Recombinant Photobacterium profundum Elongation factor G 2 (fusA2), partial

What is Elongation Factor G 2 (fusA2) in Photobacterium profundum?

Elongation Factor G 2 (EF-G 2) is a protein encoded by the fusA2 gene in Photobacterium profundum, a deep-sea bacterium belonging to the Vibrionaceae family. This protein is a paralog of the canonical Elongation Factor G (EFG), which participates in translation elongation. While EF-G 2 preserves similar domain organization and maintains significant sequence homology with EFG, it likely serves specialized functions in P. profundum's adaptation to deep-sea conditions. The protein has been cataloged in UniProt under accession number Q6LST1 and is often studied in its recombinant form for research purposes .

What is the ecological context for P. profundum and how might it influence fusA2 function?

Photobacterium profundum is a gram-negative marine bacterium adapted to deep-sea environments. Strain SS9, from which the recombinant fusA2 is often derived, exhibits optimal growth at 15°C and 28 MPa, qualifying it as both a psychrophile (cold-loving) and piezophile (pressure-loving). This extremophilic nature suggests that fusA2 may have specialized functions related to protein synthesis under high-pressure and low-temperature conditions. The extreme habitat likely drove the evolution of specialized molecular machinery, potentially including unique adaptations in translational factors like EF-G 2, to maintain protein synthesis efficiency under conditions that would normally compromise cellular processes in non-adapted organisms .

What expression systems are most effective for producing recombinant P. profundum fusA2?

While specific expression systems for P. profundum fusA2 are not extensively documented, commercial sources utilize yeast expression systems to produce the recombinant protein with high purity (>85% by SDS-PAGE). For bacterial expression, E. coli remains a viable option based on successful expression of homologous proteins. When designing expression constructs, researchers should consider:

• Codon optimization for the chosen expression host to overcome potential rare codon issues from a piezophilic source organism

• Fusion tags for improved solubility and simplified purification (His-tag, GST, MBP)

• Low-temperature induction (15-18°C) to mimic the native conditions of P. profundum and improve proper folding

• Specialized strains of E. coli designed for difficult-to-express proteins (Rosetta, Arctic Express)

What are the optimal storage conditions for maintaining recombinant fusA2 activity?

According to product specifications, recombinant P. profundum fusA2 should be stored with careful attention to stability factors. Lyophilized protein maintains stability for approximately 12 months at -20°C to -80°C, while liquid preparations have a reduced shelf life of approximately 6 months under the same conditions. For working solutions, storage at 4°C is recommended for up to one week, with repeated freeze-thaw cycles explicitly discouraged as they lead to protein degradation and activity loss. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL with the addition of 5-50% glycerol (final concentration) as a cryoprotectant for long-term storage. This approach helps maintain structural integrity and functional activity of the recombinant protein during storage periods .

What purification strategy yields the highest functional activity for recombinant fusA2?

While specific purification protocols for P. profundum fusA2 are not detailed in the available literature, efficient purification strategies can be developed based on related proteins and standard recombinant protein techniques. A multi-step purification approach is recommended:

• Initial capture: Affinity chromatography using the fusion tag (e.g., His-tag with Ni-NTA resin)

• Intermediate purification: Ion exchange chromatography (IEX) based on the protein's theoretical pI

• Polishing step: Size exclusion chromatography (SEC) to remove aggregates and ensure homogeneity

For activity preservation, all buffers should contain:

• 5-10% glycerol to maintain stability

• 1-5 mM DTT or 2-ME to protect cysteine residues

• Protease inhibitor cocktail during initial extraction steps

• Low concentrations of GTP (0.1-0.5 mM) as fusA2 has been shown to bind guanine nucleotides

Activity assays following purification should evaluate GTP binding capacity using fluorescent GTP analogs or isothermal titration calorimetry to confirm proper folding and function .

How can researchers assess the GTP-binding activity of recombinant P. profundum fusA2?

The GTP-binding activity of recombinant P. profundum fusA2 can be evaluated using several complementary approaches. Based on studies with homologous EF-G 2 proteins, researchers should implement:

• Fluorescence-based assays using fluorescent GTP analogs like BODIPY-GTP or mant-GTP, measuring fluorescence intensity changes upon binding

• Filter binding assays with radiolabeled [γ-32P]GTP to quantify binding affinities

• Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters of GTP binding

• Surface Plasmon Resonance (SPR) to evaluate binding kinetics in real-time

Critically, appropriate controls should include canonical EF-G from P. profundum or a related organism for comparative analysis. Studies on M. smegmatis EF-G 2 demonstrated that while it binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of canonical EF-G proteins. Therefore, both binding and subsequent hydrolysis activities should be assessed separately to fully characterize the functional properties of P. profundum fusA2 .

Does P. profundum fusA2 exhibit ribosome-dependent GTPase activity, and how can this be measured?

Based on studies of homologous EF-G 2 proteins, P. profundum fusA2 may lack the ribosome-dependent GTPase activity characteristic of canonical EF-G. To experimentally verify this for the P. profundum protein, researchers should implement a systematic approach:

• Ribosome-dependent GTPase assay: Measure inorganic phosphate release from GTP in the presence and absence of purified ribosomes (preferably from P. profundum or marine bacteria)

• Comparative analysis: Run parallel assays with canonical EF-G as a positive control

• Experimental conditions to test:

  • Variable pressure conditions (1 atm vs. simulated deep-sea pressure)

  • Different temperature ranges (4°C, 15°C, 25°C)

  • Various ionic conditions mimicking the marine environment

• Detection methods:

  • Malachite green assay for colorimetric detection of released phosphate

  • Radiolabeled [γ-32P]GTP hydrolysis monitoring

  • HPLC-based nucleotide analysis

Studies with M. smegmatis EF-G 2 demonstrated it lacks ribosome-dependent GTPase activity despite binding guanine nucleotides. If P. profundum fusA2 follows this pattern, researchers should explore alternative functions beyond the canonical translation elongation role .

How do high-pressure conditions affect the structure and function of recombinant fusA2?

The effects of high pressure on recombinant fusA2 structure and function represent a critical area for investigation, particularly given P. profundum's adaptation to deep-sea environments (optimal growth at 28 MPa for strain SS9). While direct experimental data on fusA2 pressure responses is limited, researchers can employ several methodologies to assess pressure effects:

• Structural analysis under pressure:

  • High-pressure circular dichroism (HP-CD) to monitor secondary structure changes

  • Fluorescence spectroscopy with intrinsic tryptophan fluorescence or applied fluorescent probes

  • FTIR spectroscopy to detect subtle conformational shifts

• Functional assays under pressure:

  • GTP binding and hydrolysis in pressure-resistant chambers

  • Ribosome interaction studies using fluorescence energy transfer techniques

• Comparative studies:

  • Analysis against canonical EF-G from pressure-sensitive organisms

  • Comparisons with homologous proteins from non-piezophilic bacteria

Expected findings might include pressure-induced conformational changes that optimize activity at high pressures, potentially reflecting evolutionary adaptations to deep-sea conditions. Research on chromosome replication in P. profundum SS9 has demonstrated pressure-sensitive adaptations in DNA metabolism, suggesting similar adaptations may exist in translation machinery components like fusA2 .

Can P. profundum fusA2 complement EF-G deficiencies in heterologous systems?

Based on studies with homologous proteins, P. profundum fusA2 likely has limited capacity to complement canonical EF-G functions in heterologous systems. Research on M. smegmatis EF-G 2 demonstrated that unlike canonical EF-G (MSMEG_1400), EF-G 2 (MSMEG_6535) failed to rescue an E. coli strain harboring a temperature-sensitive allele of EF-G at non-permissive temperatures. To experimentally verify this for P. profundum fusA2:

• Complementation assays:

  • Transform temperature-sensitive EF-G E. coli strains with P. profundum fusA2 expression constructs

  • Test growth at permissive and non-permissive temperatures

  • Compare with positive controls (canonical EF-G) and negative controls (empty vector)

• Chimeric protein approach:

  • Generate domain-swapped constructs between canonical EF-G and fusA2

  • Identify which domains contribute to functional specificity

• Cross-species complementation:

  • Test complementation in various bacterial species beyond E. coli

  • Examine whether pressure or temperature conditions affect complementation efficiency

This approach would determine whether P. profundum fusA2 has evolved specialized functions distinct from canonical translation elongation, as suggested by studies of EF-G 2 in other bacterial species .

What is the physiological relevance of fusA2 in P. profundum adaptation to deep-sea environments?

The physiological relevance of fusA2 in P. profundum adaptation to deep-sea environments likely involves specialized roles in protein synthesis under extreme conditions. Based on evolutionary patterns of fusA2 in bacteria and specific adaptations of P. profundum:

• Potential specialized functions:

  • Stress-specific translation regulation during stationary phase

  • Pressure-adapted protein synthesis mechanisms

  • Low-temperature translational adaptation

• Research approaches to elucidate physiological relevance:

  • Gene knockout or knockdown studies examining growth under various pressure and temperature conditions

  • Competition assays between wild-type and fusA2-deficient strains under environmental stress

  • Transcriptomics and proteomics analyses to identify condition-specific expression patterns

  • Ribosome profiling to detect specialized roles in translation

Studies in M. smegmatis revealed that while fusA2 disruption produced viable strains with normal growth kinetics, it conferred a fitness disadvantage in competition assays. Similar approaches with P. profundum could reveal condition-specific advantages conferred by fusA2, particularly under deep-sea pressure and temperature conditions characteristic of its native habitat .

How can fusA2 research contribute to our understanding of bacterial adaptation to extreme environments?

Research on P. profundum fusA2 offers valuable insights into bacterial adaptation to extreme environments, particularly deep-sea conditions combining high pressure and low temperature. Key contributions include:

• Molecular mechanisms of pressure adaptation:

  • Structure-function relationships in pressure-adapted proteins

  • Specialized roles of duplicated genes (fusA vs. fusA2) in environmental adaptation

  • Evolutionary pathways leading to piezophilic adaptations

• Translation machinery plasticity:

  • How translation factors evolve specialized functions in extreme conditions

  • Structural adaptations that maintain protein synthesis under pressure

  • Regulatory networks governing expression of alternative translational factors

• Biotechnological applications:

  • Design principles for pressure-stable proteins

  • Potential applications in high-pressure bioprocessing

  • Models for engineered organisms with enhanced environmental tolerance

P. profundum strain SS9, with optimal growth at 15°C and 28 MPa, serves as an excellent model organism for studying deep-sea adaptations. Understanding fusA2's role complements other studies on P. profundum adaptations, such as research on chromosome II replication origins and pressure-sensitive DNA metabolism, building a comprehensive picture of how bacteria adapt to extreme environments .

What are the major challenges in expressing and purifying functional P. profundum fusA2?

Expressing and purifying functional P. profundum fusA2 presents several technical challenges stemming from its origin in a piezophilic, psychrophilic deep-sea bacterium. These challenges include:

• Expression host compatibility:

  • Codon usage bias between deep-sea bacteria and standard expression hosts

  • Different cytoplasmic environments affecting protein folding

  • Potential toxicity when expressing heterologous translational factors

• Pressure-adapted protein stability:

  • Potential instability at atmospheric pressure due to evolutionary adaptation to high pressure

  • Altered folding pathways under standard laboratory conditions

  • Requirement for specialized purification approaches to maintain native conformation

• Activity assessment limitations:

  • Difficulty replicating deep-sea conditions for functional assays

  • Limited availability of P. profundum cellular components (ribosomes, factors) for interaction studies

  • Uncertain post-translational modifications that might occur in the native host

Recommended approaches include using low-temperature expression systems, adding stabilizing agents during purification, and developing high-pressure chambers for activity assays. Commercial sources have successfully expressed recombinant P. profundum fusA2 in yeast systems with >85% purity, suggesting that eukaryotic expression systems may offer advantages for this challenging protein .

How can researchers design experiments to distinguish the functions of fusA and fusA2 in P. profundum?

Designing experiments to distinguish the functions of fusA (encoding canonical EF-G) and fusA2 (encoding EF-G 2) in P. profundum requires a multi-faceted approach combining genetic, biochemical, and physiological analyses:

• Genetic approaches:

  • Generate clean deletions or conditional expression strains for each gene

  • Create strains with tagged versions for localization and interaction studies

  • Implement CRISPR interference for temporal regulation of expression

  • Develop dual-reporter systems to monitor expression patterns

• Physiological characterization:

  • Growth curve analysis under varying pressure, temperature, and nutrient conditions

  • Competition assays between wild-type and mutant strains

  • Stress response evaluation (oxidative, nutrient limitation, antibiotic challenge)

  • Ribosome profiling to identify mRNAs preferentially translated by each factor

• Biochemical distinction:

  • Comparative analysis of GTP binding and hydrolysis

  • Ribosome interaction assays with purified components

  • Pull-down experiments to identify unique interaction partners

  • Structure determination to identify pressure-adapted features

Studies in M. smegmatis have shown that fusA2 disruption confers a fitness disadvantage in competition assays despite normal growth kinetics, suggesting condition-specific roles. Similar approaches in P. profundum, particularly incorporating pressure variables, would help delineate the specialized functions of these paralogous genes .

What controls and validation steps are essential when working with recombinant P. profundum fusA2?

When working with recombinant P. profundum fusA2, researchers should implement rigorous controls and validation steps to ensure experimental reliability:

• Protein quality controls:

  • Multiple purity assessments (SDS-PAGE, SEC-MALS, DLS)

  • Thermal stability analysis (DSF, nanoDSF)

  • Circular dichroism to confirm secondary structure

  • Mass spectrometry verification of intact mass and sequence

• Functional validation:

  • GTP binding assays compared to canonical EF-G

  • Nucleotide specificity tests (GTP vs. ATP, GDP, GMP)

  • Negative controls using denatured protein or binding-deficient mutants

  • Positive controls with well-characterized EF-G proteins

• Experimental design considerations:

  • Inclusion of buffer-only controls in all assays

  • Testing across multiple protein preparations

  • Concentration-dependent activity assessments

  • Time-course experiments to ensure steady-state measurements

• Environmental variable controls:

  • Temperature ranges relevant to P. profundum ecology (0-25°C)

  • Pressure conditions when possible (atmospheric vs. high pressure)

  • Salt concentration controls mimicking marine environment

Commercial recombinant P. profundum fusA2 is typically available with >85% purity by SDS-PAGE, providing a baseline quality standard. Researchers should verify protein stability under their specific experimental conditions, as repeated freeze-thaw cycles are specifically noted to be detrimental to protein quality .

How might structural comparisons between P. profundum fusA and fusA2 reveal adaptive mechanisms for deep-sea environments?

Structural comparisons between P. profundum fusA (canonical EF-G) and fusA2 (EF-G 2) could reveal critical adaptive mechanisms for protein function in deep-sea environments. While specific structural data for these proteins is not yet available, an advanced research approach would include:

• Comparative structural analysis:

  • High-resolution structure determination of both proteins (X-ray crystallography or cryo-EM)

  • Molecular dynamics simulations under varying pressure conditions

  • Identification of pressure-sensitive regions and amino acid substitutions

  • Analysis of domain flexibility and interdomain interactions

• Key structural features to investigate:

  • Hydration shell and solvent-accessible surface characteristics

  • Void volume differences that would be affected by pressure

  • Ion pair networks and salt bridge distributions

  • Packing density in protein core regions

• Functional correlations:

  • Structure-guided mutagenesis to test pressure-adaptation hypotheses

  • Domain-swapping experiments between fusA and fusA2

  • Pressure-dependent conformational change mapping

This approach could reveal principles of protein adaptation to high pressure, potentially identifying conserved mechanisms across piezophilic organisms. Studies of other P. profundum proteins, such as the RctB protein involved in chromosome II replication, have already demonstrated specific adaptations to high-pressure environments that affect protein function and regulation .

What role might fusA2 play in stationary phase survival or stress response in P. profundum?

The potential role of fusA2 in stationary phase survival or stress response in P. profundum represents an important research direction based on patterns observed in homologous systems. Evidence from M. smegmatis indicates that EF-G 2 expression was detected specifically in stationary phase cultures, suggesting a specialized function during this growth phase:

• Experimental approaches to investigate stationary phase roles:

  • Temporal expression profiling across growth phases using RT-qPCR and Western blotting

  • Proteomics analysis comparing wild-type and fusA2-deficient strains during stationary phase

  • Ribosome association patterns in different growth phases

  • Stress survival assays (nutrient limitation, oxidative stress, pressure fluctuations)

• Potential specialized functions:

  • Translation of specific mRNA subsets during stress

  • Ribosome rescue or quality control under stress conditions

  • Interaction with stress-specific factors or small RNAs

  • Altered elongation kinetics optimized for stationary phase

• Comparative analysis framework:

  Growth Phase	Expected fusA Expression	Expected fusA2 Expression	Ribosome Association	Primary Function
  Exponential	High	Low/Minimal	Active translation	Bulk protein synthesis
  Early Stationary	Decreasing	Increasing	Shifting patterns	Transition to stress response
  Late Stationary	Low	Moderate/High	Specialized complexes	Stress-specific translation
  Pressure Stress	Affected	Maintained/Induced	Altered composition	Pressure-adapted translation

This research direction would connect fusA2 function to the broader stress adaptation strategies of P. profundum in its challenging deep-sea habitat .

How can advanced biophysical techniques be applied to study pressure effects on fusA2 structure and function?

Advanced biophysical techniques offer powerful approaches to study pressure effects on fusA2 structure and function, providing insights into molecular adaptations of deep-sea organisms:

• High-pressure structural biology approaches:

  • High-pressure NMR spectroscopy to monitor pressure-induced conformational changes

  • High-pressure X-ray crystallography to capture pressure-stabilized states

  • High-pressure small-angle X-ray scattering (HP-SAXS) for solution structure analysis

  • Single-molecule FRET under pressure to detect domain movements

• Pressure-modulated functional assays:

  • High-pressure stopped-flow spectroscopy for kinetic measurements

  • Pressure-jump relaxation experiments to study conformational dynamics

  • Force microscopy techniques (AFM) under variable pressure

  • Pressure-modulated binding assays using fluorescence anisotropy

• Computational approaches complementing experiments:

  • Molecular dynamics simulations under high pressure conditions

  • In silico mutational analysis to identify key pressure-sensing residues

  • Normal mode analysis to identify pressure-sensitive molecular motions

  • Quantum mechanics/molecular mechanics (QM/MM) studies of catalytic mechanisms under pressure

These techniques could reveal unique conformational states or catalytic properties that emerge specifically under high-pressure conditions resembling the native deep-sea environment of P. profundum. Such studies would provide fundamental insights into protein adaptation mechanisms in extreme environments and potentially inform protein engineering for high-pressure biotechnology applications .

How do fusA2 proteins differ across piezophilic and non-piezophilic bacterial species?

Comparative analysis of fusA2 proteins across piezophilic and non-piezophilic bacterial species can reveal evolutionary adaptations to high-pressure environments. While comprehensive comparison data is not yet available, a research framework would include:

• Sequence-level comparative analysis:

  • Multiple sequence alignment of fusA2 from piezophiles (P. profundum, Shewanella violacea) and non-piezophiles

  • Identification of pressure-correlated amino acid substitutions

  • Analysis of domain conservation and divergence patterns

  • Determination of selection pressures using dN/dS ratios

• Expected adaptations in piezophilic fusA2:

  • Increased proportion of small amino acids at core positions

  • Enhanced ion pair networks for stability

  • Reduced cavity volumes to counter pressure effects

  • Modified surface hydration patterns

• Comparative functional properties:

  Property	Piezophilic fusA2 (predicted)	Non-piezophilic fusA2	Significance
  GTP binding affinity	Pressure-modulated	Pressure-sensitive	Maintains activity at high pressure
  Conformational flexibility	Restricted at ambient pressure	Greater at ambient pressure	Adapted to function under compression
  Temperature optima	Lower (5-15°C)	Higher (20-37°C)	Psychrophilic adaptation
  Pressure stability	High (functional >50 MPa)	Low (denaturation >30 MPa)	Core adaptation to deep sea

This comparative approach would help distinguish truly pressure-adaptive features from general bacterial divergence patterns, providing insights into molecular mechanisms of deep-sea adaptation .

What can we learn about gene duplication and functional divergence from studying fusA and fusA2 in marine bacteria?

The study of fusA and fusA2 in marine bacteria offers valuable insights into evolutionary processes of gene duplication and functional divergence:

• Evolutionary trajectory analysis:

  • Phylogenetic reconstruction of fusA/fusA2 across bacterial lineages

  • Estimation of duplication timing and subsequent divergence rates

  • Identification of key mutations marking functional specialization

  • Correlation with habitat transitions (shallow to deep water)

• Functional divergence patterns:

  • Comparison of expression patterns across environmental conditions

  • Differential selective pressures on paralog domains

  • Identification of lineage-specific adaptations in fusA2

  • Analysis of co-evolving partners in the translation machinery

• Comparative genomic context:

  • Conservation of genomic neighborhood across species

  • Associated regulatory elements suggesting specialized expression

  • Presence of horizontal gene transfer signatures

  • Correlation with other duplicated translation factors

This research approach connects to broader questions about how paralogs evolve specialized functions after duplication. Studies in M. smegmatis have shown that while fusA2 is dispensable for normal growth, it confers fitness advantages in specific conditions, representing a classic case of subfunctionalization after gene duplication. Similar patterns in marine bacteria, particularly with pressure as a selective force, would provide a unique perspective on environmental adaptation driving functional divergence .

How does the evolutionary history of fusA2 relate to the adaptation of Photobacterium species to different marine niches?

The evolutionary history of fusA2 likely played a significant role in the adaptation of Photobacterium species to diverse marine niches, particularly in the colonization of deep-sea environments:

• Niche-specific adaptation signatures:

  • Correlation between fusA2 sequence features and habitat depth

  • Comparison across Photobacterium species from different depth zones

  • Analysis of selection intensity in deep-sea vs. shallow-water lineages

  • Identification of convergent adaptations in independent deep-sea lineages

• Ecological context of fusA2 evolution:

  • Correlation with other pressure-adaptive traits

  • Co-evolution with ribosomal components

  • Association with psychrophilic adaptations

  • Relationship to metabolic adaptations for deep-sea environments

• Predicted evolutionary history:

  • Ancient duplication of fusA predating Vibrionaceae diversification

  • Relaxed selection initially allowing sequence divergence

  • Intensified positive selection during deep-sea colonization

  • Maintenance through subfunctionalization in specialized niches

P. profundum strain SS9 grows optimally at 15°C and 28 MPa, marking it as both a psychrophile and a piezophile adapted to deep-sea conditions. The evolution of specialized translation factors like fusA2 likely contributed to the ability of Photobacterium lineages to exploit deep-sea niches by maintaining protein synthesis under challenging pressure and temperature conditions. Comparative genomic approaches across Photobacterium species from different depths would help reconstruct this evolutionary history and identify key adaptations enabling niche diversification .