Recombinant Staphylococcus aureus Methionyl-tRNA formyltransferase (fmt)

What is the role of Methionyl-tRNA formyltransferase (fmt) in S. aureus protein synthesis?

Methionyl-tRNA formyltransferase (fmt) catalyzes the transfer of a formyl group from 10-formyltetrahydrofolate to the amino group of methionine attached to initiator tRNA, producing formylmethionyl-tRNAifMet. This formylation is crucial for bacterial protein synthesis as it enables specific recognition by initiation factor IF2 and facilitates proper assembly of the initiation complex on the ribosome. In S. aureus, research has revealed that the T-box riboswitch mechanism regulates methionine biosynthesis, with specific interactions occurring between the met leader RNA and initiator formylmethionyl-tRNA (tRNAifMet) .

To investigate fmt function in S. aureus protein synthesis, researchers typically employ:

• In vitro formylation assays with purified components

• Pulse-chase experiments to measure protein synthesis rates

• Ribosome profiling to assess translation initiation efficiency

• Fmt depletion or mutation studies to evaluate effects on protein synthesis

How does methionine biosynthesis regulation interact with fmt activity in S. aureus?

The relationship between methionine biosynthesis and fmt activity in S. aureus involves complex regulatory mechanisms:

In S. aureus, methionine biosynthesis is regulated through a T-box riboswitch mechanism rather than the S-box riboswitches found in other Bacillales. The met leader RNA specifically interacts with uncharged initiator formylmethionyl-tRNA (tRNAifMet), suggesting a direct regulatory connection between methionine availability and protein synthesis initiation .

Experimental approaches to study this interaction include:

• RNA-protein binding assays demonstrating that the met leader RNA interacts strongly with tRNAifMet compared to other methionyl-tRNAs

• Northern blot analysis to assess transcript stability of methionine biosynthesis genes

• Mutational analysis of the T-box riboswitch to evaluate effects on fmt expression

• Metabolic labeling to track methionine flux through protein synthesis

The metICFE-mdh mRNA stability is regulated by RNase activity, with evidence suggesting RNase J2 involvement in its degradation . This post-transcriptional regulation adds another layer of control that may indirectly impact fmt activity by affecting methionine availability.

What expression systems are optimal for producing functional recombinant S. aureus fmt?

Optimizing expression of recombinant S. aureus fmt requires careful consideration of factors that maintain protein solubility and enzymatic activity:

Recommended expression systems:

• E. coli BL21(DE3) with pET vectors:

  • Culture at 18-22°C after induction with 0.1-0.5 mM IPTG

  • Include 10 μM folate in growth media to enhance cofactor incorporation

  • Codon optimization may improve expression due to different codon usage patterns

• E. coli SHuffle strains:

  • Provides improved disulfide bond formation if necessary for fmt structure

  • Useful for obtaining correctly folded protein with higher activity

Fusion tags and optimization strategies:

Critical parameters for optimal expression:

• Induction at OD600 = 0.6-0.8

• Post-induction temperature: 18-20°C

• Expression time: 16-20 hours

• Addition of 1-5% glucose to reduce basal expression

• Supplementation with folate to ensure proper cofactor incorporation

What are the key structural features and catalytic residues of S. aureus fmt?

S. aureus fmt shares structural features common to bacterial methionyl-tRNA formyltransferases, consisting of:

• N-terminal domain:

  • Contains the binding site for 10-formyltetrahydrofolate donor

  • Highly conserved across bacterial species

  • Folate binding pocket characterized by positively charged residues

• Catalytic core:

  • Contains the active site where formyl transfer occurs

  • Key catalytic residues include conserved histidine (His) and asparagine (Asn)

  • Coordination of both methionyl-tRNA and formyl donor

• C-terminal domain:

  • Responsible for tRNA recognition and binding

  • Contains specificity-determining residues that distinguish initiator tRNA

The structural features of bacterial formyltransferases have been extensively studied, as seen in related research on FmtA, another S. aureus protein that contains catalytic serine, lysine, and tyrosine residues in its active site . While FmtA is a different protein with esterase activity, it demonstrates how careful structural characterization can reveal functional mechanisms of S. aureus enzymes.

For structure-function studies, researchers employ:

• Site-directed mutagenesis of conserved residues

• X-ray crystallography of fmt alone and in complex with substrates

• Molecular dynamics simulations to understand conformational changes

• Comparative analysis with fmt from other bacterial species

What methods are used to analyze fmt enzymatic activity in vitro?

In vitro analysis of fmt activity requires sensitive and specific assays:

Radiometric assays:

• Incubation of fmt with [³H]-methionyl-tRNAifMet and 10-formyltetrahydrofolate

• Precipitation of tRNA with trichloroacetic acid and filter binding

• Quantification by scintillation counting

• Advantages: High sensitivity, direct measurement of product formation

HPLC-based methods:

• Separation of formylmethionyl-tRNA from methionyl-tRNA

• Detection by UV absorbance at 260 nm

• Quantification by peak area integration

• Advantages: No radioactivity, ability to monitor reaction progress

Mass spectrometry approaches:

• LC-MS analysis of aminoacyl-tRNA before and after fmt reaction

• Detection of mass shift corresponding to formyl addition

• Advantages: High specificity, potential for high-throughput analysis

Coupled enzyme assays:

• Link fmt activity to production of tetrahydrofolate

• Monitor through changes in absorbance or fluorescence

• Advantages: Continuous monitoring, adaptable to plate reader format

These methodological approaches are similar to those used for studying RNA-tRNA interactions in S. aureus methionine biosynthesis regulation, where binding between radioactively labeled tRNA and in vitro-transcribed met leader RNA is determined by non-denaturing polyacrylamide gel electrophoresis .

How does fmt contribute to antibiotic resistance mechanisms in S. aureus?

Fmt plays significant roles in antibiotic resistance through several mechanisms:

Direct resistance mechanisms:

• Peptidyl deformylase (PDF) inhibitor resistance:

  • Reduced fmt activity decreases dependence on formylated proteins

  • PDF inhibitors become less effective when formylation is decreased

  • Experimental approach: Compare MICs of PDF inhibitors in wild-type vs. fmt-attenuated strains

• Protein synthesis adaptation:

  • Alterations in fmt activity modify translation initiation patterns under antibiotic stress

  • May enable selective synthesis of resistance factors

  • Methodology: Ribosome profiling and proteomics to identify fmt-dependent changes

Indirect resistance contributions:

• Stress response regulation:

  • Fmt activity connects to stringent response via methionine metabolism

  • In S. aureus, methionine biosynthesis is regulated by T-box riboswitches and affected by stringent response-controlled CodY activity

  • This regulatory network may enhance survival under antibiotic pressure

• Biofilm formation:

  • Alterations in protein synthesis initiation may affect biofilm-related protein expression

  • Biofilms significantly increase antibiotic tolerance

  • Research approach: Compare biofilm formation in fmt mutants vs. wild-type

Resistance analysis methodologies include:

• Creation of fmt variants using site-directed mutagenesis

• Antibiotic susceptibility testing using broth microdilution

• Time-kill kinetics to assess dynamics of antibiotic action

• Fitness measurement in presence of subinhibitory antibiotic concentrations

• Transcriptomics to identify compensatory changes in gene expression

What approaches are effective for purifying recombinant S. aureus fmt while maintaining activity?

Purification of recombinant S. aureus fmt requires careful optimization to preserve enzymatic function:

Recommended purification workflow:

• Cell lysis buffer optimization:

  • 50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

  • Addition of protease inhibitors (PMSF, leupeptin, pepstatin A)

  • Gentle lysis using sonication (5 cycles of 10s on/30s off) or pressure-based methods

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged fmt

  • Washing with 20-50 mM imidazole to remove non-specific binding

  • Elution with 250-300 mM imidazole gradient

  • Include 1 mM DTT and 10% glycerol in all buffers to maintain stability

• Ion exchange chromatography:

  • Anion exchange (Q Sepharose) at pH 8.0 or cation exchange (SP Sepharose) at pH 6.5

  • Salt gradient elution (100-500 mM NaCl)

  • Collect fractions and test for activity

• Size exclusion chromatography:

  • Final polishing step using Superdex 75/200

  • Running buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

  • Collect monomeric protein fractions

Critical parameters for activity preservation:

Activity validation after purification:

• Formylation assay using purified S. aureus tRNAifMet

• Thermal shift assay to confirm proper folding

• Size exclusion chromatography to verify monomeric state

• Circular dichroism to assess secondary structure integrity

How can fmt-tRNA interactions be characterized at the molecular level?

Characterizing interactions between fmt and its tRNA substrate employs multiple complementary approaches:

Binding assays:

• Electrophoretic mobility shift assay (EMSA):

  • Incubate varying concentrations of fmt with radiolabeled methionyl-tRNAifMet

  • Resolve complexes on non-denaturing polyacrylamide gels

  • Quantify bound vs. unbound fractions to determine binding affinity (Kd)

  • This approach is similar to methods used for studying met leader RNA interactions with methionyl-tRNAs in S. aureus

• Surface plasmon resonance (SPR):

  • Immobilize fmt or tRNA on sensor chip

  • Flow the binding partner at varying concentrations

  • Determine association (kon) and dissociation (koff) rate constants

  • Calculate equilibrium binding constant (Kd = koff/kon)

• Microscale thermophoresis (MST):

  • Label fmt or tRNA with fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Determine binding affinity from titration curves

  • Advantages: Low sample consumption, solution-based measurement

Structural studies:

• X-ray crystallography:

  • Co-crystallize fmt with methionyl-tRNAifMet

  • Determine atomic-resolution structure

  • Identify specific residue-nucleotide interactions

  • Similar approaches have been used for structural studies of other S. aureus proteins like FmtA

• Cryo-electron microscopy:

  • Visualize fmt-tRNA complexes at near-atomic resolution

  • Capture multiple conformational states

  • Advantages: No crystallization required, conformational heterogeneity preserved

Functional interaction analysis:

• Mutagenesis studies:

  • Create fmt variants with mutations in predicted tRNA-binding residues

  • Measure effects on binding affinity and catalytic activity

  • Map the functional interaction surface

• tRNA modification analysis:

  • Prepare tRNAs with specific modifications or lacking certain modifications

  • Assess impact on fmt recognition and activity

  • Determine the contribution of tRNA modifications to specificity

• Footprinting assays:

  • Use chemical or enzymatic probes to identify tRNA regions protected by fmt binding

  • Compare accessibility patterns in free vs. fmt-bound tRNA

  • Map the interaction interface on the tRNA molecule

How can contradictions in fmt research data be reconciled across different experimental conditions?

Reconciling contradictory findings in fmt research requires systematic analysis of methodological differences:

Sources of data contradiction:

• Enzyme preparation variations:

  • Different expression systems yielding variable protein conformations

  • Presence/absence of tags affecting activity measurements

  • Variable cofactor saturation levels

• Assay condition differences:

  • Buffer composition (pH, ionic strength) affecting enzyme-substrate interactions

  • Temperature variations altering reaction kinetics

  • Differences in substrate preparation and quality

• Detection method discrepancies:

  • Varied sensitivities and dynamic ranges across methods

  • Different data normalization approaches

  • Time-dependent measurements vs. endpoint analysis

Contradiction resolution strategy:

• Standardize methodology:

  • Develop consensus protocols for fmt expression and purification

  • Establish standard assay conditions for activity measurements

  • Create reference standards for inter-laboratory comparisons

• Perform side-by-side comparisons:

  • Test multiple fmt preparations under identical conditions

  • Apply multiple detection methods to the same reaction

  • Compare across a range of conditions rather than single-point measurements

• Meta-analysis approach:

  • Systematically analyze published fmt data with attention to methodological details

  • Identify patterns in results related to specific experimental variables

  • Develop predictive models to reconcile apparently contradictory data

These approaches align with clinical contradiction detection methodologies that classify contradictions based on their source and systematically resolve them through analysis of underlying factors . The same principles can be applied to contradictions in fmt research data.

Contradiction resolution flowchart:

• Identify specific contradictory claims

• Catalog methodological differences

• Perform controlled experiments to test each variable

• Develop unified model that explains condition-dependent differences

• Validate model with new experimental data

What are the current approaches for studying fmt substrate specificity?

Studying S. aureus fmt substrate specificity employs multiple complementary methodologies:

tRNA substrate specificity analysis:

• Activity screening with diverse tRNAs:

  • Compare formylation efficiency with initiator vs. elongator tRNAs

  • Determine kinetic parameters (kcat, Km) for each substrate

  • Calculate specificity constants (kcat/Km) to quantify preference

  • Similar approaches have been used to study met leader RNA interactions with different methionyl-tRNAs in S. aureus

• tRNA structural determinant mapping:

  • Generate chimeric tRNAs with swapped domains between initiator and elongator tRNAs

  • Create point mutations in key identity elements

  • Correlate structural features with fmt recognition

  • Methodology: In vitro transcription of tRNA variants followed by aminoacylation and formylation assays

Formyl donor specificity analysis:

• Alternative donor testing:

  • Synthesize structural analogs of 10-formyltetrahydrofolate

  • Measure fmt activity with each analog

  • Structure-activity relationship analysis

  • Methodology: HPLC or radiometric assays to measure product formation

• Binding studies:

  • Isothermal titration calorimetry with different donor molecules

  • Determine binding affinity (Kd) and thermodynamic parameters

  • Correlate binding energy with catalytic efficiency

Comprehensive specificity profiling:

These methodologies enable detailed characterization of fmt substrate preferences, which is crucial for understanding its biological function and developing specific inhibitors.

How can fmt be targeted for antimicrobial development against S. aureus?

Fmt represents a promising antimicrobial target due to its essential role in bacterial protein synthesis and absence in humans:

Target validation approaches:

• Genetic validation:

  • Create conditional fmt knockdown strains

  • Demonstrate growth inhibition upon fmt depletion

  • Assess virulence attenuation in animal models

  • Methodology: Inducible antisense RNA or CRISPR interference

• Chemical validation:

  • Identify tool compounds that inhibit fmt activity

  • Demonstrate correlation between biochemical and cellular activities

  • Confirm target engagement through resistant mutant generation

  • Methodology: Enzyme assays coupled with whole-cell activity testing

Inhibitor discovery strategies:

• Structure-based design:

  • Use crystal structures of fmt to design competitive inhibitors

  • Focus on formyl donor site or tRNA binding interface

  • Employ molecular docking and virtual screening

  • Methodology: Fragment-based lead discovery followed by structure-guided optimization

• High-throughput screening:

  • Develop miniaturized fmt activity assays

  • Screen diverse chemical libraries

  • Include counterscreens to eliminate non-specific inhibitors

  • Methodology: Fluorescence-based activity assays in 384 or 1536-well format

• Natural product exploration:

  • Screen microbial extracts for fmt inhibitory activity

  • Isolate and characterize active compounds

  • Optimize leads through semi-synthetic modification

  • Methodology: Bioassay-guided fractionation coupled with structure elucidation

Lead optimization considerations:

Combination therapy potential:

• Test fmt inhibitors with established antibiotics

• Identify synergistic combinations

• Evaluate prevention of resistance emergence

• Methodology: Checkerboard assays and time-kill studies

What structural biology techniques are most effective for studying fmt-substrate complexes?

Structural characterization of S. aureus fmt in complex with its substrates requires specialized techniques:

X-ray crystallography approaches:

• Co-crystallization strategies:

  • Mix purified fmt with methionyl-tRNAifMet and non-hydrolyzable formyl donor

  • Screen crystallization conditions systematically

  • Optimize crystals for high-resolution diffraction

  • Data collection at synchrotron radiation sources

  • Similar approaches have yielded high-resolution structures of other S. aureus proteins like FmtA

• Soaking methods:

  • Obtain apo-fmt crystals, then soak with substrates or inhibitors

  • Verify ligand binding by difference electron density

  • Advantages: Higher success rate than co-crystallization for some ligands

Cryo-electron microscopy methods:

• Single-particle analysis:

  • Prepare fmt-tRNA-formyl donor complexes and vitrify

  • Collect thousands of particle images

  • Perform 2D classification and 3D reconstruction

  • Advantages: No crystallization required, conformational heterogeneity preserved

• Time-resolved cryo-EM:

  • Capture reaction intermediates by rapid mixing and freezing

  • Visualize structural changes during catalysis

  • Classify particles by conformational state

  • Advantages: Provides dynamic view of catalytic mechanism

Solution-based structural methods:

• Small-angle X-ray scattering (SAXS):

  • Collect scattering data from fmt-substrate complexes in solution

  • Generate low-resolution envelope models

  • Dock high-resolution structures into SAXS envelopes

  • Advantages: Physiological solution conditions, no crystals needed

• Nuclear magnetic resonance (NMR):

  • Prepare isotope-labeled fmt for chemical shift analysis

  • Map binding interfaces through chemical shift perturbation

  • Study dynamics of substrate recognition

  • Advantages: Provides information on protein dynamics in solution

Integrative structural biology:

Combining multiple structural techniques provides comprehensive understanding of fmt-substrate interactions:

How can modern sequencing methodologies enhance fmt functional studies?

Next-generation sequencing approaches provide powerful tools for studying fmt function and regulation:

Transcriptome analysis:

• RNA-Seq to study fmt regulation:

  • Compare transcriptome profiles under conditions affecting fmt expression

  • Identify co-regulated genes and regulatory networks

  • Discover non-coding RNAs involved in fmt regulation

  • Methodology: Strand-specific RNA-Seq with rRNA depletion

• Ribosome profiling for translation analysis:

  • Measure ribosome occupancy on mRNAs in wild-type vs. fmt-depleted cells

  • Identify genes with fmt-dependent translation efficiency

  • Characterize changes in start codon selection

  • Methodology: Isolate ribosome-protected fragments and sequence

Protein-RNA interaction analysis:

• CLIP-Seq for fmt-tRNA interactions:

  • Crosslink fmt to bound RNAs in vivo

  • Immunoprecipitate fmt and sequence associated RNAs

  • Map fmt binding sites on tRNA and potential non-tRNA targets

  • Methodology: UV crosslinking followed by immunoprecipitation and sequencing

• RNA structural probing:

  • Compare tRNA structure in presence/absence of fmt

  • Identify structural changes upon binding

  • Map protection patterns indicative of interaction sites

  • Methodology: SHAPE-Seq or DMS-Seq approaches

These approaches can complement traditional biochemical studies of RNA-protein interactions, such as those used to characterize interactions between met leader RNA and methionyl-tRNAs in S. aureus .

Genomic approaches:

• Transposon sequencing (Tn-Seq):

  • Create transposon library in S. aureus

  • Subject to growth with fmt inhibitors or under fmt depletion

  • Identify genes affecting sensitivity or resistance

  • Methodology: Transposon insertion site sequencing before and after selection

• CRISPR interference screening:

  • Construct library targeting genes throughout S. aureus genome

  • Identify genetic interactions with fmt by differential growth analysis

  • Discover synthetic lethal or suppressor relationships

  • Methodology: sgRNA abundance quantification by next-generation sequencing

Clinical application of sequencing:

• Comparative genomics:

  • Analyze fmt sequence variation across clinical S. aureus isolates

  • Correlate polymorphisms with antibiotic resistance phenotypes

  • Identify potential resistance-associated mutations

  • Methodology: Whole genome sequencing of clinical isolates

• Metatranscriptomics:

  • Analyze fmt expression in diverse infection models

  • Compare expression patterns between antibiotic-sensitive and resistant strains

  • Identify condition-specific regulation of fmt

  • Methodology: RNA-Seq from infected tissues followed by pathogen transcript mapping