Recombinant Shewanella sediminis Elongation factor Ts (tsf)

What is Shewanella sediminis and why is it significant for research?

Shewanella sediminis is a psychrophilic rod-shaped marine bacterium originally isolated from Halifax Harbour sediment. It belongs to the sodium-requiring group of Shewanella species and is particularly notable for its ability to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), making it significant for bioremediation research . Unlike other Shewanella species, S. sediminis possesses distinctive enzymatic capabilities, including lysine decarboxylase activity, which is absent in other known Shewanella species. It also differs from most Shewanella species by expressing arginine dehydrolase, ornithine decarboxylase, and chitinase, and by its ability to oxidize and ferment N-acetyl-d-glucosamine . These unique metabolic capabilities make it an important organism for understanding bacterial adaptation to marine environments and for potential biotechnological applications.

What is Elongation factor Ts and what role does it play in Shewanella sediminis?

Elongation factor Ts (EF-Ts) is a protein involved in the translation process during protein synthesis. In Shewanella sediminis, EF-Ts (encoded by the tsf gene) functions as a guanine nucleotide exchange factor that catalyzes the release of GDP from the EF-Tu-GDP complex, allowing EF-Tu to bind a new GTP molecule and continue the elongation cycle during protein synthesis . The recombinant S. sediminis EF-Ts protein consists of 282 amino acids and has a UniProt accession number of A8FY41 . As a critical component of the translation machinery, EF-Ts contributes to the adaptive capabilities of S. sediminis in its native cold marine environment, potentially supporting protein synthesis under psychrophilic conditions.

How does S. sediminis EF-Ts differ from other bacterial EF-Ts proteins?

While the fundamental function of EF-Ts is conserved across bacterial species, the S. sediminis variant exhibits specific adaptations that likely reflect the psychrophilic nature of this organism. Comparative genomic studies of Shewanella species have revealed extensive gene content diversity , suggesting that the EF-Ts from S. sediminis may contain unique structural features or post-translational modifications that optimize its function in cold environments. The primary sequence of S. sediminis EF-Ts shares homology with other bacterial EF-Ts proteins but contains specific regions that may contribute to cold adaptation. The complete amino acid sequence (282 amino acids) includes distinctive motifs that interact with EF-Tu during the nucleotide exchange process .

How can engineered modifications to S. sediminis EF-Ts affect protein synthesis efficiency?

Based on research with related species, targeted modifications to S. sediminis EF-Ts could significantly impact protein synthesis efficiency. Studies on Shewanella oneidensis have demonstrated that engineered cellular modifications can alter fundamental bacterial processes . Similar engineering approaches could be applied to S. sediminis EF-Ts to enhance stability, improve nucleotide exchange rates, or optimize interactions with EF-Tu. Potential modifications include:

• Site-directed mutagenesis of key residues in the EF-Tu binding interface

• Domain swapping with cold-adapted EF-Ts variants from other psychrophilic organisms

• Introduction of stabilizing disulfide bonds or salt bridges to enhance thermostability

When designing such modifications, researchers should consider the unique evolutionary adaptations of S. sediminis to its cold marine environment. Engineered variants should be evaluated through in vitro nucleotide exchange assays, thermal stability measurements, and in vivo complementation experiments in tsf-deficient strains.

What methodologies are optimal for studying the interaction between S. sediminis EF-Ts and EF-Tu?

Investigating the interaction between S. sediminis EF-Ts and EF-Tu requires multiple complementary approaches:

• Biochemical Interaction Assays: Nucleotide exchange assays using purified recombinant proteins can quantify the catalytic efficiency of EF-Ts in promoting GDP release from EF-Tu. Isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can determine binding kinetics and thermodynamic parameters.

• Structural Biology Approaches: X-ray crystallography or cryo-electron microscopy of the EF-Ts:EF-Tu complex can reveal atomic-level details of interaction interfaces. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map conformational changes during complex formation.

• Computational Methods: Molecular dynamics simulations can model the dynamics of the EF-Ts:EF-Tu interaction, especially adaptations for functioning at low temperatures.

When performing these studies, it's crucial to account for the temperature-dependence of these interactions, conducting experiments at temperatures relevant to S. sediminis' natural habitat (psychrophilic conditions) .

How does temperature affect the structure and function of recombinant S. sediminis EF-Ts?

As S. sediminis is a psychrophilic bacterium adapted to cold environments , its EF-Ts likely exhibits specific structural and functional adaptations for optimal activity at low temperatures. To investigate temperature effects on recombinant S. sediminis EF-Ts:

• Thermal Stability Analysis: Differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy at varying temperatures can reveal the thermal unfolding profile and secondary structure changes.

• Temperature-Dependent Activity Assays: Nucleotide exchange activity measurements across a temperature range (0-37°C) can identify optimal temperature ranges and reveal cold-adaptation features.

• Structural Flexibility Assessment: Nuclear magnetic resonance (NMR) spectroscopy or HDX-MS can detect temperature-dependent changes in protein dynamics and flexibility.

These approaches should reveal whether S. sediminis EF-Ts displays characteristic features of cold-adapted proteins, such as increased structural flexibility, reduced hydrophobic core packing, or increased surface hydrophilicity compared to mesophilic homologs.

How can transcriptomic and proteomic approaches inform our understanding of EF-Ts regulation in S. sediminis?

Integrated -omics approaches can provide comprehensive insights into EF-Ts regulation in S. sediminis:

• Transcriptomic Analysis: RNA-Seq under various environmental conditions (temperature, salinity, nutrient availability) can reveal how tsf gene expression is regulated. Comparative transcriptomics across Shewanella species can identify conserved and species-specific regulatory elements .

• Proteomic Profiling: Quantitative proteomics using techniques like LC-MS/MS can determine how EF-Ts protein levels correlate with transcriptional changes and identify post-translational modifications.

• Regulon Mapping: ChIP-Seq targeting transcription factors can identify proteins that directly regulate tsf expression. This approach has been successfully applied to map transcriptional networks in related Shewanella species .

• Protein-Protein Interaction Networks: Affinity purification coupled with mass spectrometry can identify proteins that interact with EF-Ts beyond its canonical partner EF-Tu, potentially revealing novel regulatory mechanisms.

These approaches should be conducted under conditions relevant to S. sediminis' natural environment, including appropriate temperature ranges and salt concentrations .

What are the optimal conditions for expression and purification of recombinant S. sediminis EF-Ts?

Based on the available information about recombinant S. sediminis EF-Ts , the following protocol represents an optimized approach:

Expression System Selection:

• Baculovirus expression system has been successfully employed

• Alternatively, E. coli-based expression using cold-adapted strains (ArcticExpress) may improve folding

Expression Protocol:

• Clone the full-length tsf gene (encoding all 282 amino acids) into an appropriate expression vector

• Transform/transfect the expression host

• Induce protein expression under conditions optimized for psychrophilic proteins (lower temperature, 15-20°C)

• Harvest cells and lyse using methods that preserve protein structure

Purification Strategy:

• Affinity chromatography using an appropriate tag (His-tag commonly used)

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography to obtain homogeneous protein

• Quality assessment by SDS-PAGE (target purity >85%)

Storage Recommendations:

• Store at -20°C for short-term or -80°C for extended storage

• Add 5-50% glycerol to prevent freeze-thaw damage

• Avoid repeated freeze-thaw cycles

• Work with aliquots at 4°C for up to one week

What analytical methods should be employed to verify the structure and function of purified recombinant S. sediminis EF-Ts?

A comprehensive analytical workflow should include:

Structural Verification:

• Mass Spectrometry: Accurate mass determination by ESI-MS or MALDI-TOF to confirm protein identity

• Circular Dichroism: Assessment of secondary structure elements

• Thermal Shift Assays: Determination of protein stability under various buffer conditions

• Dynamic Light Scattering: Evaluation of size distribution and aggregation state

Functional Validation:

• GDP/GTP Exchange Assay: Quantification of nucleotide exchange activity using fluorescent nucleotide analogs

• EF-Tu Binding Assays: Measurement of binding kinetics by SPR or ITC

• Translation Activity: In vitro translation assays using S. sediminis components to assess functional contribution

Quality Control Metrics:

• Purity: >85% by SDS-PAGE as minimum standard

• Homogeneity: Single peak by size exclusion chromatography

• Activity: Defined minimum specific activity in nucleotide exchange assays

• Endotoxin Levels: <1 EU/mg protein for sensitive applications

How can researchers effectively reconstitute lyophilized recombinant S. sediminis EF-Ts?

Based on the product information provided , the following reconstitution protocol is recommended:

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring contents to 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)

• Mix gently by inversion, avoiding vigorous vortexing which may denature the protein

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C for short-term or -80°C for long-term storage

Stability Considerations:

• Shelf life of liquid formulation: approximately 6 months at -20°C/-80°C

• Shelf life of lyophilized formulation: approximately 12 months at -20°C/-80°C

• For working solutions, maintain at 4°C for no more than one week

Quality Verification After Reconstitution:

• Verify protein concentration using absorbance at 280 nm or Bradford/BCA assay

• Confirm activity using a functional assay before use in critical experiments

How can S. sediminis EF-Ts be used to study bacterial adaptation to extreme environments?

S. sediminis EF-Ts offers a valuable model for studying cold adaptation mechanisms in bacterial translation machinery:

• Comparative Biochemical Analysis: Parallel characterization of EF-Ts proteins from psychrophilic (S. sediminis), mesophilic, and thermophilic bacteria can reveal temperature-specific adaptations in protein structure and function. Key parameters to compare include:

  Parameter	S. sediminis EF-Ts (Psychrophile)	Mesophilic EF-Ts	Thermophilic EF-Ts
  Activity temperature optimum	Expected: 0-15°C	Expected: 25-37°C	Expected: >50°C
  Structural flexibility	Likely higher	Moderate	Lower
  Thermal stability	Lower	Moderate	Higher
  Surface charge distribution	More negative surface charges	Balanced	More hydrophobic core

• Mutational Analysis: Creating chimeric proteins by domain swapping between S. sediminis EF-Ts and mesophilic homologs can identify specific regions responsible for cold adaptation.

• In vivo Complementation: Testing whether S. sediminis EF-Ts can functionally complement EF-Ts-deficient strains of E. coli or other model organisms at various temperatures can reveal the practical impact of cold-adaptation features.

• Evolutionary Analysis: Phylogenetic comparison of EF-Ts sequences across the Shewanella genus, which includes species adapted to different temperature regimes, can reveal evolutionary trajectories of cold adaptation .

How does S. sediminis EF-Ts compare to elongation factors from other Shewanella species?

The Shewanella genus exhibits significant genomic diversity , making comparative analysis of elongation factors particularly informative:

• Sequence Comparison: Multiple sequence alignment of EF-Ts proteins from different Shewanella species can identify conserved and variable regions that may correlate with environmental adaptation. The genus includes species from diverse environments, from cold deep-sea habitats to warmer freshwater systems .

• Structural Comparison: Homology modeling of EF-Ts proteins from different Shewanella species based on known EF-Ts structures can reveal species-specific structural adaptations.

• Functional Comparison: Biochemical characterization of recombinant EF-Ts proteins from multiple Shewanella species can correlate functional properties with environmental niches.

• Expression Pattern Analysis: Transcriptomics data from various Shewanella species can reveal whether tsf gene expression regulation differs between species in response to environmental stimuli .

This comparative approach can provide insights into how translation machinery components evolve in response to different environmental pressures within a single bacterial genus.

What insights can S. sediminis EF-Ts provide about protein synthesis in bioremediation applications?

S. sediminis is notable for its ability to degrade environmental contaminants like RDX and catalyze reductive dechlorination of tetrachloroethene (PCE) , making its protein synthesis machinery relevant to bioremediation applications:

• Translation Efficiency Under Remediation Conditions: Investigating how S. sediminis EF-Ts functions in the presence of contaminants can reveal adaptations that support protein synthesis during bioremediation. This includes:

  • Activity assays in the presence of heavy metals

  • Stability measurements in the presence of organic solvents

  • Interaction studies with other translation factors under stress conditions

• Engineered Optimization: Drawing from research on related species like S. oneidensis , engineered modifications to S. sediminis EF-Ts could potentially enhance translation efficiency under bioremediation conditions, supporting more robust degradation capabilities.

• Stress Response Integration: Understanding how EF-Ts expression and activity change during exposure to contaminants can reveal mechanisms by which S. sediminis maintains protein synthesis during environmental stress.

• Cold-Adaptation Relevance: The cold-adapted properties of S. sediminis EF-Ts may be particularly valuable for bioremediation applications in cool environments where contaminant degradation is typically slower.

What are common challenges in working with recombinant S. sediminis EF-Ts and how can they be addressed?

Researchers working with recombinant S. sediminis EF-Ts may encounter several challenges:

Challenge 1: Protein Solubility Issues

• Problem: Low solubility during expression or after purification

• Solutions:

  • Express at lower temperatures (10-15°C) to promote proper folding

  • Include solubility-enhancing tags (SUMO, MBP, etc.)

  • Optimize buffer conditions (pH 7.0-8.0, 150-300 mM NaCl, add 5-10% glycerol)

  • Consider detergents (0.05-0.1% Tween-20) for stabilization

Challenge 2: Activity Loss During Storage

• Problem: Decreased functional activity after storage

• Solutions:

  • Always store with 50% glycerol at -80°C

  • Avoid repeated freeze-thaw cycles

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

  • Consider flash-freezing in liquid nitrogen before storage

Challenge 3: Contaminating Nuclease Activity

• Problem: Degradation of nucleic acids in downstream applications

• Solutions:

  • Include additional purification steps (ion exchange chromatography)

  • Add EDTA (1-2 mM) to chelate metal ions required for nuclease activity

  • Test nuclease activity before using in sensitive applications

Challenge 4: Batch-to-Batch Variability

• Problem: Inconsistent activity between protein preparations

• Solutions:

  • Standardize expression and purification protocols

  • Implement rigorous quality control testing

  • Maintain detailed records of specific activity for each preparation

  • Create reference standards for functional comparisons

How can researchers effectively study the role of S. sediminis EF-Ts in bacterial translation systems?

To comprehensively investigate S. sediminis EF-Ts function in translation:

• Reconstituted Translation Systems:

  • Develop a minimal in vitro translation system using purified components from S. sediminis

  • Compare translation efficiency with native vs. recombinant EF-Ts

  • Assess temperature dependence of translation (4-37°C range)

• Genetic Approaches:

  • Construct tsf deletion or conditional expression mutants in S. sediminis using established genetic techniques for Shewanella species

  • Perform complementation experiments with wild-type and mutant variants

  • Use RNA-Seq to analyze global effects of tsf modulation

• Single-Molecule Techniques:

  • Apply fluorescence resonance energy transfer (FRET) to monitor EF-Ts:EF-Tu interactions in real-time

  • Use total internal reflection fluorescence (TIRF) microscopy to visualize translation dynamics

  • Employ optical tweezers to measure forces involved in EF-Ts-mediated nucleotide exchange

• Structural Biology Integration:

  • Combine cryo-EM structures of S. sediminis ribosomes with molecular dynamics simulations

  • Map the interactions between EF-Ts and other translation components

  • Identify structural adaptations specific to psychrophilic translation

What advanced techniques can reveal post-translational modifications of S. sediminis EF-Ts?

Post-translational modifications (PTMs) can significantly impact EF-Ts function but remain largely unexplored in S. sediminis:

• Mass Spectrometry-Based PTM Mapping:

  • Employ bottom-up proteomics with high-resolution MS/MS

  • Use enrichment techniques for specific modifications (phosphopeptide enrichment, etc.)

  • Apply electron transfer dissociation (ETD) or electron capture dissociation (ECD) for labile PTM preservation

• Site-Specific PTM Analysis:

  • Generate antibodies against specific PTMs predicted in S. sediminis EF-Ts

  • Use Western blotting with PTM-specific antibodies

  • Apply targeted mass spectrometry (parallel reaction monitoring, PRM) for quantitative analysis

• Functional Impact Assessment:

  • Create site-directed mutants mimicking or preventing specific PTMs

  • Compare activity profiles of modified and unmodified protein variants

  • Determine how environmental conditions affect PTM patterns

• PTM Dynamics:

  • Monitor changes in PTM status during different growth phases

  • Investigate PTM changes in response to environmental stressors

  • Apply pulse-chase experiments to determine PTM turnover rates

What are the key considerations for researchers working with recombinant S. sediminis EF-Ts?

Researchers should consider several critical factors when working with this protein:

• Cold-Adapted Properties: As a protein from a psychrophilic organism , S. sediminis EF-Ts likely has optimal activity at lower temperatures. Experimental conditions should reflect this characteristic, with assays performed across a temperature range that includes 4-15°C.

• Stability Considerations: The protein may exhibit lower thermal stability than mesophilic homologs. Storage and handling procedures should be strictly followed, including maintaining glycerol concentrations of 5-50% and avoiding repeated freeze-thaw cycles .

• Functional Context: When interpreting results, consider the native role of EF-Ts within S. sediminis' unique metabolic capabilities, including its ability to degrade environmental contaminants .

• Comparative Framework: Results should be interpreted within the broader context of the Shewanella genus, which exhibits significant genomic and functional diversity .

• Quality Control: Rigorous quality assessment is essential, with minimum purity standards of >85% by SDS-PAGE and functional validation through activity assays .