Recombinant Escherichia fergusonii Elongation factor G (fusA), partial

What is Elongation Factor G (fusA) and what is its functional role in protein synthesis?

Elongation Factor G (EF-G), encoded by the fusA gene, is a critical protein involved in the translational elongation process during protein synthesis. EF-G functions primarily in the translocation step, where it facilitates the movement of tRNAs and mRNA through the ribosome after peptide bond formation. This process requires GTP hydrolysis, with EF-G acting as a GTPase.

Specifically, EF-G:

• Binds to the ribosome in its GTP-bound state

• Catalyzes the translocation of the peptidyl-tRNA from the A-site to the P-site

• Facilitates the movement of the mRNA by one codon

• Undergoes conformational changes upon GTP hydrolysis

• Dissociates from the ribosome in its GDP-bound form

In E. fergusonii, as in other bacteria, EF-G is essential for cell viability and proper protein synthesis. Research has shown that EF-G is also involved in ribosome recycling, working with the ribosome recycling factor (RRF) to disassemble the post-termination complex .

The fusA gene in E. fergusonii is highly conserved and has been used in molecular identification and diagnostic applications due to its ubiquity and sequence conservation across bacterial species .

How does the structure of E. fergusonii EF-G differ from those of other Escherichia species?

While a high-resolution crystal structure specific to E. fergusonii EF-G has not been widely reported, structural features can be inferred from homologous EF-Gs such as that from S. aureus, crystallized to 1.9 Å resolution .

Key structural features include:

Domain organization:

• Domain I (G domain): Contains the GTPase activity center with conserved switch regions

• Domain II: Contributes to GTPase function and ribosome interaction

• Domains III-V: Involved in interactions with the ribosome during translocation

• Domain IV: Contains a critical region that mimics the anticodon stem-loop of tRNA and inserts into the decoding center

Conformational flexibility:

• EF-G undergoes dramatic conformational changes between free and ribosome-bound states

• In S. aureus EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G by about 25 Å perpendicular to that observed in previous structures

• This flexibility is likely conserved in E. fergusonii EF-G and is essential for its function

Switch regions:

• The switch I region (residues 46-56) in S. aureus EF-G is ordered in a helix and has a distinct conformation compared to EF-Tu in GDP and GTP states

• The switch II region shows conformations incompatible with fusidic acid binding when EF-G is not bound to the ribosome

Comparing across Escherichia species reveals:

• High sequence conservation in domains I and II (containing the GTPase activity)

• Greater variability in domains III, IV, and V

• Species-specific surface epitopes that could be targeted for diagnostic purposes

What are the optimal expression systems for recombinant E. fergusonii EF-G production?

For successful expression of recombinant E. fergusonii EF-G, the following systems and conditions have proven most effective:

Prokaryotic expression systems:

• E. coli BL21(DE3) and derivatives: Most commonly used for bacterial protein expression due to high yield and simplicity

• E. coli strains with rare codon supplementation: Rosetta or CodonPlus strains if E. fergusonii fusA contains rare codons

• Vector selection: T7 promoter-based vectors (pET series) provide high-level, inducible expression

Expression optimization parameters:

Fusion tags for improved expression:

• His6-tag: Most common for initial purification via IMAC

• GST-tag: Can enhance solubility but adds substantial size

• SUMO or MBP tags: Often improve folding and solubility of difficult proteins

Protocol optimization strategy:

• Clone the E. fergusonii fusA gene into multiple expression vectors with different tags

• Perform small-scale expression trials varying temperature, IPTG concentration, and induction time

• Analyze soluble vs. insoluble fractions by SDS-PAGE

• Scale up using optimized conditions

• If expression levels remain poor, consider codon optimization of the fusA sequence

What purification strategies yield highest purity and activity of recombinant E. fergusonii EF-G?

A multi-step purification strategy is recommended for obtaining high-purity, active E. fergusonii EF-G:

Standard purification workflow:

• Affinity chromatography:

  • IMAC using Ni-NTA for His-tagged EF-G

  • Gradual imidazole gradient (20-250 mM) to minimize co-purification of contaminants

  • Buffer: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol

• Tag removal (optional):

  • Protease cleavage (TEV, PreScission, or SUMO protease depending on tag)

  • Reverse IMAC to remove cleaved tag and uncleaved protein

• Ion exchange chromatography:

  • Anion exchange (Q Sepharose) at pH 7.5-8.0

  • Salt gradient elution (50-500 mM NaCl)

  • Removes nucleic acid contaminants and similarly charged proteins

• Size exclusion chromatography:

  • Superdex 200 column for final polishing

  • Buffer: 20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl2, 5% glycerol, 2 mM DTT

  • Ensures homogeneity and removes aggregates

Critical considerations:

• Maintain reducing conditions throughout purification as EF-G is sensitive to oxidation

• Include 10-50 μM GDP in buffers to stabilize protein structure

• Keep samples cold (4°C) during all purification steps

• Consider adding protease inhibitors to initial lysis buffer

Quality control metrics:

Storage conditions:

• Flash freeze in liquid nitrogen in small aliquots

• Store at -80°C in buffer containing 20% glycerol

• Avoid repeated freeze-thaw cycles

What methodologies are most reliable for assessing the activity of purified recombinant E. fergusonii EF-G?

Several complementary assays can be used to comprehensively evaluate recombinant E. fergusonii EF-G activity:

1. GTPase Activity Assays:

Malachite Green Phosphate Assay:

• Measures free phosphate released during GTP hydrolysis

• Standard reaction conditions: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.5 μM EF-G, 100 μM GTP

• Can be performed with and without ribosomes to determine intrinsic vs. ribosome-stimulated activity

• Calculation of kinetic parameters (kcat, KM) provides quantitative assessment of enzyme efficiency

Coupled Enzyme Assay:

• Uses pyruvate kinase and lactate dehydrogenase to couple GTP hydrolysis to NADH oxidation

• Continuous monitoring at 340 nm allows real-time activity measurement

• Particularly useful for kinetic studies

2. Translocation Assays:

tRNA Movement Assay:

• Dual-labeled tRNAs with fluorescent probes

• FRET changes indicate translocation events

• Can measure rate of translocation directly

mRNA Toeprinting:

• Primer extension inhibition to measure ribosome position on mRNA

• Quantitative assessment of translocation efficiency

• Detects partial as well as complete translocation events

3. Ribosome Binding Assays:

Co-sedimentation Assay:

• Mix EF-G with ribosomes and ultracentrifuge through sucrose cushion

• Quantify bound EF-G by SDS-PAGE and densitometry

• Can be performed with GTP, GDP, or non-hydrolyzable analogs

Surface Plasmon Resonance:

• Real-time binding kinetics (kon, koff)

• Determine binding affinity (KD)

• Analyze effects of nucleotides and antibiotics on binding

4. In Vitro Translation:

Poly(U)-directed Poly(Phe) Synthesis:

Coupled Transcription-Translation:

• Expression of reporter proteins (GFP, luciferase)

• Quantitative measurement of complete protein synthesis

• Can be inhibited with fusidic acid to confirm EF-G-specific effects

Comparative Activity Analysis:

How does oxidative stress affect E. fergusonii EF-G function and what are the molecular mechanisms involved?

Oxidative stress significantly impacts EF-G function, representing an important regulatory mechanism and potential antimicrobial target. Research with E. coli has demonstrated that EF-G is particularly susceptible to oxidation , and similar mechanisms likely apply to E. fergusonii EF-G.

Molecular mechanisms of oxidation:

• Disulfide bond formation: Mass spectrometry analysis of oxidized E. coli EF-G revealed the formation of an intramolecular disulfide bond between Cys114 and Cys266

• Structural implications: These cysteine residues are located in domains I and II, respectively, affecting the conformational dynamics essential for EF-G function

• Oxidant sensitivity: In vitro experiments show that as little as 0.5 mM H2O2 can induce significant oxidation and inactivation of EF-G

Functional consequences of oxidation:

Experimental approaches to study oxidative effects:

• Site-directed mutagenesis:

  • Create Cys→Ser or Cys→Ala variants

  • Measure resistance to oxidative inactivation

  • Identify specific residues responsible for redox sensitivity

• Differential oxidation analysis:

  • Expose protein to varied oxidizing conditions

  • Use mass spectrometry to identify modification sites

  • Correlate modifications with activity changes

• Structural analysis:

  • Crystallography or cryo-EM of oxidized vs. reduced forms

  • Map conformational changes induced by oxidation

  • Identify potential allosteric effects

• Comparative redox proteomics:

  • Compare oxidation susceptibility across bacterial species

  • Identify species-specific differences in redox regulation

  • Correlate with environmental adaptation

Physiological relevance:

• Oxidation of EF-G serves as a rapid mechanism to downregulate translation during oxidative stress

• This conserves energy and prevents potentially error-prone protein synthesis

• May contribute to bacterial persistence under antibiotic or immune attack

This redox sensitivity of EF-G represents both a bacterial survival mechanism and a potential target for antimicrobial development, particularly in combination with oxidative stress-inducing agents.

What are the binding dynamics between E. fergusonii EF-G and the ribosome during translocation, and how do they compare to other bacterial species?

The binding dynamics between EF-G and the ribosome involve multiple conformational changes driving translocation. While specific data for E. fergusonii is limited, comparative analysis with well-studied systems provides valuable insights:

Key stages in EF-G-ribosome interaction:

• Initial binding:

  • GTP-bound EF-G binds to pre-translocation (PRE) ribosome

  • Primary contacts through domains I, II, and V

  • Stabilizes rotated ribosomal state with hybrid tRNAs

• GTP hydrolysis and conformational changes:

  • GTP hydrolysis triggers major conformational rearrangements in EF-G

  • Switch regions reorganize, affecting interdomain relationships

  • Domain IV extends into the decoding center

  • In S. aureus EF-G, domains III-V move relative to domains I-II by approximately 25Å

• Translocation:

  • Movement of tRNAs from A/P and P/E to P and E sites

  • mRNA moves by one codon

  • Ribosome undergoes reverse rotation to post-translocation (POST) state

• Dissociation:

  • GDP-bound EF-G has reduced affinity for POST ribosome

  • Dissociation allows next elongation cycle

Comparative binding kinetics across bacterial species:

Species-specific structural features relevant to binding:

• Switch II region conformation varies across species, affecting fusidic acid binding

• Domain IV, which interacts with the decoding center, shows species-specific sequence variations

• C-terminal helix differences may affect interaction with ribosomal proteins

Advanced methodologies for studying species-specific dynamics:

• Time-resolved cryo-electron microscopy:

  • Capture multiple states of the translocation process

  • Identify species-specific conformational differences

  • Resolution now approaching 2.5-3Å for ribosome complexes

• Single-molecule FRET:

  • Strategic fluorophore placement on EF-G and ribosomal components

  • Real-time observation of conformational changes

  • Detection of potential species-specific intermediate states

• Hydrogen-deuterium exchange mass spectrometry:

  • Map conformational dynamics and binding interfaces

  • Compare exchange patterns across species

  • Identify differences in domain flexibility

• Computational molecular dynamics:

  • Simulate species-specific EF-G-ribosome interactions

  • Predict energy landscapes for conformational changes

  • Calculate transition state barriers for key steps

These studies can reveal the molecular basis for species-specific differences in antibiotic susceptibility and translational efficiency, potentially guiding the development of species-targeted antimicrobials.

How do fusidic acid resistance mechanisms involving E. fergusonii EF-G mutations compare with those in other clinical pathogens?

Fusidic acid (FA) is a bacteriostatic antibiotic that locks EF-G on the ribosome in a post-translocational state . Resistance to FA typically involves mutations in the fusA gene encoding EF-G or acquisition of resistance proteins. Understanding these mechanisms in E. fergusonii compared to other pathogens provides valuable clinical insights:

Comparative analysis of fusA mutations conferring resistance:

Based on structural and functional analyses, fusA-based resistance mutations can be categorized into several groups:

Alternative resistance mechanisms:

• FusB-family proteins:

  • Bind to domain IV of EF-G

  • Rescue FA inhibition in both elongation and recycling steps

  • Resistance mechanism: Prevent formation or facilitate dissociation of FA-locked EF-G-ribosome complex

  • Distribution: Common in S. aureus, less characterized in Escherichia species

• Impermeability and efflux mechanisms:

  • Reduced uptake through altered membrane composition

  • Enhanced efflux through upregulation of multidrug transporters

  • Often provide cross-resistance to multiple antibiotics

  • Distribution: Widespread across gram-negative bacteria including Escherichia

Structural basis for species-specific resistance profiles:

• Switch II region differences:

  • The switch II region shows distinct conformations incompatible with FA binding when EF-G is not bound to the ribosome

  • Species-specific variations in this region affect fusidic acid binding affinity

• Domain interface variations:

  • Differences in interfaces between domains I, II, and III

  • Affect how mutations propagate structural changes to the FA binding site

Methodological approaches to study resistance:

• Whole genome sequencing of resistant isolates:

  • Identify novel resistance mutations

  • Track evolutionary pathways to resistance

• Site-directed mutagenesis:

  • Introduce specific mutations to confirm their role

  • Create combinations to study epistatic effects

• MIC determination:

  • Quantify resistance levels of different mutations

  • Compare across species using isogenic backgrounds

• Fitness cost assessment:

  • Growth rate comparisons

  • Competition assays

  • Stress response profiling

• Structural biology:

  • Crystallography or cryo-EM of resistant variants

  • Map how mutations affect FA binding

Understanding these resistance mechanisms has important clinical implications for antibiotic stewardship and development of new translation-targeting antimicrobials with reduced potential for resistance.

What novel methodologies can be applied to study the interaction between E. fergusonii EF-G and fusidic acid?

Studying the interaction between E. fergusonii EF-G and fusidic acid (FA) requires sophisticated methodologies spanning structural, biochemical, and computational approaches. The following cutting-edge techniques provide comprehensive insights:

Structural biology approaches:

• Time-resolved cryo-electron microscopy:

  • Capture different states of EF-G-ribosome-FA complex

  • Visualize conformational changes upon FA binding

  • Resolution now reaching 2.5-3Å for ribosome complexes

  • Sample preparation strategy: Stalled translation complexes with FA added at defined timepoints

• X-ray crystallography with soaking experiments:

  • Crystallize EF-G in different conformational states

  • Soak crystals with FA to determine binding modes

  • Co-crystallization with FA analogs of varying affinities

  • Resolution target: <2.0Å to visualize water-mediated interactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map regions of EF-G affected by FA binding

  • Identify allosteric effects distant from binding site

  • Compare exchange patterns between sensitive and resistant variants

  • Experimental design: Short (10s) to long (1h) deuterium exposure times

Biophysical binding studies:

• Microscale thermophoresis (MST):

  • Determine binding affinities in solution

  • Minimal protein consumption (μg quantities)

  • Label-free option available

  • Advantage: Can work with crude lysates for mutant screening

• Surface plasmon resonance (SPR) with kinetic analysis:

  • Real-time association and dissociation kinetics

  • Determine kon, koff, and KD values

  • Study temperature dependence for thermodynamic parameters

  • Experimental setup: Immobilize EF-G, flow FA at multiple concentrations

• Isothermal titration calorimetry (ITC):

  • Direct measurement of binding enthalpies

  • Determine complete thermodynamic profile (ΔG, ΔH, TΔS)

  • Stoichiometry determination

  • Requires: 0.5-1mg purified EF-G per experiment

Functional assays with quantitative readouts:

• Real-time GTPase assays with FA titration:

  • Continuous monitoring of phosphate release

  • IC50 determination for FA inhibition

  • Comparison between wild-type and mutant EF-G

  • Protocol: MDCC-PBP fluorescent biosensor for real-time detection

• Single-molecule fluorescence assays:

  • Directly observe FA-induced ribosome stalling

  • Measure dwell times in different states

  • Determine concentration-dependent effects

  • Implementation: Zero-mode waveguides or TIRF microscopy

Computational approaches:

• Molecular dynamics simulations:

  • Model FA binding and induced conformational changes

  • Free energy perturbation to calculate binding energies

  • Compare wild-type and resistant mutants

  • Simulation scale: Microsecond timescale with explicit solvent

• AI-assisted binding site prediction:

  • AlphaFold-Multimer or RoseTTAFold for complex prediction

  • Deep learning-based binding site analysis

  • Virtual screening of FA derivatives

  • Validation: Experimental testing of top predictions

Comparative data table for methodology selection:

These methodologies provide complementary information about the E. fergusonii EF-G-fusidic acid interaction, enabling comprehensive characterization from binding affinity to structural mechanism.

How can post-translational modifications of E. fergusonii EF-G affect its function and antibiotic susceptibility?

Post-translational modifications (PTMs) of EF-G can dramatically alter its function, stability, and interactions with antibiotics. While specific data on E. fergusonii EF-G modifications is limited, the following represents the critical PTMs affecting bacterial translation factors:

Types of post-translational modifications affecting EF-G:

• Oxidative modifications:

  • Formation of disulfide bonds between conserved cysteine residues

  • In E. coli, oxidation leads to a disulfide bond between Cys114 and Cys266

  • Results in inactivation of EF-G and reduced translation

  • Particularly relevant during oxidative stress and antibiotic exposure

• Phosphorylation:

  • Serine/threonine/tyrosine phosphorylation by bacterial kinases

  • Affects GTPase activity and conformational dynamics

  • Can regulate interaction with the ribosome and antibiotics

  • Often responds to nutrient availability and stress conditions

• Methylation:

  • Lysine or arginine methylation

  • Alters surface properties and interaction interfaces

  • May affect protein stability and antibiotic binding

  • Often constitutive rather than regulatory

• ADP-ribosylation:

  • Target of certain bacterial toxins

  • Drastically inhibits function by preventing ribosome interaction

  • Major modification leading to translation inhibition

  • Can affect antibiotic binding sites

Impact of PTMs on antibiotic susceptibility:

Experimental approaches to study PTM effects:

• Mass spectrometry-based proteomics:

  • Bottom-up approach: Enzymatic digestion followed by LC-MS/MS

  • Top-down approach: Analysis of intact protein and modification stoichiometry

  • Targeted quantification: Multiple reaction monitoring for specific modifications

  • Comparison of modification patterns under different stress conditions

• Site-directed mutagenesis:

  • Generate mimics (e.g., Cys→Asp for phosphorylation) or prevention (Cys→Ala) mutants

  • Analyze functional consequences in vitro and in vivo

  • Determine effects on antibiotic susceptibility

  • Protocol design: Multi-site mutations may be required to observe phenotypes

• In vitro modification systems:

  • Enzymatic installation of specific PTMs

  • Chemical modification approaches for cysteine oxidation

  • Comparative functional assays before and after modification

  • Antibiotic binding studies with modified protein

• Structural studies of modified EF-G:

  • Crystallography or cryo-EM of modified protein

  • Molecular dynamics simulations to predict PTM effects

  • Hydrogen-deuterium exchange to detect conformational changes

Clinical and antimicrobial development implications:

• PTMs can significantly alter antibiotic susceptibility profiles

• Bacterial stress responses involving PTMs may contribute to tolerance phenotypes

• Understanding PTM patterns across clinical isolates may help predict treatment outcomes

• PTM-installing or PTM-preventing enzymes represent potential adjuvant targets

These studies provide valuable insights into bacterial adaptation mechanisms and may guide development of novel antimicrobial strategies that consider the PTM status of target proteins.

What are the biosafety and regulatory considerations for research involving recombinant E. fergusonii EF-G?

Research with recombinant E. fergusonii EF-G requires adherence to specific biosafety protocols and regulatory frameworks to ensure safe, compliant, and ethical research practices:

Biosafety classification and containment requirements:

• Risk assessment:

  • E. fergusonii is generally classified as a Risk Group 2 organism

  • Recombinant DNA work involving E. fergusonii genes typically requires Biosafety Level 2 (BSL-2) containment

  • According to NIH Guidelines, experiments using Risk Group 2 agents as host-vector systems require Institutional Biosafety Committee (IBC) approval before initiation

• Laboratory containment specifications:

Regulatory compliance:

• NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules :

  • Mandatory for institutions receiving NIH funding

  • Experiments with Risk Group 2 agents require IBC approval

  • Proper documentation and record-keeping essential

  • Regular training and updates on biosafety procedures

• Institutional oversight mechanisms:

  • Institutional Biosafety Committee review and approval

  • Registration of recombinant DNA protocols

  • Periodic laboratory inspections

  • Incident reporting systems

• International considerations:

  • Host country rules must be followed for international collaborations

  • Material transfer agreements for sharing recombinant materials

  • Import/export restrictions may apply to certain constructs

Responsible research practices:

• Dual-use research of concern (DURC) assessment:

  • Evaluation of potential misuse implications

  • Specific concern: Modified translation factors that could confer antibiotic resistance

  • Implementation of appropriate safeguards

  • Communication planning for sensitive findings

• Environmental impact considerations:

  • Proper containment to prevent environmental release

  • Waste management protocols for recombinant materials

  • Risk mitigation strategies for potential accidental release

• Research integrity measures:

  • Transparent reporting of methods

  • Proper validation of research findings

  • Responsible sharing of materials and data

  • Disclosure of potential conflicts of interest

Training and documentation requirements:

• Mandatory training:

  • General biosafety training

  • Specific training for recombinant DNA work

  • Pathogen-specific training for E. fergusonii

  • Documentation of completion and regular refreshers

• Standard Operating Procedures (SOPs):

  • Detailed protocols for all experimental procedures

  • Specific safety precautions for each methodology

  • Emergency response procedures

  • Regular review and updates as needed

Adherence to these biosafety and regulatory guidelines ensures that research on recombinant E. fergusonii EF-G is conducted safely, responsibly, and in compliance with applicable regulations, while minimizing potential risks to researchers, the community, and the environment.