role of recombinant Methionyl-tRNA formyltransferase (fmt) in Natranaerobius thermophilus, considering its unique extremophilic conditions?
A: To study recombinant fmt in Natranaerobius thermophilus, design an experiment that involves:
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Cloning and Expression: Clone the fmt gene into a suitable vector for expression in N. thermophilus. Use a promoter that is active under the organism's optimal conditions (e.g., high salinity and temperature).
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Growth Conditions: Grow N. thermophilus in carbonate-buffered medium with varying Na+ concentrations (e.g., 2.5 to 5.0 M) at 53°C to assess how fmt affects growth under different salinity levels.
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Enzyme Assays: Perform formyltransferase activity assays to measure the enzyme's efficiency in formylating methionyl-tRNA under these conditions.
Data Contradiction Analysis in fmt Deletion Studies
Q: How do you reconcile contradictory data regarding the essentiality of fmt in different bacterial species, such as Mycobacterium bovis vs. M. smegmatis?
A: Analyze the experimental conditions and methodologies used in each study. Consider factors like:
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Growth Conditions: Different growth conditions might affect the necessity of fmt for viability.
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Methodological Differences: Techniques used for gene deletion (e.g., allelic exchange vs. transposon mutagenesis) can influence outcomes.
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Metabolic Adaptations: Some bacteria may adapt to fmt deletion by using non-formylated methionine, which could explain variability in essentiality across species.
Advanced Research Questions: fmt in Extremophiles
Q: What advanced research questions can be explored regarding the role of fmt in extremophilic bacteria like Natranaerobius thermophilus?
A: Investigate:
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Mechanisms of Formylation Under Stress: Study how fmt functions under extreme conditions (high salinity, temperature) and its role in protein synthesis adaptation.
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Evolutionary Conservation: Compare fmt sequences across extremophiles to identify conserved regions and potential unique adaptations.
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fmt as a Drug Target: Explore whether fmt could serve as a drug target in pathogenic bacteria by comparing its function in extremophiles and pathogens.
Methodological Approaches for fmt Expression and Purification
Q: What methodological approaches are suitable for expressing and purifying recombinant fmt from Natranaerobius thermophilus?
A: Use:
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Heterologous Expression Systems: Express fmt in E. coli or other suitable hosts using vectors like pET or pBAD.
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Purification Techniques: Employ affinity chromatography (e.g., His-tag) followed by size exclusion chromatography to purify the enzyme.
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Optimization of Expression Conditions: Optimize temperature, IPTG concentration, and induction time to maximize protein yield and activity.
Data Interpretation and Statistical Analysis
Q: How do you interpret and statistically analyze data from experiments studying the effects of fmt on bacterial growth under different conditions?
A: Use:
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Growth Curve Analysis: Plot optical density (OD) over time to assess growth rates under various conditions.
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Statistical Tests: Apply ANOVA or t-tests to compare growth rates between conditions (e.g., with and without fmt).
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Data Visualization: Utilize plots to visualize differences in growth patterns and enzyme activity.
fmt in Comparative Genomics
Q: How can comparative genomics be used to study fmt across different bacterial species, including extremophiles?
A: Perform:
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Sequence Alignment: Align fmt sequences from various bacteria to identify conserved regions.
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Phylogenetic Analysis: Construct phylogenetic trees to understand evolutionary relationships among fmt genes.
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Functional Prediction: Use bioinformatics tools to predict functional differences based on sequence variations.
Challenges in Studying fmt in Extremophiles
Q: What are the challenges in studying fmt in extremophilic bacteria like Natranaerobius thermophilus, and how can they be addressed?
A: Challenges include:
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Cultivation Conditions: Maintaining optimal growth conditions (high salinity, temperature) can be difficult.
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Genetic Manipulation: Developing efficient genetic tools for extremophiles is challenging.
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Solutions: Use specialized media and equipment designed for extremophiles, and develop novel genetic manipulation techniques tailored to these organisms.
fmt as a Model for Understanding Protein Synthesis Adaptation
Q: How can studying fmt in extremophiles like Natranaerobius thermophilus contribute to understanding protein synthesis adaptation under stress?
A: Investigate:
Integration with Other Bacterial Processes
Q: How does fmt integrate with other bacterial processes, such as translation initiation and elongation, in extremophiles?
A: Study:
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Initiation Complex Formation: Investigate how fmt affects the formation of the translation initiation complex.
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Elongation Efficiency: Examine how fmt influences the efficiency of translation elongation under stress.
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Regulatory Feedback Loops: Identify potential regulatory feedback loops that modulate fmt activity based on cellular needs.
Future Directions in fmt Research
Q: What future directions in fmt research could provide new insights into bacterial physiology and potential drug targets?
A: Explore:
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Structural Biology: Determine the crystal structure of fmt to understand its mechanism and potential binding sites for inhibitors.
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In Vivo Studies: Conduct in vivo studies to assess the role of fmt in pathogenic bacteria and its potential as a drug target.
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Synthetic Biology Approaches: Use synthetic biology to engineer fmt variants with enhanced or reduced activity for biotechnological applications.
