Recombinant Photobacterium profundum Elongation factor G 1 (fusA1), partial

What is the primary function of Elongation factor G 1 in Photobacterium profundum?

Elongation factor G 1 (EF-G1A), encoded by the fusA1 gene in Photobacterium profundum, serves as a crucial component of the translational machinery. It mediates the translocation of mRNA and tRNA through the ribosome during protein synthesis and participates in the ribosome recycling process . Unlike some bacteria with redundant elongation factors, EF-G1A appears to be essential in P. profundum, as attempts to delete the fusA1 gene have been unsuccessful . This protein plays a particularly important role in adaptation to deep-sea environments, where P. profundum naturally thrives under high pressure conditions.

How is the fusA1 gene organized within the P. profundum genome?

In P. profundum, fusA1 is predicted to be cotranscribed with rpsG and tufA, which encode ribosomal protein S7 and elongation factor Tu (EF-Tu), respectively . This gene organization reflects the coordinated expression of proteins involved in the translation apparatus. The genomic context suggests that regulatory mechanisms likely control the expression of these genes as a functional unit, allowing the bacterium to modulate translation efficiency in response to environmental conditions such as pressure and temperature changes .

What is known about the domain structure of P. profundum EF-G1A?

The EF-G1A protein in P. profundum contains multiple functional domains similar to those found in other bacterial species. Based on comparison with related proteins, EF-G1A consists of five domains (I-V) . Domain I contains the GTP-binding site necessary for the protein's catalytic activity. Domains II, III, and V have been identified as regions where amino acid substitutions can significantly affect protein function, particularly in relation to antibiotic interactions . These structural features are critical for understanding how EF-G1A performs its essential role in translation and how mutations might alter its activity.

What are the optimal methods for cloning and expressing recombinant P. profundum fusA1?

For successful cloning and expression of recombinant P. profundum fusA1, researchers should consider the following methodological approach:

• Gene amplification: Use high-fidelity PCR with primers designed to incorporate appropriate restriction sites for subsequent cloning. The arbitrary PCR method used for P. profundum in prior studies can be adapted, involving two rounds of PCR with specific primers for the fusA1 gene .

• Expression vector selection: For functional studies, pET-based expression systems have proven effective for similar proteins. Include a His-tag or other affinity tag to facilitate purification.

• Expression conditions: Express in E. coli BL21(DE3) or similar strains at reduced temperatures (16-20°C) to enhance proper folding of this large protein.

• Induction protocol: Use lower IPTG concentrations (0.1-0.5 mM) and longer induction times to maximize yield of properly folded protein.

The success of expression can be verified using SDS-PAGE and Western blotting with antibodies against the affinity tag or against conserved EF-G epitopes.

How can researchers effectively generate and analyze fusA1 mutants?

To generate and analyze fusA1 mutants, researchers should implement a multi-step approach:

• Site-directed mutagenesis: Target specific amino acid residues in domains II, III, and V, which have been identified as regions where mutations can significantly affect protein function . The QuikChange method or overlap extension PCR are recommended approaches.

• Transposon mutagenesis: For random mutagenesis studies, transposable elements like mini-Tn5 (rather than mini-Tn10, which shows insertion bias) can be used as demonstrated in P. profundum studies .

• Allelic exchange: To confirm the role of specific mutations, perform allelic exchange experiments similar to those conducted with P. aeruginosa, replacing wild-type fusA1 with mutated versions to assess phenotypic changes .

• Phenotypic analysis: Examine growth under various pressure and temperature conditions to assess the impact of mutations on protein function. Particular attention should be paid to growth at high pressure, where P. profundum EF-G1A plays a specialized role .

• Biochemical characterization: Compare GTPase activity, ribosome binding, and translocation efficiency between wild-type and mutant proteins using in vitro translation systems.

What proteomics approaches are most informative for studying P. profundum EF-G1A function?

For comprehensive proteomics analysis of P. profundum EF-G1A function, researchers should consider:

• Comparative proteomics: Apply methods similar to those used in PXD017266, comparing the proteomes of wild-type and fusA1 mutant strains to identify changes in the global protein expression profile . This approach can reveal compensatory mechanisms and affected pathways.

• Sample preparation protocol:

  • Harvest cells in mid-log phase

  • Lyse cells under native conditions to preserve protein-protein interactions

  • Fractionate samples to enrich for cytoplasmic proteins

  • Perform in-solution digestion with trypsin

• Mass spectrometry: Use high-resolution instruments such as Orbitrap Fusion Lumos (as employed in PXD017266) with data-dependent acquisition for deep proteome coverage.

• Data analysis: Apply label-free quantification for comparison between wild-type and mutant strains, focusing on proteins involved in translation, stress response, and pressure adaptation.

• Interaction studies: Consider complementary approaches such as pull-down assays or cross-linking mass spectrometry to identify EF-G1A interaction partners that might be specific to P. profundum's deep-sea lifestyle.

How does P. profundum fusA1 contribute to pressure adaptation?

P. profundum fusA1 plays a significant role in adaptation to high-pressure environments through several mechanisms:

• Pressure-specific functionality: The EF-G1A from P. profundum has evolved to function optimally under high-pressure conditions typical of deep-sea environments. Research has shown that heterologous expression of P. profundum recD gene (another pressure-responsive gene) can rescue high-pressure impairment of cell division in E. coli, suggesting specialized adaptations in pressure-responsive genes .

• Structural adaptations: The protein likely contains specific amino acid substitutions that confer pressure resistance, particularly in domains involved in GTP hydrolysis and ribosome interaction.

• Regulatory response: The gene appears to be part of a pressure-responsive regulatory network. Similar to other translation components in pressure-adapted organisms, fusA1 expression may be modulated in response to pressure changes to maintain translation efficiency .

• Integration with stress responses: P. profundum adapts to high pressure through mechanisms that overlap with other stress responses. For example, the stringent response mediated by spoT (which was shown to be both cold and pressure sensitive in P. profundum) may interact with fusA1 function to coordinate cellular responses to environmental pressures .

The adaptation of P. profundum to deep-sea environments (where it experiences greater stress at atmospheric pressure than at elevated pressure) makes its fusA1 gene particularly interesting for understanding fundamental mechanisms of pressure adaptation in prokaryotes.

What is the relationship between temperature sensitivity and pressure sensitivity in P. profundum fusA1 mutants?

The relationship between temperature and pressure sensitivity in P. profundum fusA1 mutants reflects the interconnected nature of these environmental stressors:

• Overlapping effects: Research has shown that 16% of P. profundum mutants exhibited altered growth at both low temperature and high pressure, suggesting common cellular targets for these stressors .

• Mechanistic similarities: Both decreased temperature and increased pressure perturb similar cellular processes, including membrane structure/function and central dogma processes (DNA replication, transcription, and translation) .

• Differential gene expression: Studies of P. profundum have identified genes that are differentially expressed under different temperature and pressure conditions, indicating coordinated adaptive responses .

• Stringent response intersection: The stringent response appears to integrate both pressure and temperature adaptation. For example, in E. coli, temperature downshift induces a relaxed state through decreased ppGpp levels, and pressure shifts may similarly affect ppGpp metabolism . The finding that a P. profundum spoT mutant is both cold and pressure sensitive supports this connection.

This relationship suggests that researchers studying recombinant P. profundum EF-G1A should carefully control both temperature and pressure parameters in experimental designs to properly interpret phenotypic results.

How do fusA1 mutations in P. profundum compare to those identified in clinical isolates of Pseudomonas aeruginosa?

The comparison of fusA1 mutations between P. profundum and clinical isolates of P. aeruginosa reveals interesting parallels and differences:

In P. aeruginosa, single amino acid substitutions in domains II (Arg371Cys), III (Thr456Ala), and V (Arg680Cys) of EF-G1A resulted in substantial increases in aminoglycoside MICs . While the specific mutations characterized in P. profundum are different, they likely affect similar functional domains that have been adapted for high-pressure environments rather than antibiotic resistance. These comparative insights suggest that the structure-function relationship of EF-G1A is conserved across bacterial species but can be modified for different selective pressures.

What insights can be gained from studying P. profundum fusA1 that might inform understanding of translation under extreme conditions?

Studying P. profundum fusA1 provides valuable insights into translation under extreme conditions:

• Pressure-resistant translation machinery: The EF-G1A protein from P. profundum represents a naturally evolved pressure-resistant variant of a core translation component. Understanding its structural adaptations can reveal fundamental principles about how protein synthesis machinery can be modified to function under extreme conditions.

• Ribosome interactions: The interaction between EF-G1A and the ribosome under high pressure conditions may involve unique stabilizing features that maintain translational efficiency despite physical stress on macromolecular complexes .

• Energy coupling mechanisms: GTP hydrolysis by EF-G1A drives translocation during protein synthesis. How this energy coupling is maintained under high pressure, where reaction volumes and protein conformational changes are affected, represents an important area for investigation.

• Stress response integration: Research suggests connections between translation factors like EF-G1A and stress response systems. For example, in P. profundum, mutations in spoT (involved in stringent response) cause both cold and pressure sensitivity , indicating potential regulatory interactions between translation and stress adaptation mechanisms.

• Evolutionary adaptation principles: Comparing fusA1 genes across bacteria adapted to different environments can reveal convergent or divergent evolutionary strategies for maintaining translational fidelity under various stressors.

These insights have implications beyond deep-sea biology, potentially informing biotechnological applications requiring protein synthesis under extreme conditions and broadening our understanding of the limits of life.

How can recombinant P. profundum EF-G1A be utilized to study pressure effects on translation?

Recombinant P. profundum EF-G1A provides a valuable tool for investigating pressure effects on translation through several experimental approaches:

• In vitro translation systems: Researchers can develop high-pressure in vitro translation systems incorporating purified P. profundum EF-G1A to directly measure translocation rates and efficiency under varying pressure conditions. This allows for controlled experiments isolating the specific contribution of EF-G1A to pressure adaptation.

• Heterologous expression studies: Similar to studies showing that P. profundum recD expression can rescue high-pressure growth defects in E. coli , researchers can express P. profundum fusA1 in pressure-sensitive bacteria to assess its ability to confer pressure resistance to translation.

• Domain swapping experiments: By creating chimeric proteins containing domains from pressure-adapted P. profundum EF-G1A and pressure-sensitive homologs from other species, researchers can identify specific regions responsible for pressure adaptation.

• Structure-function analysis: Combining site-directed mutagenesis with high-pressure enzyme kinetics allows for determination of which amino acid residues are critical for maintaining function under pressure.

• Ribosome binding studies: Using techniques such as surface plasmon resonance adapted for high-pressure conditions, researchers can quantify how pressure affects the interaction between EF-G1A and ribosomes from different species.

These approaches can help elucidate the molecular mechanisms by which P. profundum maintains efficient translation under deep-sea conditions and potentially inform the development of pressure-resistant biotechnological systems.

What implications does research on fusA1 mutations have for understanding antibiotic resistance mechanisms?

Research on fusA1 mutations provides significant insights into antibiotic resistance mechanisms with implications for both basic research and clinical applications:

• Novel resistance mechanism: Studies in P. aeruginosa have revealed that single amino acid substitutions in fusA1 can confer 4-8 fold increased resistance to aminoglycosides . This represents a novel resistance mechanism distinct from well-known approaches like enzymatic modification of antibiotics.

• Cross-species relevance: The finding that fusA1 mutations affect aminoglycoside resistance in P. aeruginosa suggests that similar mechanisms may exist in other pathogenic bacteria. Comparisons with P. profundum fusA1 can help identify conserved functional domains susceptible to resistance-conferring mutations.

• Structural insights for drug development: Understanding which domains of EF-G1A interact with aminoglycosides provides valuable structural information for the design of new antibiotics that can overcome resistance mechanisms. The identification of mutations in domains II, III, and V of EF-G1A provides specific targets for investigation .

• Diagnostic potential: The knowledge that fusA1 mutations frequently occur in clinical isolates, particularly from cystic fibrosis patients , suggests that fusA1 genotyping could be incorporated into diagnostic workflows to predict antibiotic resistance profiles.

• Evolution of resistance: Studying how fusA1 mutations arise in different bacterial species and environments can illuminate the evolutionary pathways leading to antibiotic resistance, potentially informing strategies to limit resistance development.

This research underscores the importance of studying core cellular components like translation factors, which can develop moonlighting functions in antibiotic resistance while maintaining their essential primary roles.

What are the primary challenges in expressing and purifying active recombinant P. profundum EF-G1A?

The expression and purification of active recombinant P. profundum EF-G1A presents several technical challenges that researchers must address:

• Pressure adaptation: As a protein from a piezophilic (pressure-loving) organism, P. profundum EF-G1A may fold incorrectly or exhibit reduced stability when expressed under atmospheric pressure conditions . Researchers may need to develop expression systems that can operate under increased pressure.

• Protein size: EF-G1A is a large, multi-domain protein (approximately 75-80 kDa), which can present folding challenges in heterologous expression systems. This may lead to inclusion body formation and reduced yields of active protein.

• Codon usage bias: The different codon usage preferences between P. profundum and common expression hosts like E. coli may reduce translation efficiency. Codon optimization or expression in specialized strains with expanded tRNA repertoires may be necessary.

• GTPase activity preservation: Maintaining the native GTPase activity of EF-G1A during purification requires careful buffer optimization to preserve the active site conformation and nucleotide binding capacity.

• Structural integrity assessment: Verifying that the recombinant protein maintains its native structure under different pressure conditions requires specialized biophysical techniques adapted for high-pressure measurements.

• Functional validation: Developing appropriate assays to confirm that the purified protein retains translocation activity under various pressure conditions presents additional methodological challenges.

Researchers can address these challenges through careful optimization of expression conditions, the use of fusion tags to enhance solubility, and the development of specialized assays to verify functional activity under relevant pressure conditions.

How can researchers effectively measure the activity of recombinant P. profundum EF-G1A under varying pressure conditions?

Effectively measuring recombinant P. profundum EF-G1A activity under varying pressure conditions requires specialized methodologies:

• High-pressure enzymatic assays: Develop GTPase activity assays using high-pressure vessels with optical windows to enable real-time monitoring of GTP hydrolysis via colorimetric or fluorescent readouts. These systems must be capable of precise pressure control (from atmospheric to 40-50 MPa, typical of deep-sea environments).

• Fluorescence-based translocation assays: Adapt standard ribosomal translocation assays using fluorescently labeled tRNAs or mRNAs for use in high-pressure spectrofluorimeters. This allows direct measurement of EF-G1A's primary function under pressure.

• In vitro translation systems: Establish complete in vitro translation systems that can operate under variable pressure conditions, using purified components including ribosomes, tRNAs, mRNAs, and translation factors. The efficiency of protein synthesis can be monitored using reporters like luciferase.

• Comparative benchmarking: Include EF-G proteins from non-pressure-adapted organisms (like E. coli) as controls in all assays to establish baseline pressure sensitivity of the translation machinery.

• Structural analysis under pressure: Combine activity measurements with structural studies using techniques adapted for high-pressure conditions, such as high-pressure NMR or SAXS, to correlate functional changes with structural alterations.

• Data analysis approach: When analyzing activity data across pressure ranges, researchers should consider both absolute activity values and relative pressure stability (the percentage of activity retained as pressure increases). Both parameters provide important information about pressure adaptation mechanisms.

By implementing these methodological approaches, researchers can gain meaningful insights into how P. profundum EF-G1A maintains functionality under the high-pressure conditions encountered in its natural deep-sea habitat.

What new research approaches could advance our understanding of P. profundum fusA1 function?

Several innovative research approaches could significantly advance our understanding of P. profundum fusA1 function:

• Cryo-EM studies: Determining high-resolution structures of P. profundum EF-G1A bound to the ribosome under different pressure conditions would provide unprecedented insight into pressure-adapted translation mechanisms.

• Single-molecule techniques: Developing high-pressure single-molecule FRET or optical tweezers setups would allow real-time observation of EF-G1A-mediated translocation events under native pressure conditions.

• Computational simulations: Molecular dynamics simulations comparing EF-G1A proteins from pressure-adapted and non-adapted organisms could identify key structural elements contributing to pressure resistance.

• Directed evolution: Creating libraries of fusA1 variants and selecting for enhanced function under pressure could identify critical residues for pressure adaptation and potentially engineer improved variants.

• Systems biology approaches: Integrating transcriptomics, proteomics, and metabolomics data from P. profundum under various pressure conditions could reveal how fusA1 expression and function coordinate with global cellular responses to pressure changes .

• Synthetic biology applications: Developing minimal translation systems incorporating P. profundum EF-G1A could enable protein synthesis under high-pressure conditions for biotechnological applications.

These approaches, particularly when used in combination, have the potential to significantly expand our understanding of how translation machinery adapts to extreme environmental conditions.

How might understanding P. profundum fusA1 inform research on extremophile adaptation mechanisms?

Research on P. profundum fusA1 provides valuable insights into broader extremophile adaptation mechanisms:

• Evolutionary conservation patterns: Comparative genomic analysis of translation factors across extremophiles adapted to different stressors (pressure, temperature, pH, salinity) can reveal common and distinct adaptation strategies. P. profundum fusA1 serves as an important reference point for understanding pressure adaptation specifically .

• Stress response integration: The connection between fusA1 function and stress responses in P. profundum, such as the stringent response mediated by spoT , suggests that translation regulation may be a central hub in extremophile adaptation networks.

• Structural adaptation principles: Identifying which domains and amino acid residues in P. profundum EF-G1A are modified for pressure adaptation can reveal general principles about how proteins maintain functionality under extreme conditions. These insights may apply to other extremophiles.

• Multi-stress adaptation: The observation that P. profundum mutants often show sensitivity to both pressure and temperature indicates that adaptation mechanisms frequently address multiple stressors simultaneously, an important consideration for understanding extremophile biology.

• Biotechnological applications: Understanding how P. profundum fusA1 maintains function under pressure could inform the development of pressure-resistant enzymes and processes for industrial applications, extending the utility of extremophile research beyond basic science.

By positioning P. profundum fusA1 research within the broader context of extremophile biology, researchers can contribute to a more comprehensive understanding of life's adaptability to challenging environments, with implications ranging from astrobiology to biotechnology.