Recombinant Chromobacterium violaceum Elongation factor G (fusA), partial

What is Elongation Factor G (fusA) and what is its role in C. violaceum?

Elongation Factor G (EF-G) is a critical component of the bacterial translation machinery that catalyzes translocation of the peptidyl-tRNA from the A-site to the P-site on the ribosome during protein synthesis. In C. violaceum, EF-G functions similarly to other bacterial homologs, facilitating the movement of mRNA-tRNA complexes through the ribosome and enabling efficient protein synthesis.

Research has demonstrated that EF-G is part of the stress-responsive proteome in C. violaceum, with expression levels changing during various environmental stresses . This suggests that beyond its canonical role in translation, C. violaceum EF-G may contribute to adaptive responses that help this bacterium survive in diverse environments.

How does the structure of C. violaceum EF-G compare to homologs from other bacteria?

While the complete crystal structure of C. violaceum EF-G has not been fully characterized, comparative genomic analysis suggests structural conservation with other bacterial EF-G proteins. Like other bacterial EF-Gs, the C. violaceum protein likely contains five distinct domains, with domains I and II being responsible for GTP binding and hydrolysis.

Recombinant expression studies of other C. violaceum proteins, such as phenylalanine hydroxylase, have demonstrated that key structural and functional characteristics are maintained when expressed in heterologous systems, suggesting that similar approaches could be successful for EF-G .

How is EF-G expression regulated in C. violaceum?

Proteomic analyses have revealed that translation factors in C. violaceum, including EF-Tu and EF-P, show stress-related expression changes . While EF-G (fusA) specifically wasn't directly mentioned in these studies, its expression likely follows similar patterns given its essential role in protein synthesis.

C. violaceum has been shown to adapt to environmental stresses through complex regulatory networks. For instance, when exposed to oxidative stress with hydrogen peroxide, C. violaceum expresses proteins that protect it from damage while decreasing abundance of proteins responsible for bacterial growth and catabolism . This suggests that translation factors like EF-G may be dynamically regulated as part of this adaptive response.

What expression systems are most effective for recombinant C. violaceum EF-G?

Based on successful expression of other C. violaceum proteins, the following expression systems have proven effective and could be applied to EF-G:

E. coli-based expression systems have been successfully used for recombinant C. violaceum proteins, with specific conditions depending on the protein's characteristics. For EF-G, which is a cytoplasmic protein, E. coli BL21(DE3) with pET-based vectors would likely yield good results with IPTG induction at lower temperatures (16-25°C) to enhance proper folding.

What purification strategies work best for recombinant C. violaceum proteins?

Successful purification strategies for recombinant C. violaceum proteins that could be applied to EF-G include:

• Affinity chromatography: Utilizing His-tag or other fusion tags has proven effective for C. violaceum proteins. The choice of tag position (N- or C-terminal) should be evaluated to avoid interference with EF-G function.

• Ion exchange chromatography: This has been successful for separating C. violaceum proteins based on their charge properties, particularly using DEAE or SP sepharose depending on the protein's isoelectric point.

• Size exclusion chromatography: As a final polishing step, this helps remove aggregates and ensure homogeneity of the purified EF-G.

A typical buffer system that has worked well for C. violaceum proteins contains:

• 50 mM Tris-HCl (pH 7.5-8.0)

• 100-300 mM NaCl

• 5-10% glycerol

• 1-5 mM β-mercaptoethanol or DTT

• Protease inhibitor cocktail during initial extraction

How can researchers verify the functional activity of recombinant C. violaceum EF-G?

Functional verification of recombinant C. violaceum EF-G can be accomplished through:

• GTPase activity assay: Measuring GTP hydrolysis rates using colorimetric assays that detect inorganic phosphate release.

• In vitro translation assays: Testing the ability of purified EF-G to support protein synthesis in reconstituted translation systems.

• Ribosome binding studies: Using methods like surface plasmon resonance (SPR) to quantify binding kinetics between EF-G and ribosomes.

• Complementation studies: Determining if C. violaceum EF-G can restore growth in E. coli strains with temperature-sensitive fusA mutations.

How do environmental stresses affect elongation factors in C. violaceum?

Proteomic analysis has revealed that stress conditions significantly alter the expression of translation-related proteins in C. violaceum:

These findings suggest that translation factors, including EF-G, play important roles in the stress adaptation of C. violaceum, potentially through modulation of protein synthesis rates to conserve energy during unfavorable conditions.

How do antibiotics targeting protein synthesis affect C. violaceum?

C. violaceum responds to translation-inhibiting antibiotics in several notable ways:

• Induction of violacein production: Sublethal doses of antibiotics that target polypeptide elongation (like blasticidin S, spectinomycin, hygromycin B, tetracycline, erythromycin, and chloramphenicol) induce production of the purple pigment violacein .

• Biofilm formation: Translation-inhibiting antibiotics induce biofilm formation, potentially as a protective mechanism .

• Virulence regulation: Antibiotics targeting translation activate virulence mechanisms through the antibiotic-induced response (air) two-component regulatory system .

This complex response suggests that EF-G and other components of the translation machinery are central to C. violaceum's sensing of and adaptation to antibiotic stress.

Could EF-G be targeted for antimicrobial development against C. violaceum infections?

C. violaceum infections, while rare, have a high mortality rate of approximately 62.1% . The essential nature of EF-G for bacterial survival makes it a potential target for antimicrobial development:

• Structural differences: Bacterial EF-G differs significantly from its eukaryotic counterpart (eEF2), potentially allowing for selective targeting.

• Existing precedent: Several antibiotics already target bacterial translation, suggesting the viability of this approach.

• Critical function: As demonstrated by proteomic studies, translation factors play central roles in C. violaceum's stress response and adaptation , making interference with their function potentially highly effective.

Development of small molecule inhibitors specifically targeting unique aspects of C. violaceum EF-G structure could lead to novel therapeutic approaches for these difficult-to-treat infections.

How can molecular evolutionary analysis of C. violaceum EF-G contribute to understanding bacterial translation?

Comparative analysis of C. violaceum EF-G can provide valuable insights into:

• Evolutionary conservation: Identifying highly conserved regions across bacterial species that represent functionally critical domains.

• Environmental adaptation: Revealing unique features that might be adaptations to C. violaceum's specific ecological niche.

• Antibiotic resistance mechanisms: Understanding structural variations that might contribute to intrinsic resistance to certain antibiotics.

This approach can help identify unique features of C. violaceum translation machinery that may contribute to its environmental adaptability and pathogenicity.

What methodological approaches are most effective for studying C. violaceum protein-protein interactions?

For studying interactions involving C. violaceum EF-G, several techniques have proven effective with other C. violaceum proteins:

• Bacterial two-hybrid systems: Useful for identifying interacting partners in vivo.

• Pull-down assays with recombinant proteins: Successfully used to study C. violaceum regulatory proteins like VioS .

• Surface plasmon resonance (SPR): Allows quantitative measurement of binding kinetics.

• Cryo-electron microscopy: Particularly valuable for visualizing EF-G interactions with the ribosome during different stages of translocation.

• Crosslinking coupled with mass spectrometry: Helps identify interaction interfaces between EF-G and its binding partners.

How might recombinant C. violaceum EF-G be used as a research tool?

Recombinant C. violaceum EF-G has several potential applications in research:

• Control protein in translational studies: As shown in previous research where EF-G was used as a control .

• Antibiotic screening: For identifying compounds that specifically target bacterial translation.

• Structural biology: To understand mechanisms of bacterial translation through comparative studies.

• Evolutionary studies: As a model for investigating adaptation of translation machinery in bacteria that thrive in diverse environments.

• Development of in vitro translation systems: For biotechnological applications requiring efficient protein synthesis.

What are the common challenges in expressing and purifying active recombinant C. violaceum EF-G?

Researchers working with recombinant C. violaceum proteins have encountered several challenges that may apply to EF-G:

• Solubility issues: Large proteins like EF-G (approximately 77 kDa) can form inclusion bodies. This can be addressed by:

  • Lowering induction temperature (16-20°C)

  • Using fusion tags that enhance solubility (MBP, SUMO)

  • Adding solubility enhancers to lysis buffer (low concentrations of non-ionic detergents)

• Maintaining GTPase activity: EF-G's function depends on its GTPase activity, which can be sensitive to purification conditions. Including:

  • Mg²⁺ in all buffers (typically 5-10 mM)

  • Avoiding metal chelators like EDTA

  • Using fresh DTT or β-mercaptoethanol to prevent oxidation

• Protein stability: EF-G can be prone to degradation. Strategies to prevent this include:

  • Adding protease inhibitors during lysis

  • Maintaining low temperatures throughout purification

  • Including glycerol (10-20%) in storage buffers

How can researchers optimize codon usage for recombinant expression of C. violaceum EF-G in E. coli?

C. violaceum has a high GC content (64.83%) which can lead to codon usage bias issues when expressing its proteins in E. coli:

• Codon optimization: Adapting the fusA gene sequence to E. coli codon preferences can significantly improve expression levels.

• Use of specialized E. coli strains: Strains like Rosetta(DE3) that supply rare tRNAs can help overcome codon bias without sequence modification.

• Expression vector selection: Vectors with strong promoters but tight regulation (like pET systems with T7lac promoters) can help balance expression level with proper folding.

A comparative analysis of native versus codon-optimized gene sequences can help determine the optimal approach for a specific research application.

What strategies can address aggregation issues with recombinant C. violaceum EF-G?

Protein aggregation is a common challenge when working with recombinant bacterial proteins. For C. violaceum EF-G, effective strategies include:

• Buffer optimization:

  • Screening different pH ranges (typically 7.0-8.5)

  • Testing various salt concentrations (150-500 mM NaCl)

  • Adding stabilizing agents (glycerol, arginine, trehalose)

• Expression conditions:

  • Lower induction temperatures (16-25°C)

  • Reduced IPTG concentrations (0.1-0.5 mM)

  • Extended expression times at lower temperatures

• Purification approach:

  • Inclusion of mild detergents below their critical micelle concentration

  • Two-step affinity purification to remove partially folded intermediates

  • Size exclusion chromatography as a final polishing step

By systematically optimizing these parameters, researchers can overcome aggregation issues and obtain functionally active recombinant C. violaceum EF-G suitable for structural and functional studies.