Recombinant Prochlorococcus marinus subsp. pastoris Elongation factor P (efp)

What is Elongation factor P (EF-P) and what is its fundamental role in bacterial translation?

In bacteria like Escherichia coli, EF-P has been shown to be critical for efficient synthesis of proteins containing proline-rich sequences. Without EF-P, ribosomes pause at these sequences, leading to reduced protein output and potential downstream effects on cellular physiology .

How are recombinant proteins from Prochlorococcus marinus typically expressed and purified?

Recombinant proteins from P. marinus are typically expressed in E. coli expression systems. Based on protocols used for similar cyanobacterial proteins, the general methodology includes:

• Gene synthesis and cloning: The target gene (e.g., efp) is PCR-amplified from P. marinus genomic DNA using specific primers. The amplified gene is then subcloned into an expression vector such as pGEX-6P-1 for GST-fusion proteins or specialized vectors like pJexpress406 (with T5 promoter) .

• Expression optimization: For optimal expression:

  • Bacterial strains: E. coli BL21 cells are commonly used

  • Temperature control: Expression at 18-37°C depending on protein characteristics

  • Induction: Typically with IPTG (1 mM)

  • Duration: Protein-dependent (e.g., 21-60 hours)

• Purification:

  • Cell lysis: Using combinations of lysozyme, sonication, and nucleases

  • Column chromatography: Affinity purification (e.g., glutathione-Sepharose for GST-tagged proteins)

  • Tag removal: Using specific proteases like PreScission protease

  • Additional purification: Ion-exchange chromatography (e.g., Mono-Q column)

Protein concentration is typically estimated using methods described by Lowry or by visual comparison on Coomassie-stained SDS gels .

What are the optimal storage conditions for recombinant proteins from Prochlorococcus marinus?

Based on protocols for similar recombinant proteins from P. marinus, the following storage conditions are recommended:

• Short-term storage: Working aliquots can be stored at 4°C for up to one week

• Long-term storage: Store at -20°C; for extended storage, conserve at -20°C or -80°C

• Lyophilized form: Has a shelf life of approximately 12 months at -20°C/-80°C

• Liquid form: Generally has a shelf life of 6 months at -20°C/-80°C

For optimal stability, it is recommended to:

• Add glycerol to a final concentration of 5-50% (typically 50%)

• Aliquot the protein to avoid repeated freeze-thaw cycles

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

• Briefly centrifuge vials prior to opening to bring contents to the bottom

Repeated freezing and thawing is strongly discouraged as it can lead to protein degradation and loss of activity .

How does EF-P contribute to translational regulation in Prochlorococcus under different growth conditions?

The role of EF-P in translational regulation appears to be growth-rate dependent, which has significant implications for Prochlorococcus given its adaptation to various oceanic environments:

Growth rate dependency:
Studies in E. coli have shown that EF-P-dependent phenotypes are strongly influenced by growth conditions. When growth rate is limited (by temperature, nutrient availability, or oxygen restriction), many detrimental phenotypes associated with EF-P deficiency are suppressed . This suggests that:

• Under rapid growth conditions (high light, optimal temperature, nutrient-rich environments):

  • EF-P is critical for maintaining proteome homeostasis

  • Absence of EF-P results in significant ribosome queuing at polyproline motifs

  • Translation of proteins with PPX motifs is substantially reduced

  • Cells without EF-P have reduced fitness

• Under slow growth conditions (low light, suboptimal temperature, nutrient limitation):

  • The impact of EF-P deficiency is minimized

  • Polysome profiles become similar to wild-type

  • Colony morphology differences are less pronounced

For P. marinus, which experiences various light intensities and nutrient conditions in its natural oceanic environment, this suggests that EF-P might be particularly important during periods of rapid growth in high-light conditions near the ocean surface, but less critical in deeper waters where growth rates are naturally slower .

What methodologies can be used to investigate EF-P-dependent proteins in Prochlorococcus?

Several techniques can be employed to identify and characterize EF-P-dependent proteins in Prochlorococcus:

Proteomics approach:

• SILAC (Stable Isotope Labeling of Amino Acids in Cell Culture):

  • Grow wild-type and efp mutant Prochlorococcus in media with different isotopically labeled amino acids

  • Compare proteomes using mass spectrometry

  • Identify proteins with decreased production in the efp mutant

Genetic approaches:

• Translational fusion assays:

  • Create reporter gene constructs with suspected EF-P-dependent motifs

  • Express in wild-type and efp mutant backgrounds

  • Measure reporter activity to confirm EF-P dependence

• Ribosome profiling:

  • Map ribosome occupancy across transcripts in wild-type and efp mutant strains

  • Identify positions of ribosome pausing associated with PPX motifs

Biochemical approaches:

• In vitro translation assays:

  • Purify ribosomes and translation factors from Prochlorococcus

  • Test translation of model substrates with and without recombinant EF-P

  • Measure peptide bond formation rates at PPX motifs

Bioinformatic analysis:

• Motif identification:

  • Analyze the Prochlorococcus proteome for enrichment of PPP, PPG, APP, and other known EF-P-dependent motifs

  • Compare motif distribution with organisms having different ecological niches

When performing these analyses, special attention should be paid to proteins involved in photosynthesis, stress response, and nutrient acquisition, as these are particularly relevant to Prochlorococcus ecology .

How does the small genome of Prochlorococcus marinus influence its dependence on EF-P for protein synthesis?

P. marinus subsp. pastoris has undergone significant genome reduction with only 1,796 predicted protein-coding genes , which creates unique considerations for EF-P dependency:

Genome streamlining implications:

• Conservation of essential functions:

  • Despite genome reduction, P. marinus has retained the efp gene, suggesting its critical nature

  • This mirrors the evolutionary conservation of timing mechanisms, where despite reduction of the clock locus, P. marinus has maintained functional KaiB and KaiC proteins

• Polyproline motif distribution:

  • Genome analysis would help determine if the frequency of polyproline motifs in P. marinus differs from other cyanobacteria with larger genomes

  • A reduced occurrence of challenging polyproline sequences might represent an adaptation to minimize dependence on accessory translation factors

• Niche-specific protein requirements:

  • Proteins critical for adaptation to high-light environments might contain EF-P-dependent motifs

  • Proteins involved in photosynthesis and response to oxidative stress may be particularly affected by EF-P availability

Research approach:

• Comparative genomic analysis of polyproline motif distribution across Prochlorococcus ecotypes (high-light vs. low-light adapted)

• Correlation of motif distribution with growth rates in different environmental conditions

• Functional categorization of proteins containing EF-P-dependent motifs to identify patterns related to ecological adaptation

This type of analysis would provide insights into how genome streamlining has influenced translational regulation in this ecologically important marine cyanobacterium.

What is the relationship between EF-P function and light adaptation in Prochlorococcus strains?

Prochlorococcus strains exhibit distinct adaptations to different light environments, with high-light (HL) and low-light (LL) adapted ecotypes showing significant physiological differences :

Light adaptation and translational demands:

• Photophysiology differences:

  • HL-adapted strains like MED4 (P. marinus subsp. pastoris) show different photosystem II (PSII) quantum yield patterns compared to other cyanobacteria

  • HL strains are more sensitive to photoinactivation than strains like Synechococcus sp. WH7803

• Circadian regulation:

  • P. marinus shows biochemical evidence for timing mechanisms

  • Light/dark cycles influence photophysiology and may affect translational demands

• EF-P and oxidative stress:

  • High light conditions increase oxidative stress

  • Proteins involved in stress response may contain polyproline motifs requiring EF-P

  • The absence of EF-P could compromise cellular responses to high light conditions

Experimental approach to study this relationship:

• Generate efp knockout mutants in both HL and LL-adapted Prochlorococcus strains

• Compare growth rates, photosynthetic efficiency, and proteome composition under different light intensities

• Analyze expression patterns of EF-P and putative EF-P-dependent proteins across light/dark cycles

• Measure PSII repair rates in the presence and absence of EF-P under high light stress

A table comparing expected responses might look like:

This research would provide valuable insights into how translational regulation via EF-P contributes to the ecological success of different Prochlorococcus ecotypes across ocean depth gradients .

What techniques are available for investigating EF-P-ribosome interactions in Prochlorococcus?

Understanding the molecular details of EF-P interactions with Prochlorococcus ribosomes requires specialized techniques:

Structural approaches:

• Cryo-electron microscopy (cryo-EM):

  • Purify ribosomes from Prochlorococcus using ultracentrifugation

  • Reconstitute complexes with recombinant EF-P

  • Visualize interactions at near-atomic resolution

• X-ray crystallography:

  • Crystallize purified Prochlorococcus EF-P alone or in complex with ribosomal components

  • Determine structure and compare with known bacterial EF-P structures

Biochemical approaches:

• Ribosome binding assays:

  • Express and purify recombinant P. marinus EF-P with appropriate tags

  • Isolate ribosomes from Prochlorococcus cultures

  • Measure binding affinities under various conditions (different pH, salt, etc.)

• In vitro translation systems:

  • Develop a Prochlorococcus-specific in vitro translation system

  • Assess the impact of adding or removing EF-P

  • Use model substrates containing polyproline motifs

Genetic approaches:

• Site-directed mutagenesis:

  • Introduce specific mutations in the efp gene

  • Express mutant proteins and assess binding to ribosomes

  • Determine critical residues for EF-P function

The challenge with Prochlorococcus is obtaining sufficient biomass for these experiments. Culturing requires specialized techniques as described in research methods for P. marinus:

• Use of ultrafiltered seawater-based Pro99 media

• Careful monitoring of growth using bulk fluorescence

• Growth under defined light and temperature conditions

These approaches would provide valuable insights into any unique features of EF-P-ribosome interactions in this ecologically important marine organism.

How can recombinant Prochlorococcus EF-P be used to study polyproline motif translation in heterologous systems?

Recombinant Prochlorococcus EF-P can serve as a valuable tool for studying translation mechanisms across systems:

Expression and purification protocol:

• Codon-optimized gene synthesis:

  • Synthesize the P. marinus efp gene with codon optimization for E. coli

  • Clone into an expression vector with an appropriate promoter (e.g., T5)

  • Include a purification tag (His-tag) and remove any signal sequences

• Expression conditions:

  • Transform into E. coli expression strain (e.g., BL21)

  • Grow in rich media (e.g., TB medium) with appropriate antibiotics

  • Induce with IPTG and optimize temperature for maximum yield

• Purification strategy:

  • Lyse cells in appropriate buffer (e.g., 50 mM Tris HCl [pH 8.0], 150 mM NaCl, etc.)

  • Purify using affinity chromatography followed by size exclusion

  • Assess purity by SDS-PAGE (>85% purity)

Applications in heterologous systems:

• Comparative analysis of EF-P function:

  • Complement efp-deficient E. coli with Prochlorococcus EF-P

  • Assess rescue of polyproline translation defects

  • Compare efficiency with EF-P from other organisms

• In vitro translation assays:

  • Add purified Prochlorococcus EF-P to E. coli cell-free translation systems

  • Test translation of reporters containing various polyproline motifs (PPP, PPG, APP)

  • Measure effects on translation rate and efficiency

• Structural studies:

  • Use purified protein for crystallization and structure determination

  • Compare with EF-P structures from other organisms to identify unique features

This approach would provide insights into how EF-P function has evolved in Prochlorococcus, potentially revealing adaptations specific to its unique marine environment and streamlined genome.