Recombinant Prochlorococcus marinus subsp. pastoris Elongation factor G (fusA), partial

What is Elongation factor G (fusA) and what role does it play in Prochlorococcus marinus?

Elongation factor G (EF-G), encoded by the fusA gene, is a critical protein involved in the translocation step of bacterial protein synthesis. In Prochlorococcus marinus, this protein facilitates the movement of tRNAs and mRNA through the ribosome during translation elongation. Studies have shown that EF-G is essential for protein synthesis and cellular metabolism in this cyanobacterium, which is fundamental to its survival in marine environments . The protein functions as a GTPase, using the energy from GTP hydrolysis to catalyze the translocation process, making it an integral part of the cellular machinery in this ecologically significant organism.

How is recombinant Prochlorococcus marinus EF-G typically produced for research purposes?

Recombinant Prochlorococcus marinus EF-G can be produced using several expression systems, with E. coli being the most common. The production typically follows this methodology:

• The fusA gene sequence is cloned from Prochlorococcus marinus genomic DNA

• The gene is inserted into an appropriate expression vector containing desired tags

• The recombinant vector is transformed into expression hosts

• Protein expression is induced under optimized conditions

• The protein is purified using affinity chromatography based on the fusion tag

Based on available data, multiple expression systems have been developed for this protein:

For optimal research applications, the lyophilized protein is typically reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage at -20°C to -80°C .

What are the structural characteristics of Prochlorococcus marinus EF-G protein?

Prochlorococcus marinus EF-G is characterized by multiple functional domains that contribute to its role in protein translation. While the partial recombinant protein may not contain all domains, the complete EF-G typically contains:

• Domain I (G domain): Contains the GTP binding and hydrolysis site

• Domain II: Interacts with the 30S ribosomal subunit

• Domains III-V: Interact with the ribosomal A site during translocation

The protein has structural homology with other bacterial EF-G proteins but may contain unique features that reflect the evolutionary adaptation of Prochlorococcus to its ecological niche. The genomic context of fusA in Prochlorococcus marinus shows it is part of the core genome maintained across different ecotypes, highlighting its essential role .

How should researchers optimize experimental conditions when working with recombinant Prochlorococcus marinus EF-G?

When working with recombinant Prochlorococcus marinus EF-G, researchers should consider the following methodological approaches:

• Buffer optimization: The protein functions optimally in buffers mimicking physiological conditions (pH 7.0-8.0) with appropriate ionic strength.

• GTP concentration: As EF-G is a GTPase, experiments should include GTP at physiologically relevant concentrations (typically 0.1-1mM).

• Mg²⁺ concentration: Mg²⁺ is critical for GTPase activity, and concentrations between 5-10mM are typically used in functional assays.

• Temperature considerations: Given that Prochlorococcus thrives in specific temperature ranges in oceanic environments, activity assays should be performed at relevant temperatures (20-25°C).

• Storage stability: To maintain protein activity, store in small aliquots with glycerol as a cryoprotectant at -80°C and avoid multiple freeze-thaw cycles .

For functional studies, researchers should implement a fractional factorial design approach to efficiently test multiple parameters simultaneously rather than varying one factor at a time . This experimental design strategy significantly reduces the number of experiments needed while identifying optimal conditions.

What methods are most effective for assessing the functionality of recombinant Prochlorococcus marinus EF-G?

Several complementary approaches can be used to assess the functionality of recombinant EF-G:

• GTPase activity assay: Measuring the rate of GTP hydrolysis using colorimetric methods (malachite green assay) or radioactive [γ-³²P]GTP.

• In vitro translation systems: Reconstituted translation systems can evaluate the ability of EF-G to promote translocation using fluorescently labeled tRNAs or mRNAs.

• Ribosome binding assays: Surface plasmon resonance (SPR) or microscale thermophoresis (MST) can measure binding kinetics to ribosomes.

• Structural integrity assessment: Circular dichroism (CD) spectroscopy provides information about secondary structure elements, while thermal shift assays can evaluate protein stability.

• Polysome profile analysis: Assess the impact of EF-G on polysome formation and stability through sucrose gradient centrifugation followed by fraction analysis.

For comprehensive functional characterization, combine multiple methodologies to establish both binding and catalytic properties of the recombinant protein.

What troubleshooting strategies should be employed when recombinant EF-G shows poor activity?

When recombinant Prochlorococcus marinus EF-G exhibits suboptimal activity, consider these methodological solutions:

• Verify protein integrity: Run SDS-PAGE to check for degradation or aggregation; consider Western blotting if appropriate antibodies are available.

• Check for proper folding: Use circular dichroism or fluorescence spectroscopy to assess secondary structure.

• Optimize buffer conditions: Systematically test different buffer compositions, pH values, and salt concentrations.

• Evaluate the impact of additives: Test the effect of stabilizing agents such as glycerol, reducing agents (DTT, β-mercaptoethanol), or nucleotides.

• Consider tag interference: If the recombinant protein contains tags, determine whether they interfere with activity by comparing tagged and cleaved versions.

• Assess protein oligomerization: Use size exclusion chromatography to determine if the protein forms inactive oligomers.

• Review expression system limitations: Different expression systems may produce proteins with varying post-translational modifications that affect activity .

How can recombinant Prochlorococcus marinus EF-G be used to study antibiotic resistance mechanisms?

Recombinant Prochlorococcus marinus EF-G represents a valuable tool for studying antibiotic resistance mechanisms, particularly those targeting protein synthesis. Several methodological approaches are recommended:

• Binding studies with translation-targeting antibiotics: Use fluorescence quenching or isothermal titration calorimetry to measure binding of antibiotics that target EF-G (e.g., fusidic acid).

• Site-directed mutagenesis: Create specific mutations in the fusA gene corresponding to known resistance mutations in other bacteria, then evaluate how these affect antibiotic binding and GTPase activity.

• Structural studies: Employ X-ray crystallography or cryo-EM to determine the structure of EF-G in complex with antibiotics, providing insights into binding modes and resistance mechanisms.

• Comparative studies: Compare the properties of Prochlorococcus EF-G with those from antibiotic-resistant bacteria like those mentioned in research on Corynebacterium glutamicum and Brevibacterium flavum resistance to fusidic acid .

• In vitro translation inhibition assays: Assess how antibiotics affect translation rates in the presence of wild-type versus mutant EF-G proteins.

This research direction becomes particularly relevant as studies have demonstrated connections between EF-G mutations and drug resistance in various bacterial species .

What role might Prochlorococcus marinus EF-G play in horizontal gene transfer in marine ecosystems?

Recent research suggests potential connections between Prochlorococcus proteins and horizontal gene transfer (HGT) in marine ecosystems:

• Cyanophage-mediated transduction: Studies have shown that cyanophages can mispackage Prochlorococcus DNA during infection, potentially facilitating HGT between marine bacteria . While not directly related to EF-G, this mechanism establishes a precedent for genetic exchange.

• Conservation analysis: Comparative genomics approaches can assess whether the fusA gene shows evidence of HGT by analyzing its sequence conservation, GC content, and phylogenetic distribution across different Prochlorococcus ecotypes and related cyanobacteria.

• Experimental approaches: Researchers can study the potential for fusA gene transfer using laboratory-controlled microcosms that simulate natural marine conditions, tracking gene movement through molecular methods.

• Ecological significance: The ubiquity of Prochlorococcus in marine environments (being the most abundant photosynthesizing organism on the planet) suggests that any genes transferred from this organism could have significant ecological impacts .

Research has revealed that the frequency of DNA mispackaging in cyanophages infecting Prochlorococcus can reach detectable levels (comparable to approximately 10 PPM reported for marine Synechococcus cyanophage S-PM2), suggesting potential mechanisms for gene transfer in these ecosystems .

How can recombinant EF-G be applied in structural biology studies to understand Prochlorococcus adaptation to extreme environments?

Prochlorococcus marinus thrives in various marine environments, including nutrient-poor, high-light, and sometimes extreme conditions. Structural biology approaches using recombinant EF-G can provide insights into adaptations:

• Comparative structural analysis: Determine the 3D structure of Prochlorococcus EF-G using X-ray crystallography or cryo-EM and compare it to EF-G from organisms in different environments.

• Stability studies under extreme conditions: Assess protein stability and activity under various conditions (temperature, pressure, salinity) that mimic different ocean environments:

• Molecular dynamics simulations: Perform in silico experiments to model how the protein structure responds to different environmental stressors.

• Mutational studies: Create variants mimicking sequence differences found in Prochlorococcus ecotypes from different ocean regions and assess their functional properties.

• Protein-protein interaction studies: Investigate interactions between EF-G and other components of the Prochlorococcus translational machinery using pull-down assays, surface plasmon resonance, or crosslinking mass spectrometry.

These approaches can reveal adaptive features that contribute to Prochlorococcus' remarkable success across diverse marine environments .

What are the optimal experimental designs for studying the interactions between Prochlorococcus marinus EF-G and the ribosome?

When investigating interactions between Prochlorococcus marinus EF-G and ribosomes, researchers should consider these methodological approaches:

• Ribosome source selection: Determine whether to use homologous (Prochlorococcus) ribosomes or heterologous (E. coli) ribosomes. Homologous systems provide more biologically relevant data but may be technically challenging to obtain in sufficient quantities.

• Binding kinetics analysis: Employ biophysical techniques such as:

  • Surface plasmon resonance (SPR)

  • Microscale thermophoresis (MST)

  • Bio-layer interferometry (BLI)

  • Fluorescence anisotropy with labeled EF-G

• Functional assays: Implement in vitro translation systems to assess:

  • Translocation rates using fluorescently labeled tRNAs

  • GTP hydrolysis rates in the presence of ribosomes

  • Effects on translation fidelity using reporter systems

• Structural approaches: Utilize cryo-electron microscopy to capture different states of the EF-G-ribosome complex during translocation.

• Experimental design strategy: Apply fractional factorial design to efficiently test multiple variables simultaneously, including:

  • GTP/GDP concentrations

  • Mg²⁺ and monovalent ion concentrations

  • Temperature

  • pH

  • Presence of translation factors

This approach significantly reduces the number of experiments needed compared to varying one factor at a time while providing valuable information about factor interactions .

How should researchers address potential contamination issues when working with recombinant Prochlorococcus proteins?

Contamination concerns are particularly relevant when working with Prochlorococcus-derived proteins, as highlighted by challenges in obtaining axenic cultures . Researchers should implement these methodological approaches:

• Expression system selection: Choose expression systems that minimize contamination with host proteins. E. coli systems with high-specificity tags like His6 or GST can be effective for initial purification .

• Multi-step purification strategy: Implement sequential purification steps:

  • Initial affinity chromatography based on fusion tags

  • Ion exchange chromatography to separate based on charge differences

  • Size exclusion chromatography as a final polishing step

• Purity assessment methods:

  • SDS-PAGE with sensitive staining methods (silver stain)

  • Western blotting with specific antibodies

  • Mass spectrometry for contaminant identification

  • Activity assays specific to EF-G function

• Contamination mitigation for functional studies:

  • Include appropriate controls with known contaminants

  • Use highly purified components in reconstituted systems

  • Consider the impact of potential contaminants on experimental readouts

• DNA contamination considerations: When isolating recombinant protein from expression systems, be aware of potential DNA contamination, which has been documented in studies of Prochlorococcus . Implement DNase treatment steps if necessary for downstream applications.

What statistical approaches are most appropriate for analyzing data from experiments involving Prochlorococcus marinus EF-G?

When analyzing experimental data related to Prochlorococcus marinus EF-G, researchers should consider these statistical approaches:

• Experimental design statistics:

  • Implement fractional factorial design to efficiently test multiple variables with minimal experimental runs

  • Use response surface methodology (RSM) to optimize multiple parameters simultaneously

  • Apply Taguchi methods for robust parameter design when environmental factors cannot be controlled

• Kinetic data analysis:

  • Fit GTPase activity data to appropriate enzyme kinetic models (Michaelis-Menten, allosteric models)

  • Use global fitting approaches for complex kinetic schemes

  • Apply statistical tests (F-test) to determine which model best describes the data

• Binding data analysis:

  • Use appropriate binding models (one-site, two-site, cooperative) for equilibrium binding data

  • Implement global analysis for data from multiple techniques

  • Calculate confidence intervals for derived parameters

• Structural data statistics:

  • Apply proper statistical frameworks for structural comparisons

  • Use bootstrap analysis for phylogenetic comparisons with other EF-G proteins

• Robust statistical practices:

  • Include biological replicates (n≥3) to account for variability

  • Apply appropriate statistical tests based on data distribution

  • Use power analysis to determine adequate sample sizes

  • Implement multiple comparison corrections when testing numerous hypotheses

How might the study of Prochlorococcus marinus EF-G contribute to understanding global carbon cycling in marine ecosystems?

Prochlorococcus marinus is the most abundant photosynthetic organism on Earth, collectively fixing as much carbon as all terrestrial crops combined . Research on its EF-G protein connects to carbon cycling in several ways:

• Metabolic efficiency: EF-G's role in translation directly impacts the metabolic efficiency of Prochlorococcus, which affects carbon fixation rates. Studies using recombinant EF-G can help quantify translation rates under different environmental conditions.

• Cross-feeding networks: Recent research indicates that Prochlorococcus acts as a "conductor in the daily symphony of ocean metabolism," with its metabolic products synchronizing the activities of other marine microbes . Investigating how translational regulation through EF-G impacts these cross-feeding relationships can reveal mechanisms of ecosystem-level carbon cycling.

• Climate change adaptation: As ocean conditions change (temperature, pH, light penetration), understanding how these factors affect EF-G function could predict impacts on Prochlorococcus populations and consequently global carbon fixation.

• Methodological approaches:

  • Combine recombinant protein studies with oceanographic measurements

  • Integrate findings into biogeochemical models

  • Develop in situ probes for translation activity in natural populations

This research direction connects molecular mechanisms to global-scale processes, highlighting the ecological significance of this abundant marine organism .

What are the current challenges in comparing the function of EF-G across different Prochlorococcus ecotypes?

Prochlorococcus exists as multiple ecotypes adapted to different ocean conditions, including high-light and low-light adapted forms . Comparing EF-G function across these ecotypes presents several challenges:

• Sequence variability: The degree of conservation in fusA genes across ecotypes may impact protein function and necessitates careful sequence analysis before functional studies.

• Culturing difficulties: Many Prochlorococcus ecotypes are difficult to maintain in laboratory culture, particularly in axenic conditions , making it challenging to obtain native protein for comparison or to validate recombinant protein function in vivo.

• Methodological approach: To address these challenges, researchers should:

  • Perform comprehensive sequence analysis across available genomes

  • Use recombinant expression of EF-G variants from different ecotypes

  • Develop standardized assays for comparative functional analysis

  • Implement statistical methods to account for experimental variability

• Experimental design considerations: Use factorial designs to efficiently test multiple variables simultaneously when comparing EF-G from different ecotypes .

• Bioinformatic analysis: Integrate genomic, transcriptomic, and proteomic data to contextualize observed functional differences within the broader cellular metabolism of each ecotype.

Understanding these differences could provide insights into the evolutionary adaptations that allow Prochlorococcus to dominate diverse marine environments.

How can systems biology approaches integrate EF-G function into models of Prochlorococcus cellular metabolism?

Systems biology offers powerful approaches to contextualize EF-G function within the broader cellular metabolism of Prochlorococcus:

This systems approach can leverage data from recombinant protein studies to develop predictive models of how Prochlorococcus responds to environmental changes, potentially informing broader ecosystem models of marine carbon cycling .