Recombinant Geobacter uraniireducens Elongation factor Ts (tsf)

What is Geobacter uraniireducens and why is it of scientific interest?

Geobacter uraniireducens is a gram-negative anaerobic bacterium belonging to the Geobacteraceae family, notable for its distinctive extracellular electron transfer (EET) capabilities. Unlike other Geobacter species which typically require conductive pili for efficient EET, G. uraniireducens possesses nonconductive pili yet demonstrates exceptional ability to reduce iron oxide even without direct contact with the oxide . This microorganism has garnered scientific interest due to its unique electron transfer mechanisms, particularly its ability to secrete high concentrations of riboflavin (up to 270 nM at stationary phase) which facilitates electron transfer to external acceptors . These properties make G. uraniireducens valuable for studying alternative EET pathways and potential applications in bioremediation and microbial fuel cells.

What are the optimal storage and handling conditions for recombinant G. uraniireducens EF-Ts?

For optimal preservation of recombinant G. uraniireducens Elongation Factor Ts activity and stability, researchers should adhere to the following storage and handling protocols:

Long-term Storage:

• Store the protein at -20°C for regular storage

• For extended storage periods, maintain at -20°C or preferably at -80°C

• The shelf life of the lyophilized form is approximately 12 months when stored at -20°C/-80°C, while the liquid form typically remains stable for 6 months

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to ensure contents are at the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Prepare working aliquots to minimize freeze-thaw cycles

• Store working aliquots at 4°C for short-term use (up to one week)

Researchers should avoid repeated freezing and thawing cycles as this significantly compromises protein integrity and activity. For experimental work requiring multiple uses, it is advisable to prepare smaller working aliquots from the main stock to preserve the remainder of the sample in optimal condition.

How can researchers verify the purity and activity of recombinant G. uraniireducens EF-Ts?

Verification of recombinant G. uraniireducens Elongation Factor Ts purity and activity requires a multi-faceted approach combining analytical and functional assays:

Purity Assessment:

• SDS-PAGE Analysis: Run the protein on a 10-15% polyacrylamide gel under denaturing conditions and stain with Coomassie blue. Commercial preparations typically achieve >85% purity . Expected molecular weight should correspond to approximately 23-24 kDa based on the 216 amino acid sequence.

• Western Blot Analysis: Using antibodies specific to the tag employed in the recombinant protein production or to conserved EF-Ts epitopes.

• Mass Spectrometry: For precise molecular weight determination and verification of protein identity.

Activity Verification:

• GDP/GTP Exchange Assay: Measure the ability of EF-Ts to catalyze nucleotide exchange on EF-Tu using radiolabeled or fluorescently-labeled nucleotides.

• In vitro Translation Assay: Assess the protein's ability to enhance translation efficiency in a cell-free protein synthesis system. Active EF-Ts should increase the rate of protein synthesis compared to control reactions lacking the factor.

• Binding Affinity Measurements: Determine the interaction strength between EF-Ts and EF-Tu using techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), or microscale thermophoresis (MST).

For researchers working with this protein, it is advisable to include both positive controls (verified active EF-Ts from a related organism) and negative controls (heat-inactivated protein) in activity assays to establish reliable activity benchmarks.

What expression systems are most effective for producing recombinant G. uraniireducens EF-Ts?

Based on the available information and general practices in recombinant protein production, the following expression systems are recommended for producing functional G. uraniireducens Elongation Factor Ts:

Yeast Expression System:
The commercial recombinant G. uraniireducens EF-Ts is produced in a yeast expression system , which offers several advantages:

• Post-translational modifications that may enhance protein folding and stability

• Lower endotoxin levels compared to bacterial systems

• Potential for higher yield of soluble protein

Alternative Expression Systems:

• E. coli Expression System:

  • Advantages: Rapid growth, high protein yields, simple cultivation

  • Considerations: May require optimization of codon usage for G. uraniireducens genes

  • Best suited for: High-throughput screening and initial characterization studies

• Insect Cell Expression:

  • Advantages: More complex post-translational modifications, often better folding

  • Considerations: Longer production time, higher cost

  • Best suited for: Structural studies requiring highly pure and properly folded protein

Expression Optimization Parameters:

• Induction temperature: 16-30°C (lower temperatures often favor proper folding)

• Induction duration: 4-24 hours depending on expression system

• IPTG concentration (for E. coli): 0.1-1.0 mM

• Addition of folding enhancers: 2-10% sorbitol, 0.5-2.5% glycine betaine

Researchers should consider testing multiple expression constructs with different affinity tags (His, GST, MBP) to identify the optimal configuration for obtaining soluble, active protein. Purification strategies should be designed based on the selected tag system, with immobilized metal affinity chromatography (IMAC) being the most common first step for His-tagged proteins.

How does G. uraniireducens EF-Ts compare structurally and functionally with EF-Ts from other bacterial species?

While the search results don't provide direct comparative data for EF-Ts across species, a structure-function analysis can be inferred from the protein sequence and general knowledge about bacterial elongation factors:

Sequence Homology Analysis:
The G. uraniireducens EF-Ts (UniProt: A5G7W7) exhibits the typical domain architecture of bacterial elongation factors. Key regions likely include:

• N-terminal domain (involved in EF-Tu binding)

• Core domain (catalyzes nucleotide exchange)

• C-terminal domain (structural stability)

Functional Conservation and Divergence:
The primary function of EF-Ts in nucleotide exchange is highly conserved across bacterial species, but subtle variations may exist in:

• Binding affinity to EF-Tu

• Rate of nucleotide exchange catalysis

• Thermostability profiles

• Interaction with other translation factors

Potential Unique Adaptations:
Given G. uraniireducens' specialized metabolism involving extracellular electron transfer and metal reduction, its EF-Ts might possess adaptations for functioning under the organism's specific growth conditions. For instance, the protein may have evolved features that enable efficient translation under the anaerobic, metal-rich environments where this organism typically thrives.

Researchers conducting comparative studies should employ techniques such as:

• Multiple sequence alignment with EF-Ts proteins from related Geobacter species and other bacteria

• Homology modeling based on available crystal structures of bacterial EF-Ts

• Thermal shift assays to compare stability under various conditions

• Kinetic studies of nucleotide exchange rates across species

What role might EF-Ts play in the unique electron transfer capabilities of G. uraniireducens?

Indirect Contributions to Electron Transfer Metabolism:

• Translational Regulation of EET Components:
  EF-Ts, through its role in translation elongation, influences the synthesis rate of proteins involved in extracellular electron transfer (EET). G. uraniireducens employs a unique EET mechanism that relies heavily on secreted riboflavin rather than conductive pili . The efficient translation of proteins involved in riboflavin synthesis, secretion, and redox cycling would depend on optimal EF-Ts function.

• Adaptation to Environmental Stress:
  During environmental transitions or stress conditions, G. uraniireducens must rapidly adjust its proteome. EF-Ts may serve as a control point for modulating translation rates in response to changing electron acceptor availability or redox conditions.

• Potential Specialized Interactions:
  Though speculative without direct experimental evidence, G. uraniireducens EF-Ts might have evolved specific interactions with ribosomes translating mRNAs encoding EET components, potentially providing a mechanism for coordinated expression of this specialized metabolic pathway.

Research approaches to investigate these potential connections could include:

• Proteomic analysis comparing wild-type and EF-Ts mutant strains under various electron acceptor conditions

• Ribosome profiling to identify mRNAs whose translation is particularly sensitive to EF-Ts levels

• Protein-protein interaction studies to identify potential non-canonical partners of EF-Ts in G. uraniireducens

The unique electron transfer capabilities of G. uraniireducens, particularly its reliance on secreted riboflavin (reaching concentrations up to 270 nM compared to only 70 nM in G. sulfurreducens) , suggest specialized metabolic adaptations that likely extend to the translation machinery components including EF-Ts.

How can researchers investigate the interaction between G. uraniireducens EF-Ts and EF-Tu?

Investigating the interaction between G. uraniireducens EF-Ts and EF-Tu requires a multi-technique approach that addresses both structural and functional aspects of this critical protein-protein interaction:

Analytical Methods for Interaction Characterization:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified EF-Ts on a sensor chip

  • Flow various concentrations of EF-Tu over the surface

  • Analyze association/dissociation kinetics to determine:

    • kon (association rate constant)

    • koff (dissociation rate constant)

    • KD (equilibrium dissociation constant)

• Isothermal Titration Calorimetry (ITC):

  • Provides thermodynamic parameters of the interaction

  • Directly measures:

    • Binding stoichiometry

    • Enthalpy change (ΔH)

    • Entropy change (ΔS)

    • Binding constant (Ka)

• Structural Analysis Techniques:

  • X-ray crystallography of the EF-Ts:EF-Tu complex

  • Cryo-electron microscopy for visualization of conformational states

  • NMR spectroscopy for mapping interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify regions protected upon binding

Functional Assays:

• Nucleotide Exchange Assay:

  • Monitor the rate of mant-GDP release from EF-Tu in the presence of varying concentrations of EF-Ts

  • Quantify the catalytic efficiency (kcat/KM) of the exchange reaction

• Mutagenesis Studies:

  • Generate site-directed mutants of predicted interface residues

  • Assess the impact on binding affinity and nucleotide exchange activity

  • Create chimeric proteins with domains from other bacterial EF-Ts to identify species-specific determinants

• In vivo Complementation:

  • Test whether G. uraniireducens EF-Ts can functionally replace EF-Ts in other bacterial species

  • Assess growth rates and protein synthesis capacity

Data Analysis and Modeling:

• Construct binding isotherms from interaction data

• Develop kinetic models of the multi-step nucleotide exchange process

• Use molecular dynamics simulations to predict structural changes during complex formation

A comprehensive study would integrate these approaches to develop a complete mechanistic understanding of how G. uraniireducens EF-Ts interacts with its cognate EF-Tu and how this interaction contributes to the organism's unique physiology and electron transfer capabilities.

What are the major challenges in expressing and purifying functional G. uraniireducens EF-Ts?

Researchers working with G. uraniireducens Elongation Factor Ts face several technical challenges that must be addressed to obtain high-quality, functional protein:

Expression Challenges:

• Codon Usage Optimization:
  G. uraniireducens has a significantly different codon usage pattern compared to common expression hosts, potentially leading to translational pausing and truncated products. Researchers should consider codon-optimized synthetic genes for expression in E. coli or yeast systems.

• Solubility Issues:
  Bacterial elongation factors can form inclusion bodies when overexpressed, particularly at higher temperatures. Strategies to address this include:

  • Expression at lower temperatures (16-20°C)

  • Use of solubility-enhancing fusion partners (MBP, SUMO, TRX)

  • Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Post-translational Modifications:
  If G. uraniireducens EF-Ts requires specific post-translational modifications for activity, researchers should select expression systems capable of providing these modifications (e.g., yeast systems as used in commercial production ).

Purification Challenges:

• Maintaining Native Conformation:
  Preserving the native structure throughout purification is critical for functional studies. Consider:

  • Including stabilizing agents in purification buffers (5-10% glycerol, 1-2 mM DTT or TCEP)

  • Minimizing exposure to extreme pH conditions

  • Using rapid purification protocols to reduce time at room temperature

• Protein-Protein Interactions:
  EF-Ts may co-purify with endogenous EF-Tu or other translation factors from the expression host. High-stringency washing steps during affinity purification or additional purification techniques (ion exchange, size exclusion) may be necessary to obtain pure protein.

• Protein Verification:
  Confirming that the purified protein is both structurally intact and functionally active requires multiple analytical approaches:

  • Mass spectrometry for accurate molecular weight confirmation

  • Circular dichroism for secondary structure analysis

  • Activity assays measuring nucleotide exchange on EF-Tu

Storage Stability:
As indicated in the product information, recombinant G. uraniireducens EF-Ts has defined stability limitations. The liquid form has a shelf life of approximately 6 months at -20°C/-80°C, while the lyophilized form maintains stability for up to 12 months . Researchers should aliquot the protein to avoid freeze-thaw cycles and consider adding stabilizing agents like glycerol to a final concentration of 5-50% .

How can researchers investigate the role of EF-Ts in G. uraniireducens' adaptation to different electron acceptors?

Investigating the role of Elongation Factor Ts in G. uraniireducens' adaptation to different electron acceptors requires integrated experimental approaches that connect translation regulation with electron transfer mechanisms:

Transcriptomic and Proteomic Approaches:

• Differential Expression Analysis:

  • Compare EF-Ts expression levels when G. uraniireducens is grown with different electron acceptors (Fe(III) oxide vs. electrode)

  • Techniques: RT-qPCR for transcripts, Western blot for protein levels

  • Expected outcome: Potential correlation between EF-Ts abundance and specific electron acceptor conditions

• Ribosome Profiling:

  • Map ribosome occupancy on mRNAs under different electron acceptor conditions

  • Analyze translation efficiency of genes involved in extracellular electron transfer

  • Determine if EF-Ts availability becomes limiting for translation of specific mRNAs

Genetic Manipulation Strategies:

• Conditional EF-Ts Expression:

  • Create strains with titratable EF-Ts levels

  • Assess growth rates and electron transfer capabilities with different electron acceptors at varying EF-Ts concentrations

  • Measure riboflavin production (which reaches up to 270 nM in wild-type G. uraniireducens) as a function of EF-Ts levels

• Domain Swapping:

  • Generate chimeric EF-Ts proteins containing domains from EF-Ts of other Geobacter species with different electron transfer mechanisms

  • Evaluate impacts on protein synthesis rates and specificity

  • Correlate with electron transfer efficiency to Fe(III) oxide or electrodes

Biochemical and Biophysical Characterization:

• Translation Kinetics:

  • Establish an in vitro translation system using components from G. uraniireducens

  • Compare translation rates of EET-related mRNAs versus housekeeping genes

  • Assess the impact of varying EF-Ts concentrations on these differential translation rates

• Protein-Protein Interaction Network:

  • Identify interaction partners of EF-Ts using pull-down assays coupled with mass spectrometry

  • Look for unexpected interactions with components of electron transfer pathways

  • Compare interaction networks under Fe(III) oxide versus electrode growth conditions

Correlation with Electron Transfer Modes:
G. uraniireducens employs different modes of riboflavin-mediated electron transfer depending on the electron acceptor: free riboflavin shuttles for Fe(III) oxide reduction versus bound riboflavin cofactors for electrode reduction . Researchers should investigate whether EF-Ts plays a role in modulating the translation of proteins involved in these distinct mechanisms.

What potential biotechnological applications exist for recombinant G. uraniireducens EF-Ts?

Recombinant G. uraniireducens Elongation Factor Ts has several potential biotechnological applications that leverage its fundamental role in protein synthesis and the unique adaptations of G. uraniireducens to various environmental conditions:

Enhancement of Cell-Free Protein Synthesis Systems:

• Specialized Translation Systems:

  • Development of cell-free protein synthesis platforms optimized for expression of difficult-to-produce proteins

  • Incorporation of G. uraniireducens EF-Ts along with other translation factors could enhance translation efficiency under non-standard conditions

  • Potential applications in synthesizing proteins that require anaerobic or metal-rich environments for proper folding

• Thermostability Engineering:

  • G. uraniireducens thrives in subsurface environments with specific temperature conditions

  • Its EF-Ts may possess stability characteristics that could be valuable for engineering more robust cell-free translation systems

Bioelectrochemical Applications:

• Protein Components for Microbial Fuel Cells:

  • G. uraniireducens demonstrates unique electron transfer capabilities, particularly its use of riboflavin as an electron shuttle

  • Understanding how translation factors like EF-Ts support these specialized metabolic pathways could aid in designing more efficient bioelectrochemical systems

  • Potential for engineering enhanced protein production systems for key components of electron transfer chains

• Biosensors for Environmental Monitoring:

  • Development of translation-based biosensors that utilize G. uraniireducens EF-Ts

  • Applications in detecting specific environmental conditions relevant to metal reduction or electron transfer processes

Structural Biology and Drug Discovery:

• Comparative Structural Studies:

  • G. uraniireducens EF-Ts could serve as a model for understanding bacterial translation factor adaptations to specialized metabolic conditions

  • Potential insights into designing antimicrobials that target translation in related pathogenic species

• Protein Engineering Platform:

  • Using the structural and functional insights from G. uraniireducens EF-Ts to engineer translation factors with novel properties

  • Applications in synthetic biology for creating organisms with expanded metabolic capabilities

Environmental Bioremediation Technologies:

• Enhanced Metal Reduction Systems:

  • Understanding how EF-Ts supports the unique riboflavin-mediated electron transfer in G. uraniireducens

  • Applications in engineering more efficient systems for uranium and other heavy metal bioremediation

  • Potential for developing optimized protein expression systems for key components in metal reduction pathways

These applications would require detailed characterization of G. uraniireducens EF-Ts structure, function, and interaction network, combined with protein engineering approaches to enhance desired properties for specific biotechnological applications.

How does understanding G. uraniireducens EF-Ts contribute to knowledge about bacterial adaptation to extreme environments?

Studying Elongation Factor Ts from Geobacter uraniireducens provides valuable insights into how protein synthesis machinery adapts to support life in specialized environmental niches:

Adaptations to Redox-Active Environments:

G. uraniireducens thrives in subsurface environments where it must interact with insoluble electron acceptors like Fe(III) oxide. Its protein synthesis machinery, including EF-Ts, likely contains adaptations that enable efficient translation under these challenging conditions. Research shows that G. uraniireducens has evolved unique extracellular electron transfer mechanisms, notably its high production of riboflavin (reaching 270 nM compared to only 70 nM in G. sulfurreducens) , which compensates for its nonconductive pili. This adaptation suggests potential specialization in translation factors to support this distinctive metabolism.

Insights into Translation Under Energy Limitation:

Extracellular electron transfer to insoluble metals represents an energy-constrained form of respiration. Studying how EF-Ts functions in G. uraniireducens can reveal mechanisms for maintaining protein synthesis efficiency under energy-limited conditions, potentially including:

• Optimized binding kinetics with EF-Tu

• Enhanced recycling efficiency of translation factors

• Specialized interactions with ribosomes to maximize energy utilization

Environmental Stress Response Mechanisms:

G. uraniireducens must adapt to fluctuating environmental conditions, including changes in available electron acceptors. Research shows it employs different modes of riboflavin-mediated electron transfer depending on whether Fe(III) oxide or an electrode serves as the electron acceptor . Translation factors like EF-Ts likely play critical roles in modulating the proteome during these transitions, potentially through:

• Altered expression levels under different growth conditions

• Post-translational modifications that change activity

• Specialized interactions with regulatory RNA-binding proteins

Evolutionary Insights:

Comparative analysis of EF-Ts across Geobacter species with different electron transfer mechanisms (conductive pili in G. sulfurreducens versus riboflavin shuttling in G. uraniireducens) can illuminate how fundamental cellular machinery co-evolves with specialized metabolic pathways. This evolutionary perspective provides a framework for understanding bacterial adaptation to extreme environments more broadly.

By integrating structural, functional, and systems-level analyses of G. uraniireducens EF-Ts, researchers can develop a more comprehensive understanding of how protein synthesis machinery adapts to support specialized metabolic capabilities in challenging environmental niches.

What methodological approaches can integrate G. uraniireducens EF-Ts research with extracellular electron transfer studies?

Integrating research on G. uraniireducens Elongation Factor Ts with studies of extracellular electron transfer (EET) requires multidisciplinary approaches that connect translation processes with the organism's unique electron transfer capabilities:

Systems Biology Approaches:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from G. uraniireducens grown with different electron acceptors

  • Construct regulatory networks connecting translation factors like EF-Ts with EET components

  • Identify potential feedback mechanisms between electron transfer status and translation regulation

• Flux Analysis:

  • Track carbon and electron flow through central metabolism to protein synthesis and EET pathways

  • Quantify how translation resource allocation shifts when G. uraniireducens transitions between different electron acceptors

  • Determine if EF-Ts becomes rate-limiting under specific electron transfer conditions

Molecular and Biochemical Methods:

• Ribosome Profiling with EET Focus:

  • Analyze translation efficiency of EET components in wild-type versus EF-Ts mutant strains

  • Compare ribosome occupancy patterns under Fe(III) oxide versus electrode growth conditions

  • Identify potential translational "regulons" associated with different EET modes

• In vitro Reconstitution Experiments:

  • Establish a minimal in vitro system combining translation components (including EF-Ts) with EET components

  • Test how translation rates of specific mRNAs respond to redox conditions

  • Investigate potential direct interactions between translation machinery and EET components

Advanced Biophysical Techniques:

• Single-Molecule Approaches:

  • Track individual ribosomes during translation of EET component mRNAs

  • Measure the impact of varying EF-Ts concentrations on translation dynamics

  • Correlate with electron transfer activity in real-time

• Structural Biology Integration:

  • Solve structures of translation complexes from G. uraniireducens under conditions mimicking different electron acceptor environments

  • Compare with structures of EET components to identify potential co-evolutionary patterns

Experimental Design Framework:

By implementing these integrated approaches, researchers can develop a comprehensive understanding of how fundamental cellular processes like translation, mediated by factors like EF-Ts, are coordinated with specialized metabolic capabilities like the unique riboflavin-mediated EET systems in G. uraniireducens .

How can structural insights into G. uraniireducens EF-Ts inform the development of novel antimicrobial strategies?

Structural analysis of Geobacter uraniireducens Elongation Factor Ts can provide valuable insights for antimicrobial drug development, particularly for targeting related bacterial pathogens while offering perspectives on resistance mechanisms:

Structural Basis for Antimicrobial Target Identification:

• Comparative Structural Analysis:

  • Determine high-resolution structures of G. uraniireducens EF-Ts using X-ray crystallography or cryo-EM

  • Compare with structures from pathogenic bacteria to identify:

    • Conserved binding pockets suitable for broad-spectrum antibiotics

    • Unique structural features for species-selective targeting

    • Conformational dynamics during the nucleotide exchange cycle

• EF-Ts:EF-Tu Interface Analysis:

  • Map the interaction surface between EF-Ts and EF-Tu in detail

  • Identify critical residues that could be targeted to disrupt this essential protein-protein interaction

  • Design peptidomimetics or small molecules that compete with natural binding

Structure-Based Drug Design Strategies:

• Virtual Screening Approaches:

  • Use the G. uraniireducens EF-Ts structure as a template for in silico screening

  • Identify compounds predicted to bind at functional interfaces

  • Prioritize molecules that target conserved pockets across multiple bacterial species

• Fragment-Based Drug Discovery:

  • Screen fragment libraries against specific structural domains of EF-Ts

  • Develop composite inhibitors by linking fragments that bind to adjacent sites

  • Optimize for properties that enhance selectivity for bacterial over human translation factors

Resistance Mechanism Insights:

• Natural Variation Analysis:

  • Compare EF-Ts sequences and structures across diverse bacterial species

  • Identify naturally occurring variations in drug-binding regions

  • Predict potential resistance mechanisms based on structural plasticity

• Rational Design of Dual-Targeting Inhibitors:

  • Develop compounds that simultaneously target EF-Ts and other components of the translation machinery

  • Create inhibitors that bind to multiple conformational states to reduce resistance potential

Experimental Validation Framework: