Recombinant Prochlorococcus marinus Elongation factor Ts (tsf)

What is Elongation factor Ts (EF-Ts) in Prochlorococcus marinus?

Elongation factor Ts (EF-Ts) in Prochlorococcus marinus is a protein involved in the translational machinery that functions as a guanine nucleotide exchange factor. It facilitates protein synthesis by catalyzing the release of GDP from elongation factor Tu (EF-Tu), allowing EF-Tu to bind GTP and subsequently interact with aminoacyl-tRNA. This cycling between GDP and GTP-bound states is essential for the elongation phase of protein synthesis in these marine photosynthetic prokaryotes. In Prochlorococcus marinus, EF-Ts is encoded by the tsf gene, with the full-length protein consisting of 218 amino acids . The protein plays a critical role in the highly efficient cellular machinery that has evolved to support Prochlorococcus' remarkable ability to thrive in nutrient-limited marine environments where it dominates photosynthetic biomass .

Which Prochlorococcus marinus strains have been used for recombinant production of EF-Ts?

Based on the available data, recombinant Prochlorococcus marinus EF-Ts has been produced from at least two different strains:

These strains represent different ecotypes of Prochlorococcus, which is significant as Prochlorococcus is known to have genetically distinct ecotypes with different ecophysiological characteristics adapted to various ocean depths . The use of different expression systems (baculovirus versus E. coli) reflects different approaches to optimizing recombinant protein production while maintaining native structure and function.

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

According to the product data sheets, the optimal storage conditions for recombinant Prochlorococcus marinus EF-Ts are:

The shelf life depends on the formulation:

• Liquid form: Approximately 6 months at -20°C/-80°C

• Lyophilized form: Approximately 12 months at -20°C/-80°C

It's important to note that repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity . Therefore, preparing small aliquots for storage is advisable to minimize freeze-thaw cycles, which can compromise protein integrity and experimental reproducibility.

What considerations should be made when choosing between baculovirus and E. coli expression systems for Prochlorococcus marinus EF-Ts?

The data sheets indicate that EF-Ts from Prochlorococcus marinus strain MIT 9515 was expressed using a baculovirus system, while the protein from strain MIT 9215 was expressed in E. coli . This difference highlights important considerations for researchers:

For Prochlorococcus marinus EF-Ts, the choice between these systems might depend on:

• Required protein yield for specific applications

• Downstream application sensitivity to protein folding

• Budget and time constraints

• Need for specific post-translational modifications

The successful expression in both systems suggests that EF-Ts is relatively robust and can be produced in either system, giving researchers flexibility based on their specific needs and available resources .

How can recombinant Prochlorococcus marinus EF-Ts be used to study translation mechanisms in marine cyanobacteria?

Recombinant Prochlorococcus marinus EF-Ts can serve as a valuable tool for investigating translation mechanisms in marine cyanobacteria through several methodological approaches:

• Reconstituted Translation Systems: Purified EF-Ts can be combined with other translation factors (EF-Tu, EF-G, ribosomes) from Prochlorococcus or related cyanobacteria to establish in vitro translation systems. This allows direct measurement of translation rates and fidelity under defined conditions that mimic the oceanic environment where Prochlorococcus thrives.

• Structure-Function Studies: Crystal structures of EF-Ts in complex with EF-Tu would reveal species-specific interactions and conformational changes that might be adapted to marine environments. This is particularly relevant given that Prochlorococcus has evolved by "reducing its cell and genome sizes" as an adaptation to oligotrophic conditions .

• Comparative Analysis: By comparing the activity and properties of EF-Ts from different Prochlorococcus ecotypes (surface vs. deep-water adapted strains), researchers can identify adaptations in translation machinery that correspond to ecological niches. This is significant because Prochlorococcus has "genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, present at depth and in surface waters" .

• Temperature and Salt Dependence: Assaying EF-Ts activity across temperature and salt gradients relevant to oceanic conditions can reveal adaptations to the marine environment. This is particularly relevant as Prochlorococcus inhabits ocean waters "from the surface down to depths of 200 m" , experiencing significant environmental gradients.

• Light-Dependent Translation Regulation: Since Prochlorococcus is photosynthetic with distinct diurnal patterns, investigating how translation factors might be regulated by light could reveal unique regulatory mechanisms that coordinate protein synthesis with photosynthetic output.

These approaches could provide insights into how translation in these globally important marine prokaryotes has adapted to their specific ecological constraints and resource limitations.

What is the recommended protocol for reconstitution of recombinant Prochlorococcus marinus EF-Ts?

According to the product data sheets, the recommended protocol for reconstitution of Prochlorococcus marinus EF-Ts is as follows :

Materials Required:

• Recombinant EF-Ts protein (lyophilized or frozen)

• Deionized sterile water

• Glycerol (molecular biology grade)

• Microcentrifuge

• Sterile microcentrifuge tubes

• Pipettes and sterile tips

Protocol:

• Briefly centrifuge the vial containing protein prior to opening to bring the contents to the bottom of the tube.

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

• Add glycerol to a final concentration of 5-50% (the default recommendation is 50%).

• Aliquot the reconstituted protein into smaller volumes to minimize freeze-thaw cycles.

• Store aliquots at -20°C or -80°C for long-term storage.

Additional Considerations:

• Working aliquots can be stored at 4°C for up to one week.

• Avoid repeated freezing and thawing as it can lead to protein denaturation and loss of activity.

• The exact reconstitution buffer may be optimized based on the specific downstream application.

This protocol ensures that the reconstituted protein maintains its stability and activity for the intended experimental use while minimizing degradation during storage and handling .

How can the activity of recombinant Prochlorococcus marinus EF-Ts be assayed in vitro?

The activity of recombinant Prochlorococcus marinus EF-Ts can be assayed through several in vitro methods that measure its primary function as a guanine nucleotide exchange factor for EF-Tu:

• Fluorescent Nucleotide Exchange Assay

Principle: EF-Tu bound to fluorescent GDP analogues (mant-GDP) exhibits high fluorescence. Upon EF-Ts-catalyzed exchange with non-fluorescent GDP/GTP, fluorescence decreases.

Protocol:

• Preload EF-Tu with mant-GDP by incubating EF-Tu with excess mant-GDP and removing free nucleotide

• In a fluorimeter cuvette, combine EF-Tu- mant-GDP complex (typically 0.1-1 μM)

• Record baseline fluorescence (excitation 355 nm, emission 440 nm)

• Add varying concentrations of EF-Ts (0.01-1 μM)

• Add excess non-fluorescent GDP or GTP (50-200 μM)

• Monitor fluorescence decrease over time

• Calculate exchange rates from exponential decay curves at different EF-Ts concentrations

• EF-Ts-Dependent Poly(Phe) Synthesis Assay

Principle: Measures the functional impact of EF-Ts on in vitro translation.

Protocol:

• Set up an in vitro translation system with ribosomes, mRNA (poly(U)), aminoacylated tRNA^Phe, EF-Tu, and GTP

• Add varying concentrations of EF-Ts

• Measure poly(Phe) synthesis by incorporation of radiolabeled Phe

• Compare translation rates with and without EF-Ts

• Surface Plasmon Resonance (SPR) Nucleotide Exchange Assay

Principle: Measures real-time binding and dissociation of EF-Tu to immobilized GDP/GTP in the presence of EF-Ts.

Protocol:

• Immobilize biotinylated GDP on a streptavidin sensor chip

• Flow EF-Tu over the surface to form EF-Tu- GDP complex

• Introduce EF-Ts and monitor the dissociation of EF-Tu from the surface

• Calculate exchange rates from the dissociation curves

These assays allow researchers to quantitatively measure EF-Ts activity, determine kinetic parameters, and assess the effects of mutations, environmental conditions, or potential inhibitors on its function.

What analytical methods are suitable for characterizing the structure and function of Prochlorococcus marinus EF-Ts?

A comprehensive characterization of Prochlorococcus marinus EF-Ts structure and function requires multiple complementary analytical methods:

Structural Characterization:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-260 nm): Provides information about secondary structure content (α-helices, β-sheets)

  • Near-UV CD (250-350 nm): Reveals tertiary structure fingerprint through aromatic residue environment

  • Thermal melting curves: Determines protein stability and unfolding transitions

• X-ray Crystallography:

  • Provides atomic-resolution 3D structure

  • Can capture different functional states if crystallized with binding partners or nucleotides

  • Protocol: Purify protein to >95% homogeneity, screen crystallization conditions, collect diffraction data, solve phase problem, build and refine model

• Small-Angle X-ray Scattering (SAXS):

  • Provides low-resolution envelope of protein shape in solution

  • Useful for analyzing conformational changes upon binding

  • Complements high-resolution structural methods

Functional Characterization:

• Nucleotide Exchange Assays:

  • Fluorescence-based assays using mant-GDP or mant-GTP

  • Measures kinetics of EF-Ts-catalyzed nucleotide exchange on EF-Tu

  • Protocol: Preload EF-Tu with fluorescent nucleotide, add EF-Ts, monitor fluorescence decrease

• Thermal Shift Assays (Differential Scanning Fluorimetry):

  • Measures protein stability and ligand binding

  • Protocol: Mix protein with SYPRO Orange dye, perform temperature gradient, monitor fluorescence increase upon unfolding

• Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molecular weight and oligomeric state

  • Verifies homogeneity and complex formation

• In Vitro Translation Assays:

  • Functional assays in reconstituted translation systems

  • Measures the effect of EF-Ts on translation rate and accuracy

  • Particularly relevant for understanding Prochlorococcus' adaptation to "typically divide once a day in the subsurface layer of oligotrophic areas"

These methods provide complementary data that together yield a comprehensive understanding of both structural features and functional properties of Prochlorococcus marinus EF-Ts, contributing to our understanding of how this critical protein functions in an organism that has evolved for extreme efficiency in nutrient-limited environments.

How does the function of EF-Ts in Prochlorococcus marinus relate to its ecological adaptation in nutrient-deprived marine environments?

The function of EF-Ts in Prochlorococcus marinus likely plays a crucial role in its remarkable adaptation to nutrient-deprived marine environments through several mechanisms:

• Translation Efficiency in Resource-Limited Conditions:

  Prochlorococcus has evolved to thrive in oligotrophic (nutrient-poor) oceanic regions where it dominates the photosynthetic biomass . In such resource-limited environments, efficient protein synthesis is critical. EF-Ts, by catalyzing the recycling of EF-Tu during translation elongation, ensures optimal translation rates even when energy resources are scarce.

  Research has shown that Prochlorococcus typically divides once per day in subsurface layers of oligotrophic areas , suggesting precisely regulated growth and protein synthesis. The efficient function of translation factors like EF-Ts would be essential for maintaining this controlled growth rate under nutrient limitation.

• Genome Streamlining and Protein Optimization:

  Prochlorococcus has undergone genome reduction as an adaptation to its environment, evolving "from an ancestral cyanobacterium by reducing its cell and genome sizes" . This genomic streamlining extends to its protein machinery, where each protein must function optimally with minimal resources.

  Comparative analysis of EF-Ts sequences from different Prochlorococcus strains (MIT 9515 and MIT 9215) reveals high conservation with only minor variations , suggesting strong selective pressure to maintain its essential function while potentially fine-tuning its performance for specific ecological niches.

• Adaptation to Environmental Gradients:

  Prochlorococcus inhabits ocean waters from the surface down to depths of 200m , experiencing gradients of light, temperature, and pressure. EF-Ts must maintain activity across these environmental ranges, particularly as Prochlorococcus includes "genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, present at depth and in surface waters" .

Understanding how EF-Ts contributes to Prochlorococcus' ecological success could provide insights into fundamental mechanisms of cellular adaptation to extreme environments and resource limitations, with potential applications in synthetic biology and biotechnology.

What is the evolutionary significance of EF-Ts conservation across different Prochlorococcus marinus ecotypes?

The evolutionary significance of EF-Ts conservation across different Prochlorococcus marinus ecotypes reveals important insights into both molecular evolution and ecological adaptation:

Understanding the evolutionary constraints on EF-Ts provides insights into both the fundamental mechanisms of molecular evolution and the specific adaptations that have allowed Prochlorococcus to become "the most abundant photosynthetic organism on Earth" despite its resource-limited environment.

How might the study of Prochlorococcus marinus EF-Ts contribute to our understanding of translation efficiency in photosynthetic prokaryotes?

Studying Prochlorococcus marinus EF-Ts can significantly advance our understanding of translation efficiency in photosynthetic prokaryotes through several research avenues:

• Integration of Translation with Photosynthetic Metabolism:

  Prochlorococcus marinus has evolved highly efficient cellular machinery to thrive in nutrient-limited environments where it "typically divides once a day in the subsurface layer of oligotrophic areas" . The coordination between photosynthesis and protein synthesis is likely critical for this efficiency.

  Research on EF-Ts could reveal:

  • How translation factors respond to changing energy availability during light/dark cycles

  • Potential regulatory mechanisms that coordinate protein synthesis with photosynthetic output

  • Unique adaptations in translation machinery that optimize resource utilization

• Adaptations to Extreme Resource Limitations:

  Prochlorococcus represents an extreme case of adaptation to nutrient limitation, having evolved "by reducing its cell and genome sizes" . Its translation machinery must function with minimal resource investment.

  Comparison of photosynthetic parameters and oxygen evolution rates between different marine picocyanobacteria strains has shown significant variations that correspond to their ecological niches . Similar variations might exist in translation efficiency, with EF-Ts playing a key role in these adaptations.

• Insights into Translational Adaptations to Environmental Challenges:

  Prochlorococcus exhibits negative net O2 evolution rates at low irradiances encountered in minimum oxygen zones , suggesting metabolic adaptations to challenging environments. The translation machinery, including EF-Ts, must maintain function across these environmental gradients.

  Potential research approaches include:

  • Comparing kinetic parameters of EF-Ts from different ecotypes under varying temperature, pressure, and light conditions

  • Analyzing translation rates in reconstituted systems under conditions mimicking different ocean depths

  • Structural studies of EF-Ts under conditions representing ecological extremes

• Evolution of Minimal Translation Systems:

  As a model of genome streamlining, Prochlorococcus provides insights into the minimal requirements for efficient translation.

  Research Approach	Potential Insights
  Structure-function analysis	Identify essential vs. dispensable regions of EF-Ts
  Minimal reconstituted translation systems	Determine minimal components required for efficient translation
  Computational modeling	Predict translation efficiency based on EF-Ts kinetic parameters
  Comparative genomics	Identify evolutionary patterns in translation machinery across ecotypes

These research directions would not only advance our understanding of Prochlorococcus biology but also provide broader insights into how photosynthetic prokaryotes optimize their translation machinery for survival in challenging environments, with potential applications in biotechnology and synthetic biology.