Recombinant Prochlorococcus marinus Elongation factor Tu (tuf)

What is Elongation factor Tu (tuf) in Prochlorococcus marinus and what is its function?

Elongation factor Tu (EF-Tu) is a crucial protein involved in the elongation phase of protein synthesis. In Prochlorococcus marinus, this protein (encoded by the tuf gene) facilitates the binding of aminoacyl-tRNA to the ribosome during translation. The full-length protein consists of 399 amino acids and has a molecular weight of approximately 43-45 kDa .

Beyond its canonical role in translation, recent research in related bacteria suggests EF-Tu may have moonlighting functions. For instance, in certain bacteria like Lactobacillus johnsonii, EF-Tu can act as a cell surface-associated protein mediating attachment to intestinal epithelial cells and mucins . Whether Prochlorococcus marinus EF-Tu serves similar multifunctional roles remains an active area of investigation.

What are the optimal storage conditions for recombinant Prochlorococcus marinus EF-Tu?

For short-term storage (up to one week), recombinant Prochlorococcus marinus EF-Tu can be maintained at 4°C in working aliquots. For extended storage, the protein should be stored at -20°C or -80°C with the addition of glycerol (typically 5-50% final concentration, with 50% being standard) .

To minimize protein degradation, it is advisable to:

• Centrifuge the vial briefly before opening to bring contents to the bottom

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

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Store in low-protein-binding tubes to prevent adsorption to container surfaces

The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for up to 12 months under the same conditions .

What reconstitution protocols are recommended for optimal activity of recombinant Prochlorococcus marinus EF-Tu?

For optimal reconstitution of recombinant Prochlorococcus marinus EF-Tu, follow this methodological approach:

• Centrifuge the vial briefly (30 seconds at 10,000 × g) to collect the lyophilized product 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% (with 50% being standard) for cryoprotection

• Mix gently by inversion or low-speed vortexing until completely dissolved

• Prepare small aliquots (10-20 μL) in low-protein-binding microcentrifuge tubes

• Flash-freeze aliquots in liquid nitrogen before transferring to -20°C or -80°C for long-term storage

Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity. Working aliquots can be maintained at 4°C for up to one week .

How can researchers verify the purity and integrity of recombinant Prochlorococcus marinus EF-Tu?

Researchers can employ several complementary analytical techniques to verify the purity and integrity of recombinant Prochlorococcus marinus EF-Tu:

• SDS-PAGE analysis: Commercial preparations typically show >85% purity by SDS-PAGE . Run a protein sample alongside molecular weight markers to confirm the expected size of approximately 43-45 kDa.

• Western blotting: Using antibodies specific to EF-Tu or to an incorporated tag (if present).

• Mass spectrometry: Tandem mass spectrometry can verify the intact status of the protein, as demonstrated in studies of surface-expressed EF-Tu in other bacteria .

• Functional assays: GDP/GTP binding assays or aminoacyl-tRNA binding assays can confirm biological activity.

• Circular dichroism (CD) spectroscopy: To assess proper protein folding and secondary structure.

Data analysis should include densitometry of SDS-PAGE gels to quantify purity percentage and mass spectrometry data to confirm the expected molecular weight and sequence coverage.

How does salt stress affect EF-Tu expression and function in Prochlorococcus marinus?

Proteomic analyses have revealed that EF-Tu expression in cyanobacteria is responsive to salt stress, with studies showing a 1.73-fold reduction of EF-Tu protein under 6% salt conditions . This suggests that salt stress may influence translation efficiency through modulation of EF-Tu levels.

For Prochlorococcus marinus specifically, growth experiments demonstrate:

• The optimal salinity range for growth is 30-40 psu (practical salinity units)

• High-light adapted strain MED4 can tolerate lower salinity (down to 22 psu) compared to low-light adapted strain NATL1A (minimum 26 psu)

• After acclimation, MED4 can grow at salinity as low as 25 psu, while NATL1A requires at least 28 psu

Transcriptomic analyses of salt-stressed Prochlorococcus cells show strain-specific responses:

• NATL1A exhibits 525 differentially expressed genes under low salinity (28 psu)

• MED4 shows more stability with only 277 differentially expressed genes under similar conditions

• In MED4, translation-related genes (including those involved in ribosomal structure and biogenesis) tend to be induced under low salinity stress

• In NATL1A, these same genes tend to be repressed

This differential regulation may reflect the ecological niches of these strains and their evolutionary adaptations to varying oceanic conditions.

What methodologies are most effective for studying the interaction of Prochlorococcus marinus EF-Tu with other cellular components?

Several methodological approaches can effectively investigate interactions between Prochlorococcus marinus EF-Tu and other cellular components:

• Pull-down assays: Using tagged recombinant EF-Tu to identify binding partners from cell lysates, followed by mass spectrometry identification.

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics with potential interacting partners.

• Microscale thermophoresis (MST): To characterize molecular interactions under near-native conditions.

• Bacterial two-hybrid system: For in vivo validation of protein-protein interactions.

• Immunofluorescence microscopy: To visualize subcellular localization and potential surface expression, as observed in other bacterial species .

• Cryo-electron microscopy: To determine structural details of EF-Tu in complex with ribosomes or other binding partners.

For investigating potential moonlighting functions (as seen in other bacteria), methodological approaches might include:

• Cell surface protein extraction and analysis

• Enzyme-linked immunosorbent assays (ELISA) with live bacteria

• Electron microscopy with immunogold labeling

These techniques would help determine whether Prochlorococcus marinus EF-Tu, like that of Lactobacillus johnsonii, can function at the cell surface and potentially interact with environmental factors.

How might Prochlorococcus marinus EF-Tu expression patterns relate to environmental adaptation?

Prochlorococcus marinus comprises multiple ecotypes adapted to different light regimes and oceanic niches. Research indicates that translation-related proteins, including EF-Tu, may play significant roles in adaptation to varying environmental conditions .

Climate change is predicted to significantly affect Prochlorococcus distribution, with models suggesting:

• A net global increase of 29% in cell abundances

• Poleward shifts of at least 10° in marine phytoplankton niches by century's end

• Increases of approximately 50% in Prochlorococcus abundance in more poleward regions of their distribution

These changes in distribution will expose Prochlorococcus populations to new environmental stressors, potentially affecting EF-Tu expression and function. The differential regulation of translation machinery (including EF-Tu) between strains may contribute to their distinct capacities to adapt to changing conditions.

What role might EF-Tu play in Prochlorococcus marinus adaptation to different light intensities?

Prochlorococcus marinus exhibits remarkable adaptations to varying light intensities, with distinct high-light (HL) and low-light (LL) adapted ecotypes. Research on responses to light and oxygen provides insights into potential roles of translation machinery, including EF-Tu:

• Different growth responses to light conditions between strains correlate with differences in gene expression related to:

  • Protein turnover

  • Oxygen-dependent enzymes

  • DNA repair enzymes

• Exposure to high light and UV leads to:

  • DNA lesions

  • Replication fork arrests

  • Delayed chromosome replication

• Reactive oxygen species (ROS) generated under high light conditions can:

  • Cause DNA damage

  • Create imbalances between protein synthesis and DNA replication

  • Potentially affect translation efficiency

While specific EF-Tu expression patterns under different light regimes aren't directly reported in the search results, the general pattern shows that translation machinery responds to environmental stressors. In low-light adapted strains (NATL1A), translation-related genes tend to be repressed under stress conditions, while in high-light adapted strains (MED4), they tend to be induced .

This divergent regulation of translation machinery may contribute to the different capacities of Prochlorococcus ecotypes to thrive in their respective light niches, with EF-Tu potentially serving as a key component in this adaptation process.

What are common challenges in working with recombinant Prochlorococcus marinus EF-Tu and how can they be addressed?

Researchers working with recombinant Prochlorococcus marinus EF-Tu may encounter several technical challenges:

• Protein stability issues:

  • Challenge: EF-Tu may exhibit limited stability during storage or experimental manipulation.

  • Solution: Add 5-50% glycerol as a cryoprotectant, store at -80°C in small aliquots, and avoid repeated freeze-thaw cycles .

• Solubility limitations:

  • Challenge: Recombinant EF-Tu may form aggregates or precipitate during reconstitution.

  • Solution: Reconstitute slowly at low concentrations (0.1-1.0 mg/mL) in appropriate buffers, and consider adding low concentrations of non-ionic detergents if necessary .

• Functional activity assessment:

  • Challenge: Confirming that recombinant EF-Tu retains native functionality.

  • Solution: Implement GDP/GTP binding assays or in vitro translation systems to verify activity.

• Expression system limitations:

  • Challenge: Mammalian cell expression systems (as used for commercial preparations) may introduce post-translational modifications that differ from the native cyanobacterial protein.

  • Solution: Compare results with EF-Tu expressed in bacterial systems or validate key findings using native protein extracted from Prochlorococcus cultures.

• Strain-specific variations:

  • Challenge: Different Prochlorococcus strains may exhibit variations in EF-Tu sequence and function.

  • Solution: Consider strain-specific differences when designing experiments, and clearly document which strain's EF-Tu is being used (e.g., MIT 9215) .

How can researchers optimize experimental design when investigating potential moonlighting functions of Prochlorococcus marinus EF-Tu?

Based on findings that EF-Tu can serve as a cell surface protein in some bacteria , researchers investigating potential moonlighting functions of Prochlorococcus marinus EF-Tu should consider these methodological approaches:

• Surface localization studies:

  • Develop a surface protein extraction protocol specific for Prochlorococcus

  • Use immunofluorescence microscopy with anti-EF-Tu antibodies

  • Employ surface biotinylation followed by pull-down and Western blotting

• Functional binding assays:

  • Test pH-dependent binding properties, as seen with Lactobacillus EF-Tu

  • Develop competition assays to assess binding specificity

  • Use recombinant tagged EF-Tu in binding studies with potential interaction partners

• Structural biology approaches:

  • Compare the Prochlorococcus EF-Tu structure with surface-expressed EF-Tu from other bacteria

  • Identify potential surface-exposed domains that might facilitate moonlighting functions

• Environmental condition considerations:

  • Test for differential surface expression under varying conditions (light intensity, salinity, temperature)

  • Correlate any surface expression with environmental adaptation patterns

• Controls and validation:

  • Include appropriate negative controls using other abundant cytoplasmic proteins

  • Validate findings using multiple complementary techniques

  • Consider the natural context of Prochlorococcus in designing physiologically relevant assays

By implementing these methodological approaches, researchers can systematically investigate whether Prochlorococcus marinus EF-Tu exhibits moonlighting functions similar to those observed in other bacterial species, potentially revealing novel aspects of Prochlorococcus ecology and adaptation.

How might studying Prochlorococcus marinus EF-Tu contribute to understanding global ocean ecology in the context of climate change?

As climate change alters oceanic conditions, understanding the molecular mechanisms of adaptation in key photosynthetic organisms like Prochlorococcus becomes increasingly important:

• Biomarker potential: Changes in EF-Tu expression patterns could serve as molecular indicators of adaptation to changing oceanic conditions. Research shows that translation-related genes respond differently to stress in different Prochlorococcus ecotypes .

• Distribution prediction models: Understanding the relationship between EF-Tu function and environmental adaptation could improve models predicting Prochlorococcus distribution changes:

  • Current models predict a 29% global increase in Prochlorococcus abundance

  • Poleward shifts of at least 10° in marine phytoplankton niches

  • Approximately 50% increases in more poleward regions

• Strain-specific responses: Research on strain differences in stress responses (including differential regulation of translation machinery) provides insight into how different Prochlorococcus populations might respond to climate change:

  • High-light adapted strain MED4 shows greater tolerance to low salinity than low-light adapted strain NATL1A

  • MED4 induces translation genes under stress while NATL1A represses them

• Oxygen Minimum Zone adaptation: Prochlorococcus occupies distinct niches including Oxygen Minimum Zones, which are predicted to expand with climate change. Understanding how EF-Tu function relates to oxygen adaptation could provide insights into future ecological shifts .

Methodological approaches should include comparative transcriptomics and proteomics across strains and conditions, coupled with functional studies of EF-Tu under simulated future ocean conditions.

What novel experimental approaches could advance our understanding of Prochlorococcus marinus EF-Tu structure-function relationships?

Several cutting-edge experimental approaches could significantly advance our understanding of Prochlorococcus marinus EF-Tu:

• Cryo-electron microscopy (Cryo-EM):

  • Determine high-resolution structures of EF-Tu in complex with ribosomes

  • Visualize conformational changes during GTP/GDP cycling

  • Compare structures across different Prochlorococcus ecotypes

• Single-molecule techniques:

  • Apply Förster resonance energy transfer (FRET) to monitor EF-Tu conformational dynamics

  • Use optical tweezers to study force generation during translocation

  • Implement single-molecule tracking in vivo to analyze EF-Tu mobility and localization

• Integrative structural biology:

  • Combine X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations

  • Develop EF-Tu structural models under various environmental conditions

  • Map functional domains to specific ecological adaptations

• CRISPR-based approaches:

  • Develop CRISPR interference (CRISPRi) systems for Prochlorococcus

  • Create site-specific mutations to test structure-function hypotheses

  • Engineer strain-specific EF-Tu variants to test adaptation hypotheses

• In situ studies:

  • Develop methods to analyze EF-Tu expression and activity in natural oceanic samples

  • Correlate with environmental parameters using autonomous sampling platforms

  • Implement metaproteomics to study natural variation in EF-Tu across oceanic regions

These advanced methodological approaches would provide unprecedented insights into how this key translational protein contributes to Prochlorococcus adaptation and success as the most abundant photosynthetic organism in nutrient-poor regions of the world's oceans.