Recombinant Synechococcus elongatus Elongation factor Tu (tuf)

What is Elongation factor Tu (tuf) in Synechococcus elongatus and what is its role in protein synthesis?

Elongation factor Tu (EF-Tu) is a highly conserved GTP-binding protein that plays a crucial role in the elongation phase of protein synthesis in Synechococcus elongatus. During translation, EF-Tu delivers aminoacyl-tRNAs to the ribosome A-site in a GTP-dependent manner. Once codon-anticodon recognition occurs, GTP is hydrolyzed, and EF-Tu:GDP is released from the ribosome. EF-Tu then undergoes nucleotide exchange to regenerate the active EF-Tu:GTP form.

In cyanobacteria such as Synechococcus, EF-Tu is particularly important due to its dual role in translation and stress response mechanisms. Evidence indicates that EF-Tu in cyanobacteria is a direct target of reactive oxygen species (ROS), becoming inactivated via oxidation of conserved cysteine residues, which serves as a mechanism to rapidly inhibit protein synthesis during oxidative stress conditions . This regulatory mechanism is part of a broader stress response system connected to photosynthetic activity.

What expression systems are effective for producing recombinant Elongation factor Tu in Synechococcus elongatus?

For recombinant expression of EF-Tu in Synechococcus elongatus, several approaches have proven effective:

• Native promoter systems: Utilizing native cyanobacterial promoters like psbA2, which responds to stress conditions, has shown success in driving recombinant protein expression in S. elongatus PCC 7942 . This approach eliminates the need for costly exogenous inducers and reduces potential cell stress.

• Inducible promoter systems: IPTG-inducible promoters such as Ptrc can be used for controlled expression, as demonstrated in studies with other recombinant proteins in S. elongatus . This system allows for temporal control of expression.

• Integration sites: Neutral sites in the S. elongatus genome (NSI or NSII) can be targeted for stable integration of expression cassettes encoding EF-Tu .

A typical expression cassette would include:

• A strong or inducible promoter (psbA2 or Ptrc)

• The tuf gene sequence optimized for S. elongatus codon usage

• A selection marker (streptomycin or kanamycin resistance)

• Flanking sequences for homologous recombination at neutral sites

What experimental techniques are commonly used to verify and quantify recombinant EF-Tu expression?

Several complementary techniques are essential for robust verification and quantification of recombinant EF-Tu expression:

• Western blotting: Using antibodies specific to EF-Tu or to an epitope tag fused to the recombinant protein.

• RT-qPCR: Quantifying tuf gene transcript levels, as demonstrated in studies of gene expression in S. elongatus . This approach allows measurement of transcriptional changes in response to different conditions.

• Fluorescence measurements: If using a fluorescent protein fusion or reporter system under the same promoter.

• Activity assays: Measuring GTPase activity using colorimetric or fluorescent assays.

• Mass spectrometry: For precise identification and quantification of the recombinant protein.

How does Synechococcus elongatus growth conditions affect recombinant EF-Tu expression?

Growth conditions significantly impact recombinant protein expression in S. elongatus, including EF-Tu production:

• Light intensity: Light is a critical parameter affecting gene expression in photosynthetic organisms. Different S. elongatus strains exhibit varying sensitivities to light intensity, with growth responses categorized as:

  • Moderate light (ML): ~50 μmol m⁻² s⁻¹

  • High light (HL): ~400 μmol m⁻² s⁻¹

  • Very high light (VHL): ~850 μmol m⁻² s⁻¹

• Culture medium: Liquid versus solid medium can result in different growth behaviors and protein expression levels. S. elongatus shows greater sensitivity to excess light on solid media compared to liquid cultures .

• Temperature: S. elongatus UTEX 2973, a fast-growing strain, shows pronounced tolerance to high temperatures, which may affect recombinant protein expression .

• Stress conditions: As observed with the psbA2 promoter, certain stress conditions can enhance recombinant protein expression .

How does oxidative stress affect the function of Elongation factor Tu in Synechococcus elongatus?

Oxidative stress profoundly impacts EF-Tu function in cyanobacteria through redox-based regulation. In Synechocystis sp. PCC 6803, a related cyanobacterium, ROS directly inhibits protein synthesis at the elongation step by targeting translation factors:

• Mechanism of inhibition: EF-Tu contains conserved cysteine residues that undergo oxidation during oxidative stress, resulting in functional inactivation .

• Regulatory significance: This oxidation constitutes a post-translational regulatory mechanism that rapidly inhibits protein synthesis during stress conditions, particularly affecting the synthesis of photosystem components like D1 .

• Experimental approaches to study this phenomenon include:

  • Site-directed mutagenesis of specific cysteine residues

  • Redox proteomics to identify oxidation sites

  • In vitro translation assays under controlled redox conditions

  • Comparative analysis with other translation factors like EF-G, which is also subject to redox regulation

• Physiological implications: The redox sensitivity of EF-Tu represents an important mechanism linking photosynthetic activity, ROS production, and translational regulation in cyanobacteria .

What promoter systems optimize recombinant EF-Tu production in Synechococcus elongatus?

Selecting the optimal promoter system is critical for successful recombinant EF-Tu production:

• Native photosynthetic promoters: The psbA2 promoter, which responds to stress conditions, has shown effectiveness for recombinant protein expression in S. elongatus PCC 7942 .

• Inducible systems: IPTG-inducible promoters like Ptrc provide temporal control but require exogenous inducers that may stress cells .

• Promoter strength optimization: dRNA-Seq techniques have enabled genome-wide identification of transcription start sites (TSSs) in S. elongatus UTEX 2973, revealing 4,808 TSSs that can inform promoter selection .

• Experimental evaluation: Different promoters should be systematically compared using reporter systems before use with EF-Tu. Parameters to assess include:

What methodological approaches are effective for studying the interaction between EF-Tu and the ribosome in Synechococcus elongatus?

Investigating EF-Tu-ribosome interactions in S. elongatus requires specialized approaches:

• Cryo-electron microscopy: Enables visualization of EF-Tu binding to ribosomes at near-atomic resolution.

• Ribosome profiling: Provides genome-wide information on ribosome positioning and translation efficiency, revealing the impact of EF-Tu modifications on translational dynamics.

• In vitro reconstitution systems: Using purified components to study the kinetics of EF-Tu-mediated aminoacyl-tRNA delivery.

• Fluorescence-based approaches:

  • Förster resonance energy transfer (FRET) between labeled EF-Tu and ribosomal proteins

  • Single-molecule techniques to track individual EF-Tu molecules during translation

• Crosslinking mass spectrometry: Identifies specific contact points between EF-Tu and ribosomal components.

• Genetic approaches: Creating EF-Tu variants with altered ribosome interaction properties and assessing their impact on translation and cell growth.

How can magnetic field application enhance recombinant EF-Tu production in Synechococcus elongatus?

Recent research has demonstrated that magnetic field application (MF) can significantly enhance recombinant protein production in S. elongatus:

• Optimal field strength: Exposure to 30 mT (MF30) has been shown to increase transcription of recombinant genes under the psbA2 promoter in S. elongatus PCC 7942 .

• Mechanism of action: MF appears to influence the cyanobacterial photosynthetic machinery, likely through:

  • Stress-induced shifts in gene expression

  • Altered enzyme activity

  • Positive impact on photosystem II (PSII) without disrupting the electron transport chain

• Quantum-mechanical mechanism: The effects align with the "quantum-mechanical mechanism" theory of magnetic field interactions with biological systems .

• Experimental implementation:

  • Expose cultures to controlled magnetic fields during growth

  • Monitor photosynthetic parameters alongside protein expression

  • Assess both transcriptional and translational impacts

• Advantages: This approach is non-invasive and does not require chemical additives, making it particularly suitable for applications requiring high purity of the final product.

What role does the stringent response play in regulating translation and EF-Tu function in Synechococcus elongatus?

The stringent response, mediated by (p)ppGpp, represents a critical regulatory system affecting translation in S. elongatus:

• Triggering factors: Unlike most bacteria where nutrient limitation induces (p)ppGpp accumulation, in cyanobacteria, (p)ppGpp accumulates primarily in response to absence of photosynthetic activity, such as during night periods .

• Key enzymes: The enzyme mediating this response in cyanobacteria is the bifunctional (p)ppGpp synthetase/hydrolase Rel .

• Impact on translation: (p)ppGpp accumulation affects multiple aspects of translation, including:

  • Ribosome biogenesis

  • Translation factors (including EF-Tu)

  • Global protein synthesis rates

• Experimental approaches:

  • Measure (p)ppGpp levels under different conditions using thin-layer chromatography

  • Investigate the impact of RelQ overexpression on EF-Tu activity

  • Examine changes in EF-Tu expression and modification during stringent response

• Connections to cellular morphology: Interestingly, overexpression of RelQ increases cell size in S. elongatus , suggesting complex relationships between translation regulation, growth control, and cell morphology.

How can proteomic approaches be used to identify post-translational modifications of EF-Tu in Synechococcus elongatus?

Post-translational modifications (PTMs) significantly impact EF-Tu function and can be comprehensively characterized using advanced proteomic approaches:

• Sample preparation strategies:

  • Affinity purification of tagged recombinant EF-Tu

  • Enrichment for specific modifications (phosphopeptides, redox-modified peptides)

  • Fractionation to increase detection sensitivity

• Mass spectrometry techniques:

  • Bottom-up proteomics for comprehensive PTM mapping

  • Top-down proteomics for intact protein analysis

  • Targeted approaches for quantification of specific modifications

• Key PTMs to investigate:

  • Oxidation of cysteine residues (particularly relevant given EF-Tu's redox sensitivity)

  • Phosphorylation

  • Methylation

  • Acetylation

• Comparative analysis:

  • Different growth conditions (light intensities, nutrient availability)

  • Stress responses (oxidative stress, high light)

  • Different strains (PCC 7942 vs. UTEX 2973)

• Functional validation of identified PTMs:

  • Site-directed mutagenesis to mimic or prevent modifications

  • In vitro activity assays with modified vs. unmodified EF-Tu

  • Structural analysis to determine impact on protein conformation

What approaches can resolve contradictory data regarding EF-Tu function under different experimental conditions?

Researchers frequently encounter contradictory results when studying EF-Tu function across different experimental setups. Resolving these discrepancies requires systematic approaches:

• Standardize experimental conditions:

  • Light quality and intensity should be precisely controlled and reported

  • Growth media composition must be consistent

  • Cell density and growth phase must be matched across experiments

• Multi-method validation:

  • Employ complementary techniques to verify key findings

  • Combine in vivo and in vitro approaches to distinguish direct vs. indirect effects

  • Use genetic and biochemical methods in parallel

• Strain authentication:

  • Verify strain identity through genomic analysis

  • Consider genetic drift in laboratory strains

  • Maintain rigorous strain validation procedures

• Environmental variables control:

  • Monitor and report CO₂ levels

  • Control temperature fluctuations

  • Account for circadian effects

• Statistical considerations:

  • Ensure adequate biological and technical replicates

  • Use appropriate statistical tests for data analysis

  • Report effect sizes alongside significance values

The contradictory results often stem from the complex interplay between photosynthesis, redox regulation, and translation in cyanobacteria, where subtle experimental differences can significantly impact outcomes .

How does EF-Tu interact with the photosynthetic apparatus in Synechococcus elongatus?

The interaction between EF-Tu and the photosynthetic machinery represents a critical regulatory nexus in cyanobacteria:

• Co-regulation with photosystems:

  • Oxidative damage from photosynthesis directly impacts EF-Tu through cysteine oxidation

  • Inhibition of EF-Tu by ROS affects synthesis of photosystem components, particularly D1 protein of PSII

• Spatial organization:

  • Translation machinery may be spatially organized near thylakoid membranes to facilitate co-translational insertion of membrane proteins

  • This organization creates microenvironments where EF-Tu is exposed to varying redox conditions

• Light-dependent regulation:

  • High light conditions affect both translation factors and photosystems

  • The recovery of translation after high light stress involves reactivation of oxidized EF-Tu

• Experimental approaches:

  • Co-localization studies using fluorescently tagged EF-Tu

  • Membrane fractionation to detect membrane-associated EF-Tu pools

  • Analysis of EF-Tu oxidation state in relation to photosynthetic activity

• Functional significance: This interaction represents a key mechanism by which cells rapidly adjust protein synthesis in response to changing light conditions, indicating that EF-Tu functions not just as a translation factor but as a sensor integrating photosynthetic activity with cellular protein synthesis .

Research Methodology Tables

Methods for Assessing EF-Tu Oxidation State

What emerging technologies hold promise for studying EF-Tu function in cyanobacteria?

The next generation of research into cyanobacterial EF-Tu will benefit from several cutting-edge approaches:

• CRISPR-Cas9 genome editing: Precise modification of the native tuf gene to create variants with altered redox sensitivity, GTPase activity, or ribosome interactions.

• Single-cell analysis: Technologies for examining translation dynamics and EF-Tu activity at the single-cell level, revealing cell-to-cell heterogeneity in responses to environmental changes.

• In situ structural biology: Techniques like cryo-electron tomography that can visualize translation complexes within their native cellular context.

• Synthetic biology approaches: Orthogonal translation systems incorporating engineered EF-Tu variants with novel properties.

• Systems biology integration: Multi-omics approaches combining transcriptomics, proteomics, and metabolomics to create comprehensive models of how EF-Tu links photosynthesis, translation, and stress responses.