Recombinant Synechococcus sp. Elongation factor Tu (tuf)

What is the canonical function of Elongation Factor Tu in cyanobacteria?

Elongation Factor Thermo Unstable (EF-Tu) is one of the most abundant proteins in bacterial cells, including cyanobacteria like Synechococcus sp. Its primary canonical function is to transport aminoacylated tRNAs to the ribosome during protein translation . EF-Tu forms a ternary complex with GTP and aminoacyl-tRNA, which then associates with the ribosomal A-site. Once the incoming aminoacyl-tRNA correctly docks with the mRNA codon, GTPase activity induces a conformational change enabling the release of EF-Tu from the ribosome . This process is critical for ensuring the fidelity and efficiency of protein translation in cyanobacterial cells.

How does the molecular structure of EF-Tu facilitate its function in Synechococcus sp.?

The molecular architecture of Synechococcus sp. EF-Tu consists of three distinct domains, conventionally referred to as domains i, ii, and iii, which have evolved a high degree of molecular flexibility . To perform its canonical function, EF-Tu must form a functional binding pocket for an aminoacyl-tRNA, which requires domain i to align more closely with domains ii and iii by moving approximately 90° . The extent of this intramolecular movement is significant, comprising about one-third of the protein's total diameter, highlighting the dramatic conformational change necessary for proper function . This structural flexibility is essential for the protein's ability to bind GTP, interact with aminoacyl-tRNAs, and engage with the ribosome during translation.

How many copies of the tuf gene are present in Synechococcus sp. and how does this compare to other bacteria?

Unlike many enteric bacteria that carry two copies of the tuf gene (tufA and tufB), low G+C Gram-positive bacteria typically carry only a single copy of tuf . While the search results don't explicitly state the number of tuf copies in Synechococcus sp., we can infer from comparative genomic studies that cyanobacteria generally possess fewer tuf gene copies than enteric bacteria. In species with two copies, the nucleotide sequences differ by less than 1.4% . The presence of multiple tuf genes in some bacterial species appears to be an ancient feature that evolved before the branching of eubacteria, with some lineages subsequently losing the second copy through random events rather than lateral gene transfer .

What factors influence the expression levels of EF-Tu in Synechococcus sp. under different environmental conditions?

Expression levels of EF-Tu in Synechococcus sp. are significantly influenced by environmental stress conditions, particularly temperature and light intensity. Research has shown that during acclimatization to environmentally relevant conditions such as low temperature or high light, there is an upregulation of translation factors including EF-Tu . This upregulation likely serves as a protective mechanism, as translation factors are particularly vulnerable to oxidative damage under stress conditions. The regulation appears to involve complex signaling pathways that help coordinate the cellular response to environmental changes, ensuring that protein synthesis can continue effectively despite challenging conditions .

What expression systems are most effective for producing recombinant Synechococcus sp. EF-Tu?

For optimal expression of recombinant Synechococcus sp. EF-Tu, the implementation of cyanobacterial expression systems offers significant advantages over heterologous systems. Research has demonstrated success using the Ptrc promoter system in Synechococcus sp., which allows for controlled induction with IPTG . This approach enables precise regulation of expression levels, which is critical for functional studies. The expression system should incorporate:

• A strong, inducible promoter (such as Ptrc)

• The complete coding sequence of the tuf gene

• A repressor system (such as LacI) for tight control of expression

• Appropriate selection markers for stable maintenance

What purification strategies yield the highest quality recombinant EF-Tu protein for structural and functional studies?

Purification of high-quality recombinant EF-Tu from Synechococcus sp. requires a multi-step approach that preserves both structure and function. Based on research methodologies used for elongation factors, an effective purification protocol should include:

• Initial extraction using a buffer containing 50 mM Tris/HCl pH 7.4, 4 mM EDTA, with protease inhibitors (0.5 mM PMSF, 0.5 mM benzamidine) and reducing agents (1 mM DTT)

• Cell disruption via mechanical methods, such as bead beating with 0.1 μm glass beads

• Clarification by centrifugation at 5,500 g for 5 min at 4°C

• Column chromatography steps, potentially including:

  • Affinity chromatography (if using tagged constructs)

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

This approach minimizes protein degradation and oxidation, which is particularly important given EF-Tu's sensitivity to oxidative damage . The inclusion of reducing agents throughout the purification process is critical to maintain EF-Tu in its functional state.

How does oxidative stress affect the function of EF-Tu in Synechococcus sp., and what are the molecular mechanisms involved?

Elongation factors, including EF-Tu, are primary targets of reactive oxygen species (ROS) in cyanobacteria like Synechococcus sp. . Oxidative stress inhibits protein synthesis by targeting the translation machinery, particularly at the elongation phase. The molecular mechanism involves the oxidation of conserved cysteine residues in EF-Tu, which impairs its function in delivering aminoacyl-tRNAs to the ribosome . This sensitivity to oxidation makes EF-Tu a critical regulatory point where cellular redox status can modulate protein synthesis rates.

Research has demonstrated that in cyanobacterial translation systems, the addition of reduced forms of elongation factors can reverse the inhibition of translation caused by oxidative stress, while oxidized forms fail to restore translational activity . This suggests that the redox state of EF-Tu directly influences its functionality in protein synthesis during stress conditions.

What role does EF-Tu play in the adaptation of Synechococcus sp. to high light and oxidative stress conditions?

EF-Tu plays a crucial role in the adaptation of Synechococcus sp. to high light and resulting oxidative stress conditions. Under high light, cyanobacteria experience increased production of ROS, which can inhibit the repair of photodamaged photosystem II (PSII) by suppressing protein synthesis at the translational elongation level . The upregulation of EF-Tu appears to be part of the cellular defense mechanism against this stress.

Experimental evidence shows that overexpression of elongation factors in Synechococcus sp. increases tolerance to H₂O₂ in terms of protein synthesis . Additionally, growth assays demonstrate that strains with reduced ability to upregulate elongation factors show decreased growth rates under high light conditions compared to control strains . This supports the hypothesis that the regulation of EF-Tu levels is important for growth under stress conditions, as shown in the following experimental data:

These data highlight the protective role of EF-Tu against oxidative damage in the translation machinery, allowing cells to maintain protein synthesis rates even under stressful conditions.

What are the optimal conditions for assessing EF-Tu activity in vitro for Synechococcus sp.?

Assessing EF-Tu activity in vitro from Synechococcus sp. requires careful consideration of experimental conditions to maintain its functionality. Based on research methodologies, an optimal in vitro translation system should include:

• Cell extract preparation:

  • Cultures harvested during logarithmic growth phase

  • Extraction in buffer containing 50 mM Tris/HCl pH 7.4, 4 mM EDTA, with protease inhibitors and reducing agents

  • Cell disruption via mechanical methods while maintaining low temperature

• Reaction conditions:

  • Temperature: 30°C for standard conditions, 18°C for cold stress studies

  • Buffer components: Mg²⁺ (critical for GTP binding), K⁺, and NH₄⁺ ions

  • Reducing environment: DTT or similar to prevent oxidation

  • ATP regeneration system

  • Complete set of amino acids, tRNAs, and ribosomes

• Activity monitoring:

  • Incorporation of radiolabeled amino acids to track protein synthesis

  • GTPase activity measurement

  • Formation of ternary complexes with aminoacyl-tRNAs

This experimental setup allows for the assessment of how different conditions, particularly oxidative stress, affect EF-Tu function in protein synthesis .

How can researchers effectively detect and quantify EF-Tu in Synechococcus sp. cellular extracts?

For accurate detection and quantification of EF-Tu in Synechococcus sp. cellular extracts, a systematic approach combining multiple techniques yields the most reliable results:

• Immunoblotting protocol:

  • Sample collection: Harvest 10 mL of culture by centrifugation (7,300 g for 6 min at 4°C)

  • Cell lysis: Use lysis buffer (50 mM Tris/HCl pH 7.4, 4 mM EDTA, 0.5 mM PMSF, 0.5 mM benzamidine, 1 mM DTT) with 0.1 μm glass beads

  • Disruption: Three cycles of 60 s at 5 m/s in a high-speed homogenizer with 60 s rest at 4°C

  • Clarification: Centrifuge at 5,500 g for 5 min at 4°C

  • Protein quantification: Bradford or BCA assay

  • SDS-PAGE: Load equal protein amounts (typically 10-20 μg)

  • Transfer: To PVDF or nitrocellulose membrane

  • Blocking: 5% non-fat milk in TBST

  • Primary antibody: Anti-EF-Tu (specific to conserved regions)

  • Detection: HRP-conjugated secondary antibody with chemiluminescence

  • Quantification: Densitometry analysis using appropriate software

• Mass spectrometry-based quantification:

  • Digest samples with trypsin

  • Select signature peptides unique to Synechococcus sp. EF-Tu

  • Use targeted MS approaches (MRM/PRM) for accurate quantification

  • Include isotopically labeled standards for absolute quantification

These methods enable researchers to track changes in EF-Tu levels under different growth conditions or genetic manipulations .

What are the known moonlighting functions of EF-Tu in cyanobacteria, and how do they differ from its canonical role?

Beyond its canonical role in translation, EF-Tu exhibits several moonlighting functions in bacteria, including cyanobacteria like Synechococcus sp. These additional functions include:

• Cell surface localization: Despite lacking conventional secretion signal motifs, EF-Tu can traffic to and be retained on cell surfaces where it can interact with membrane receptors and extracellular matrix components

• Stress response: EF-Tu appears to contribute to cellular responses to various stress conditions, including temperature changes, oxidative stress, and high light intensity

• Potential role in pathogenesis: In pathogenic bacteria, surface-exposed EF-Tu can mediate interactions with host molecules, though this function is likely less relevant for non-pathogenic cyanobacteria

These moonlighting functions are distinct from EF-Tu's canonical role in translation and appear to be mediated by short linear motifs (SLiMs) in surface-exposed, non-conserved regions of the protein . These SLiMs allow EF-Tu to evolve additional functions without compromising its essential role in protein synthesis. The evolutionary conservation of these moonlighting capabilities suggests they provide significant adaptive advantages to the organism.

What protein-protein interactions have been identified for EF-Tu in Synechococcus sp., and what methods are most effective for studying these interactions?

While the search results don't provide specific details about EF-Tu protein-protein interactions in Synechococcus sp., we can infer potential interactions based on general knowledge of EF-Tu function and the limited information provided:

• Known and potential interactions:

  • EF-Ts: For nucleotide exchange and recharging of EF-Tu after GTP hydrolysis

  • Ribosomes: Particularly the A-site during translation

  • Aminoacyl-tRNAs: Forms ternary complexes during translation

  • PipX: A regulatory protein mentioned in the context of translation factors in Synechococcus

  • Components of redox signaling pathways: Given EF-Tu's sensitivity to oxidation

• Recommended methodologies for studying these interactions:

  • Co-immunoprecipitation: Using anti-EF-Tu antibodies to pull down interaction partners

  • Bacterial two-hybrid assays: For testing specific protein-protein interactions

  • Mass spectrometry-based interactomics: For unbiased identification of the EF-Tu interactome

  • Surface plasmon resonance: For determining binding kinetics and affinities

  • Molecular dynamics simulations: To predict potential interaction interfaces

  • FRET/BRET approaches: For monitoring interactions in vivo

These methodologies can help identify and characterize the full range of EF-Tu interactions in Synechococcus sp., providing insights into both its canonical and moonlighting functions.

How can researchers design experiments to distinguish between the canonical and moonlighting functions of EF-Tu in Synechococcus sp.?

Designing experiments to distinguish between canonical and moonlighting functions of EF-Tu in Synechococcus sp. requires sophisticated approaches that can separate these overlapping roles:

• Domain-specific mutations strategy:

  • Identify conserved residues essential for canonical function versus surface-exposed regions likely involved in moonlighting functions

  • Generate point mutations that selectively disrupt one function while preserving others

  • Create a matrix of mutations targeting different domains and functions

  • Assess both translation efficiency and specific moonlighting functions for each mutant

• Compartment-specific detection approach:

  • Develop tools to independently monitor cytoplasmic versus surface-localized EF-Tu

  • Use cell fractionation techniques to separate cytoplasmic, membrane, and extracellular fractions

  • Employ non-permeabilizing immunofluorescence to detect only surface-exposed EF-Tu

  • Compare EF-Tu distribution under various stress conditions

• Functional complementation experiments:

  • Create conditional knockdown strains with controlled expression of native EF-Tu

  • Complement with variants engineered to perform only canonical or only moonlighting functions

  • Assess the ability of each variant to rescue specific phenotypes

This experimental framework allows researchers to methodically separate and characterize the distinct functions of this multifaceted protein in Synechococcus sp.

What are the implications of EF-Tu's sensitivity to oxidation for experimental design and data interpretation?

EF-Tu's demonstrated sensitivity to oxidation has significant implications for experimental design and data interpretation in Synechococcus sp. research:

• Critical considerations for experimental design:

  • Sample preparation: All buffers should contain reducing agents (DTT or β-mercaptoethanol) to preserve EF-Tu's native state

  • Storage conditions: Samples should be maintained under oxygen-limited conditions when possible

  • Time management: Minimize the time between cell disruption and analysis to prevent artificial oxidation

  • Controls: Include parallel samples with and without oxidizing/reducing treatments

  • Oxidation state analysis: Consider incorporating techniques to assess the oxidation state of EF-Tu's cysteine residues

• Implications for data interpretation:

  • Translation assays: Reduced activity might reflect oxidation of EF-Tu rather than true biological regulation

  • Stress response studies: Distinguish between direct effects of stress conditions and secondary effects due to EF-Tu oxidation

  • Interaction studies: Protein-protein interactions may be redox-sensitive and change depending on EF-Tu's oxidation state

  • In vivo versus in vitro discrepancies: Differences may reflect the more oxidizing environment typically present in in vitro systems

• Experimental validation approaches:

  • Parallel assays with reduced and oxidized forms of EF-Tu to establish functional differences

  • Site-directed mutagenesis of redox-sensitive cysteine residues to create oxidation-resistant variants

  • Comparison of results under aerobic versus anaerobic conditions

Understanding these implications allows researchers to design more robust experiments and avoid misinterpreting data due to artifacts of EF-Tu oxidation .

How do researchers reconcile contradictory findings regarding EF-Tu function across different cyanobacterial species?

Reconciling contradictory findings regarding EF-Tu function across different cyanobacterial species requires a systematic approach that considers multiple factors:

• Methodological standardization:

  • Establish consensus protocols for EF-Tu isolation, activity assays, and oxidation state assessment

  • Document experimental conditions comprehensively (pH, temperature, buffer composition, etc.)

  • Develop reference standards for comparing results across laboratories

• Species-specific considerations:

  • Sequence alignment analysis to identify critical differences in EF-Tu between cyanobacterial species

  • Consideration of genomic context, including the number of tuf gene copies and their regulation

  • Assessment of physiological differences between species that might impact EF-Tu function

• Integration framework:

  • Meta-analysis of published data with attention to methodological differences

  • Collaborative cross-laboratory studies using standardized materials and methods

  • Development of mathematical models that can account for species-specific variables

• Resolution strategies for specific contradictions:

  • Direct side-by-side comparisons of EF-Tu from different species under identical conditions

  • Chimeric protein studies to identify domains responsible for species-specific differences

  • Heterologous expression to test if species-specific effects are intrinsic to EF-Tu or due to cellular context

By implementing these approaches, researchers can build a coherent understanding of EF-Tu function that accommodates genuine species-specific differences while eliminating artifacts arising from methodological variations.

What emerging technologies hold promise for advancing our understanding of EF-Tu function in Synechococcus sp.?

Several cutting-edge technologies show particular promise for deepening our understanding of EF-Tu function in Synechococcus sp.:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of EF-Tu conformational changes during different stages of translation

  • Allows for structural determination of EF-Tu in complex with various interaction partners

  • Provides insights into the structural basis of EF-Tu's moonlighting functions

• Redox proteomics:

  • Enables precise mapping of oxidation-sensitive residues in EF-Tu

  • Allows quantification of the oxidation state of specific cysteine residues under different conditions

  • Provides temporal resolution of oxidative modifications during stress responses

• Single-molecule techniques:

  • FRET-based approaches to track individual EF-Tu molecules during translation

  • Optical tweezers to measure forces involved in EF-Tu-ribosome interactions

  • Super-resolution microscopy to visualize EF-Tu localization within cyanobacterial cells

• CRISPR-based technologies:

  • CRISPRi for precise temporal control of EF-Tu expression

  • Base editing for introducing specific mutations without disrupting the entire gene

  • APEX2 proximity labeling to identify context-specific interaction partners

These technologies will enable researchers to address fundamental questions about EF-Tu's multifunctional nature in Synechococcus sp. and other cyanobacteria.

What are the key unresolved questions regarding EF-Tu function in Synechococcus sp. that should be prioritized in future research?

Several critical questions about EF-Tu function in Synechococcus sp. remain unresolved and merit prioritization in future research:

• Regulatory mechanisms:

  • What transcriptional and post-translational mechanisms regulate EF-Tu levels in response to different environmental stressors?

  • How is the balance between cytoplasmic and surface-localized EF-Tu controlled?

  • What signaling pathways coordinate EF-Tu activity with other cellular processes?

• Oxidation and redox regulation:

  • Which specific cysteine residues in Synechococcus sp. EF-Tu are most sensitive to oxidation?

  • How do these oxidation events specifically impair EF-Tu function?

  • What cellular mechanisms protect or repair oxidized EF-Tu?

• Structural dynamics:

  • How do the conformational changes of EF-Tu differ in Synechococcus sp. compared to model organisms?

  • What structural features enable EF-Tu to perform its diverse functions?

  • How does the structure of EF-Tu allow it to adapt to extreme environmental conditions?

• Evolutionary considerations:

  • Why have some cyanobacterial species retained multiple tuf genes while others have only one?

  • How have the moonlighting functions of EF-Tu evolved in cyanobacteria?

  • What selective pressures have shaped EF-Tu's dual role in translation and stress response?