Recombinant Rhodobacter sphaeroides Methionyl-tRNA formyltransferase (fmt)

What is the primary function of Rhodobacter sphaeroides Methionyl-tRNA formyltransferase?

Methionyl-tRNA formyltransferase (Fmt) catalyzes the formylation of methionyl-tRNA^fMet (Met-tRNA^fMet) to form formylmethionyl-tRNA^fMet (fMet-tRNA^fMet). This formylation is essential for efficient translation initiation in bacteria like R. sphaeroides and in eukaryotic organelles. The formylation process significantly enhances the fidelity of translation initiation by ensuring proper start codon recognition .

Recent research has demonstrated that Fmt-mediated formylation is not merely a vestigial process but plays a critical role in maintaining translational accuracy. Studies have shown that Fmt deletion strains (Δfmt) exhibit reduced growth rates and altered protein expression profiles, indicating its importance in cellular metabolism .

What substrates can R. sphaeroides Fmt utilize for the formylation reaction?

R. sphaeroides Fmt exhibits substrate flexibility that may contribute to its metabolic versatility. While 10-formyltetrahydrofolate (10-CHO-THF) is the canonical formyl group donor, recent research has revealed that Fmt can also utilize 10-formyldihydrofolate (10-CHO-DHF) as an alternative substrate for formylating Met-tRNA^fMet. This finding is significant as it demonstrates previously unrecognized metabolic flexibility in the formylation reaction .

The dual substrate capability has been confirmed through both in vivo and in vitro approaches. In vitro assays have demonstrated the formation of dihydrofolate (DHF) as a by-product when 10-CHO-DHF serves as the formyl donor, which was verified by LC-MS/MS analysis .

How can I determine the kinetic parameters of R. sphaeroides Fmt with different substrates?

To determine kinetic parameters of recombinant Fmt with different substrates, researchers should follow this methodology:

• Protein Preparation: Purify recombinant R. sphaeroides Fmt to >85% homogeneity using affinity chromatography.

• Substrate Preparation: Prepare varying concentrations of substrates (10-CHO-THF and 10-CHO-DHF, typically ranging from 5-200 µM).

• Reaction Setup:

  • Prepare Met-tRNA^fMet by charging deacylated tRNAs with methionine using methionyl-tRNA synthetase (MetRS)

  • Incubate Met-tRNA^fMet with recombinant Fmt (0.2 µg) and varying concentrations of formyl donors

  • Conduct reactions in aminoacylation buffer at room temperature

• Activity Measurement: Determine formylation by acid urea PAGE and Northern blotting with tRNA^fMet-specific probes, or by direct measurement of DHF formation using LC-MS/MS.

• Data Analysis: Calculate kinetic parameters (Km, kcat, Vmax) using Lineweaver-Burk or Eadie-Hofstee plots.

Typical reaction conditions include: aminoacylation buffer (100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 0.1% BSA), 25-100 µM formyl donor, 0.2 µg Fmt, and 10 min incubation at room temperature .

How can I perform an in vitro formylation assay with recombinant R. sphaeroides Fmt?

In vitro formylation assays with recombinant R. sphaeroides Fmt require careful preparation of both the enzyme and substrates. The following methodological approach is recommended:

• tRNA Preparation:

  • Isolate total tRNA from a Δfmt strain (to ensure absence of pre-formylated tRNA^fMet)

  • Alternatively, use recombinant expression systems overexpressing initiator tRNA^fMet

• Aminoacylation:

  • Charge the tRNA^fMet with methionine using purified MetRS (approximately 180 ng)

  • Incubate total tRNA preparations (10 µg) with MetRS in aminoacylation buffer (100 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 0.1% BSA, 2 mM methionine) for 1 hour

• Formylation Reaction:

  • Add recombinant Fmt (0.2 µg) and formyl donors (10-CHO-THF or 10-CHO-DHF, 25-100 µM)

  • Incubate for 10 minutes at room temperature

  • Stop reaction with 0.1 M HCl and 0.1 M β-mercaptoethanol

• Analysis Method:

  • Resolve samples on acid urea PAGE

  • Perform Northern blotting using 5'-32P end-labeled DNA oligomers complementary to tRNA^fMet

  • Expose to phosphor-imager screen and analyze on a Bio Image analyzer

For optimal activity, store recombinant Fmt at -20°C and avoid repeated freeze-thaw cycles. Working aliquots may be stored at 4°C for up to one week .

How can I detect the formylation status of tRNA^fMet in vivo?

To detect the formylation status of tRNA^fMet in bacterial cells, Northern blotting under specific conditions is recommended:

• RNA Isolation:

  • Extract total RNA under cold and acidic conditions to preserve the ester bond linking amino acids to tRNA

  • Perform extractions at 4°C using acidic phenol (pH 4.5-5.0)

• Selective Deacylation:

  • For aminoacyl-tRNA (Met-tRNA^fMet): Deacylate with 10 mM CuSO4 in 100 mM Tris-HCl (pH 8.0)

  • For both formylaminoacyl- and aminoacyl-forms (fMet-tRNA^fMet and Met-tRNA^fMet): Deacylate with 100 mM Tris-HCl (pH 9.0)

• Electrophoretic Separation:

  • Resolve tRNAs on acid urea polyacrylamide gel electrophoresis

  • Use 6.5% polyacrylamide gels containing 8 M urea and 0.1 M sodium acetate (pH 5.0)

• Northern Blotting Analysis:

  • Transfer RNA to nylon membranes

  • Hybridize with a 5'-32P end-labeled DNA oligomer complementary to positions 25-39 of tRNA^fMet

  • Quantify formylation status by measuring the mobility shift between formylated and non-formylated species

The formylated species exhibits a slightly lower electrophoretic mobility compared to the non-formylated form, allowing for quantitative assessment of in vivo formylation levels.

How does Fmt's utilization of 10-CHO-DHF impact antifolate drug sensitivity?

The discovery that Fmt can utilize 10-CHO-DHF as an alternative substrate has significant implications for antifolate drug sensitivity. Research has demonstrated that:

• Increased Trimethoprim Sensitivity: FolD-deficient mutants and Fmt over-expressing strains show increased sensitivity to trimethoprim (TMP) compared to Δfmt strains. This suggests that Fmt's activity contributes to antifolate drug action through a domino effect that ultimately inhibits protein synthesis .

• Mechanistic Basis: When TMP inhibits dihydrofolate reductase (DHFR), it leads to accumulation of DHF and depletion of reduced folate species. Fmt's ability to utilize 10-CHO-DHF may exacerbate this effect by:

  • Consuming available 10-CHO-DHF

  • Producing additional DHF as a byproduct

  • Creating a metabolic bottleneck in folate metabolism

• Folate Pool Dynamics: Antifolate treatment in E. coli leads to depletion of reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF, and 5-CHO-THF) and increases oxidized folate species (folic acid and DHF). In stationary phase, 10-CHO-DHF and 10-CHO-folic acid are enriched .

This research suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway that contributes to generating other folate intermediates and fMet-tRNA^fMet, potentially providing a new target for antimicrobial development.

What is the role of R. sphaeroides Fmt in bacterial adaptation to different growth conditions?

R. sphaeroides is a metabolically versatile bacterium that can adapt to diverse growth conditions. The role of Fmt in this adaptation process appears multifaceted:

• Core Proteome Component: Methionyl-tRNA formyltransferase is part of the core proteome of R. sphaeroides, suggesting its expression is largely ubiquitous, abundant, and likely independent of culture condition. Analysis of the R. sphaeroides proteome identified Fmt among the core proteins observed across different growth conditions .

• Transcriptional Regulation: Genome-wide transcriptome analysis of R. sphaeroides under three diverse growth modes (aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis) revealed significant differences in gene expression patterns. While specific data on Fmt expression changes wasn't provided, proteins involved in translation initiation may be regulated as part of these adaptive responses .

• Metabolic Versatility: R. sphaeroides can utilize different metabolic pathways, including the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and mevalonate (MVA) pathway for isoprenoid biosynthesis. The formylation of methionyl-tRNA may interact with these pathways through one-carbon metabolism connections .

• Stress Response: Under conditions like stationary phase growth where 10-CHO-DHF levels are enriched, Fmt's ability to utilize this alternative substrate may provide metabolic flexibility that supports adaptation to nutrient limitation or other stresses .

Understanding these adaptive roles could provide insights into bacterial persistence mechanisms and potential targets for antimicrobial development targeting metabolically flexible bacteria.

How does R. sphaeroides Fmt compare with other bacterial formyltransferases?

A comparative analysis of R. sphaeroides Fmt with formyltransferases from other bacterial species reveals important evolutionary and functional insights:

*Sequence identity compared to R. sphaeroides ATCC 17029 Fmt

Key observations from comparative analysis:

• Conservation Pattern: The formyltransferase domain is highly conserved across bacterial species, reflecting the essential nature of this enzyme in protein synthesis initiation.

• Substrate Flexibility: While R. sphaeroides Fmt demonstrates flexibility in utilizing both 10-CHO-THF and 10-CHO-DHF, the relative efficiency with these substrates varies across species. This flexibility may represent an evolutionary adaptation to different metabolic conditions .

• Structural Features: R. sphaeroides Fmt contains the characteristic SPDFSV motif near the N-terminus that is involved in substrate binding, though with species-specific variations that may account for differences in substrate preference.

• Evolutionary Context: As part of the core bacterial proteome, Fmt has been conserved throughout bacterial evolution while acquiring species-specific adaptations that may reflect the ecological niche and metabolic capabilities of each organism .

These comparative insights can guide structure-function studies and inform strategies for developing species-specific inhibitors targeting bacterial translation initiation.

What experimental approaches can determine if R. sphaeroides Fmt functions differently from E. coli Fmt?

To determine functional differences between R. sphaeroides and E. coli Fmt, several experimental approaches can be employed:

• Heterologous Complementation Studies:

  • Generate Δfmt mutants in both R. sphaeroides and E. coli

  • Express each Fmt ortholog in both deletion backgrounds

  • Assess growth rates, translation efficiency, and stress response

  • Measure formylation levels under identical conditions

• Comparative Enzymatic Assays:

  • Purify recombinant Fmt from both species

  • Determine kinetic parameters (Km, kcat, Vmax) with various substrates

  • Compare temperature and pH optima, metal ion requirements

  • Measure activity with 10-CHO-THF versus 10-CHO-DHF

• Structural Studies:

  • Perform X-ray crystallography or cryo-EM on both proteins

  • Compare substrate binding pockets and active site architecture

  • Conduct molecular dynamics simulations to identify structural determinants of substrate preference

• Targeted Mutagenesis:

  • Create chimeric enzymes with domains swapped between species

  • Perform site-directed mutagenesis of non-conserved residues

  • Test mutants for altered substrate specificity or kinetic parameters

• Metabolomic Profiling:

  • Monitor folate metabolite profiles in wild-type and Fmt-modified strains

  • Quantify 10-CHO-THF, 10-CHO-DHF, and other folate species

  • Assess metabolic changes following antifolate treatment

These approaches can reveal the molecular basis for any functional differences between the Fmt enzymes from these evolutionarily distinct bacterial species, potentially uncovering principles of enzymatic adaptation to different metabolic environments .

How can recombinant R. sphaeroides Fmt be optimally expressed and purified for experimental use?

For optimal expression and purification of recombinant R. sphaeroides Fmt, researchers should consider the following methodological approach:

• Expression System Selection:

  • Mammalian cell systems provide proper folding and post-translational modifications for R. sphaeroides ATCC 17029 Fmt

  • Yeast expression systems work well for R. sphaeroides ATCC 17025 Fmt

  • E. coli systems may be used but might require optimization of codon usage and growth conditions

• Construct Design:

  • Include the full-length protein (302 amino acids)

  • Consider adding a purification tag (His-tag is commonly used)

  • Use appropriate promoter systems (inducible promoters allow controlled expression)

• Expression Conditions:

  • For mammalian systems: Maintain cells at 37°C, 5% CO₂

  • For yeast systems: Optimal growth at 30°C

  • Induction parameters should be optimized based on expression system

• Purification Protocol:

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Consider ion exchange chromatography as a secondary purification step

  • Aim for >85% purity as assessed by SDS-PAGE

• Storage Recommendations:

  • Store at -20°C for regular use, or -80°C for extended storage

  • Add 5-50% glycerol (final concentration) to prevent freeze-thaw damage

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

The reconstitution of lyophilized protein should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL with appropriate glycerol addition for stability.

What are potential applications of recombinant R. sphaeroides Fmt in studying bacterial adaptation mechanisms?

Recombinant R. sphaeroides Fmt offers several research applications for studying bacterial adaptation mechanisms:

• Metabolic Flexibility Studies:

  • Investigate how Fmt's dual substrate utilization contributes to metabolic resilience

  • Study adaptation to folate-limited environments

  • Examine the role of formylation in response to antifolate treatments

• Comparative Systems Biology:

  • Use recombinant Fmt to study differences in translation initiation across species

  • Compare formylation efficiency under various stress conditions

  • Investigate species-specific adaptations in the folate metabolism network

• Antibiotic Resistance Research:

  • Evaluate how Fmt activity influences sensitivity to trimethoprim and other antifolates

  • Study the relationship between translation initiation and antibiotic tolerance

  • Develop combination therapies targeting both Fmt and other folate metabolism enzymes

• Synthetic Biology Applications:

  • Engineer translation initiation systems with modified Fmt proteins

  • Design Fmt variants with altered substrate specificity

  • Create biosensors for folate metabolism perturbations

• Environmental Adaptation Studies:

  • Investigate how R. sphaeroides maintains translation under varying growth modes (aerobic, anaerobic, photosynthetic)

  • Study the role of Fmt in stationary phase survival

  • Examine formylation patterns in response to environmental stressors

These applications leverage R. sphaeroides' remarkable metabolic versatility and the dual substrate capability of its Fmt enzyme to provide insights into bacterial adaptation strategies that could inform both fundamental microbiology and applied biotechnology.

What are common challenges in working with recombinant R. sphaeroides Fmt and how can they be addressed?

Researchers working with recombinant R. sphaeroides Fmt may encounter several challenges. Here are common issues and recommended solutions:

• Low Enzymatic Activity:

  • Problem: Purified Fmt shows reduced or no activity in formylation assays.

  • Solutions:

    • Verify protein folding integrity through circular dichroism

    • Ensure proper storage conditions with glycerol addition

    • Check for inhibitory contaminants in the reaction buffer

    • Confirm tRNA substrate is properly aminoacylated

    • Add reducing agents (e.g., DTT or β-mercaptoethanol) to prevent oxidation

• Substrate Availability:

  • Problem: Difficulty obtaining or synthesizing 10-CHO-THF and 10-CHO-DHF substrates.

  • Solutions:

    • Use enzymatic synthesis with purified FolD to generate 10-CHO-THF

    • Consider commercial sources for folate derivatives

    • Implement HPLC purification to ensure substrate quality

    • Verify substrate integrity through LC-MS/MS before use

• tRNA Substrate Preparation:

  • Problem: Inefficient aminoacylation of tRNA^fMet.

  • Solutions:

    • Optimize MetRS concentration and reaction conditions

    • Ensure tRNA is properly folded by heating and cooling

    • Check for inhibitory contaminants in tRNA preparations

    • Consider using in vitro transcribed tRNA^fMet for consistency

• Detection Sensitivity:

  • Problem: Difficulty detecting formylated tRNA^fMet.

  • Solutions:

    • Implement acid urea PAGE with optimized conditions

    • Use highly sensitive Northern blotting techniques

    • Consider radioactive labeling for increased sensitivity

    • Optimize probe design for tRNA^fMet detection

• Protein Stability:

  • Problem: Rapid loss of enzymatic activity during storage.

  • Solutions:

    • Store at -20°C or -80°C with 50% glycerol

    • Avoid repeated freeze-thaw cycles

    • Store working aliquots at 4°C for maximum one week

    • Consider adding stabilizing agents like BSA

Careful optimization of each step in the experimental workflow can significantly improve success when working with this enzyme.

How can I design experiments to investigate the relationship between Fmt activity and antifolate drug resistance?

To investigate the relationship between R. sphaeroides Fmt activity and antifolate drug resistance, consider the following experimental design approaches:

• Genetic Manipulation Studies:

  • Generate strains with varying Fmt expression levels (knockout, wild-type, overexpression)

  • Create point mutants with altered substrate specificity

  • Introduce heterologous Fmt enzymes from different bacterial species

• Dose-Response Analysis:

  • Determine minimum inhibitory concentrations (MICs) of trimethoprim and other antifolates

  • Generate comprehensive dose-response curves for each strain

  • Analyze growth kinetics using automated plate readers for high temporal resolution

• Combinatorial Drug Testing:

  • Test synergy between antifolates and other antibiotics

  • Evaluate effects of combining DHFR inhibitors with FolD inhibitors

  • Assess impact of sequential versus simultaneous drug administration

• Metabolomic Profiling:

  • Quantify folate metabolites before and after antifolate treatment

  • Monitor 10-CHO-THF/10-CHO-DHF ratios in different genetic backgrounds

  • Measure flux through folate-dependent pathways

• Translation Fidelity Assessment:

  • Analyze protein synthesis rates using radioactive amino acid incorporation

  • Measure translation initiation efficiency with reporter constructs

  • Quantify mistranslation rates using specialized reporter systems

• Evolution Experiments:

  • Subject strains to gradually increasing antifolate concentrations

  • Sequence evolved strains to identify resistance mutations

  • Characterize Fmt activity changes in resistant isolates

• Experimental Controls:

  • Include control strains with mutations in other folate metabolism genes

  • Test non-antifolate antibiotics to confirm specificity

  • Use defined media to control folate availability

This systematic approach can reveal causal relationships between Fmt activity, folate metabolism, and antifolate resistance mechanisms, potentially identifying new therapeutic strategies targeting bacterial translation initiation .