Recombinant Chromobacterium violaceum Elongation factor P (efp)

What is Chromobacterium violaceum and why is it significant in research?

Chromobacterium violaceum is a Gram-negative, facultative anaerobe bacillus belonging to the Neisseriaceae family that predominantly inhabits tropical and subtropical regions. This β-proteobacterium has gained significant research attention since the 1970s due to its diverse biotechnological applications in producing antibiotics, anti-tumor compounds, and biopolymers . C. violaceum is also noteworthy as an opportunistic pathogen that can cause severe infections leading to sepsis in immunocompromised individuals . The bacterium's genome was fully sequenced in 2003, revealing numerous genes related to stress adaptability, which has spurred extensive research into its environmental resilience mechanisms . The distinctive purple pigment violacein produced by C. violaceum is regulated through quorum sensing and serves as a valuable indicator in anti-quorum sensing research .

What is Elongation Factor P (EF-P) and what is its function in C. violaceum?

Elongation Factor P (EF-P) is a highly conserved bacterial protein that plays a crucial role in efficient protein synthesis. In C. violaceum, as in other bacteria, EF-P functions primarily as a translation factor that facilitates the formation of peptide bonds during protein synthesis, particularly when ribosomes encounter consecutive proline codons. This helps prevent ribosomal stalling and ensures efficient translation of proteins with polyproline motifs. The full-length EF-P protein in C. violaceum (strain ATCC 12472) consists of 187 amino acids with a defined sequence beginning with MKTAQELRAG and ending with NEFKKRA . Proteomic analyses have demonstrated that EF-P expression in C. violaceum changes in response to various stress conditions, suggesting its importance in the bacterium's adaptive responses to environmental challenges .

How is recombinant C. violaceum EF-P typically produced and purified?

Recombinant C. violaceum EF-P is typically produced in heterologous expression systems, with yeast being a common host organism as indicated in product specifications . The production process generally follows these methodological steps:

• Gene cloning: The C. violaceum efp gene (corresponding to UniProt accession Q7NY96) is amplified and inserted into an appropriate expression vector.

• Transformation: The recombinant vector is introduced into the yeast expression host.

• Expression induction: Culture conditions are optimized to induce high-level expression of the target protein.

• Cell harvesting and lysis: Cells are collected and disrupted to release the intracellular recombinant protein.

• Purification: Typically involves affinity chromatography using a fusion tag, followed by additional purification steps.

• Quality control: The final product is assessed for purity (>85% by SDS-PAGE for commercially available products) and proper folding .

For research applications, the recombinant protein is available in either liquid or lyophilized forms, with shelf lives of approximately 6 months and 12 months respectively when stored at -20°C/-80°C .

How can recombinant C. violaceum EF-P be used to study bacterial stress responses?

Proteomic studies have identified EF-P as one of several proteins in C. violaceum that display altered expression patterns under different stress conditions . To effectively use recombinant EF-P in stress response studies, researchers can:

• Perform comparative expression analysis: Generate antibodies against the recombinant EF-P to track native protein levels in C. violaceum under various stress conditions (pH stress, nutrient starvation, oxidative stress, etc.).

• Conduct functional complementation assays: Use the recombinant protein to complement EF-P-deficient bacterial strains to assess its ability to restore normal growth and stress tolerance.

• Design in vitro translation assays: Add purified recombinant EF-P to cell-free translation systems containing polyproline-rich mRNAs to evaluate how stress-related post-translational modifications might affect its activity.

• Analyze protein-protein interactions: Use the recombinant EF-P as bait in pull-down assays or two-hybrid systems to identify stress-specific interaction partners.

These approaches can help elucidate how C. violaceum modulates translation in response to environmental challenges, as the proteomic signature of stressed C. violaceum indicates significant changes in proteins involved in biosynthetic pathways, molecule recycling, and energy production .

What are the optimal storage and handling conditions for maintaining recombinant C. violaceum EF-P activity?

To maintain optimal activity of recombinant C. violaceum EF-P, researchers should follow these evidence-based protocols:

• Reconstitution: Centrifuge the product vial briefly before opening. Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage buffer optimization: Add glycerol to a final concentration of 5-50% (with 50% being recommended) to prevent protein denaturation during freeze-thaw cycles .

• Aliquoting: Divide the reconstituted protein into single-use aliquots to avoid repeated freeze-thaw cycles.

• Storage temperature: Store at -20°C/-80°C for long-term storage (6 months for liquid form, 12 months for lyophilized form) .

• Working storage: For active experiments, store working aliquots at 4°C for no more than one week .

• Freeze-thaw management: Minimize freeze-thaw cycles as repeated freezing and thawing is not recommended and can lead to significant loss of activity .

These handling procedures are critical for maintaining the structural integrity and functional activity of the protein for experimental applications.

How can recombinant C. violaceum EF-P be used in studies of bacterial translation mechanisms?

Recombinant C. violaceum EF-P serves as a valuable tool for investigating bacterial translation mechanisms through several methodological approaches:

• In vitro translation assays: The purified recombinant protein can be added to cell-free translation systems to directly measure its effect on the synthesis of polyproline-containing proteins. This allows researchers to quantify translation efficiency and fidelity.

• Structural studies: The availability of highly purified recombinant EF-P enables X-ray crystallography or cryo-EM studies to determine its three-dimensional structure, providing insights into its interaction with the ribosome and other translation factors.

• Site-directed mutagenesis: Researchers can create variants of the recombinant EF-P with specific amino acid substitutions to identify critical residues for function. The amino acid sequence provided in the product specifications (starting with MKTAQELRAG) can guide the selection of conserved regions for mutation.

• Comparative studies: C. violaceum EF-P can be compared with EF-P proteins from other bacterial species to investigate evolutionary conservation and specialization of function.

These approaches contribute to our understanding of how bacterial elongation factors overcome translation challenges, particularly at challenging mRNA sequences.

How does C. violaceum EF-P expression change under different environmental stresses, and what methodologies best capture these changes?

Proteomic analysis of C. violaceum has revealed that EF-P expression levels change in response to environmental stresses . To effectively characterize these changes, researchers should consider these methodological approaches:

• Quantitative proteomics: Employ techniques like 2D-PAGE combined with mass spectrometry, as described in previous studies where C. violaceum was subjected to nutritional and pH stresses . This approach allows visualization of protein expression patterns across different conditions.

• RNA-seq and qRT-PCR: Complement protein-level studies with transcriptomic analysis to determine whether EF-P regulation occurs at the transcriptional level.

• Western blotting with phospho-specific antibodies: Develop antibodies that recognize specific post-translational modifications of EF-P to track not just expression levels but also activation states.

• Time-course experiments: Monitor EF-P expression at different time points during stress exposure to capture the dynamics of the stress response.

Research has shown that while stressed C. violaceum cultures produce higher total protein mass than reference cultures, they show reduced diversity in protein expression . This suggests that stress induces a focused response involving specific proteins, including translation factors like EF-P, to maintain cellular function under adverse conditions.

What role does EF-P play in C. violaceum virulence and adaptation to host environments?

C. violaceum is an opportunistic pathogen associated with lethal sepsis in immunocompromised individuals . The role of EF-P in virulence can be investigated through these advanced research approaches:

• Infection models: Use EF-P deletion or overexpression strains in infection models to assess how altered EF-P levels affect C. violaceum pathogenicity.

• Virulence factor expression analysis: Determine whether EF-P is specifically required for the efficient translation of virulence factors, particularly those containing polyproline motifs.

• Host-mimicking stress conditions: Expose C. violaceum to conditions that mimic the host environment (oxidative stress, nutrient limitation, antimicrobial peptides) and assess EF-P's contribution to survival.

• Comparative analysis with related pathogens: Compare the function and regulation of EF-P between C. violaceum and closely related pathogenic bacteria to identify conserved mechanisms.

The fact that C. violaceum has evolved mechanisms to cope with various environmental challenges suggests that EF-P might be a key factor in its adaptive responses, potentially contributing to its ability to transition from an environmental microbe to a human pathogen under specific conditions.

How can recombinant C. violaceum EF-P be used to develop targeted antimicrobial strategies?

Given the importance of EF-P in bacterial translation and potential role in stress adaptation, it represents a promising target for novel antimicrobial development. Researchers can employ these methodologies:

• High-throughput screening assays: Develop assays using recombinant EF-P to screen compound libraries for molecules that specifically inhibit its function.

• Structure-based drug design: Utilize the amino acid sequence of C. violaceum EF-P (MKTAQELRAG...NEFKKRA) as a basis for structural modeling and rational design of inhibitors targeting critical functional domains.

• Post-translational modification inhibitors: Identify and target the enzymes responsible for EF-P post-translational modifications, which are essential for its function in many bacteria.

• Combinatorial approaches: Test EF-P inhibitors in combination with existing antibiotics, particularly those that target translation, to identify synergistic effects.

• C. violaceum reporter systems: Leverage the violacein pigment production system of C. violaceum to develop whole-cell screening assays for compounds that might affect EF-P function indirectly.

This research direction is particularly relevant given the increasing problem of antimicrobial resistance and the need for novel targets and mechanisms of action.

What are the critical quality control parameters for recombinant C. violaceum EF-P preparations?

To ensure experimental reproducibility when working with recombinant C. violaceum EF-P, researchers should verify these key quality control parameters:

• Purity assessment: Confirm protein purity using SDS-PAGE, with commercial preparations typically guaranteeing >85% purity . Consider higher purity requirements for sensitive applications like crystallography.

• Identity verification: Validate protein identity through mass spectrometry, comparing results to the expected molecular weight calculated from the amino acid sequence (MKTAQELRAG...NEFKKRA) .

• Functional activity: Develop assays to confirm that the recombinant EF-P can perform its native function, such as in vitro translation assays with polyproline-containing templates.

• Endotoxin testing: For applications involving cell culture or in vivo experiments, verify that the preparation is endotoxin-free or below acceptable thresholds.

• Post-translational modification status: Determine whether the recombinant EF-P (produced in yeast) lacks bacterial-specific modifications that might be essential for full activity.

These quality control measures are essential for ensuring that experimental results accurately reflect the biological properties of EF-P rather than artifacts introduced by the recombinant production process.

How can researchers differentiate between the functions of EF-P and other elongation factors in C. violaceum?

Distinguishing the specific functions of EF-P from other elongation factors (like EF-Tu, which is also stress-responsive in C. violaceum) requires sophisticated experimental approaches:

• Specific inhibition studies: Develop or identify inhibitors that selectively target EF-P but not other elongation factors.

• Selective depletion: Use RNA interference or CRISPR-based approaches to selectively deplete EF-P while monitoring the effects on different types of translation events.

• Reporter constructs: Design reporter gene constructs with and without polyproline sequences to specifically monitor EF-P-dependent translation.

• In vitro reconstitution: Perform in vitro translation experiments with defined components, systematically adding or removing different elongation factors to dissect their individual contributions.

• Ribosome profiling: Use ribosome profiling techniques to identify translation pause sites genome-wide in wild-type and EF-P-deficient C. violaceum to map EF-P-dependent translation events.

These approaches help delineate the unique role of EF-P in facilitating translation of specific protein sequences that may be particularly important during stress conditions or pathogenesis.

How might high-throughput approaches advance our understanding of C. violaceum EF-P function?

Several emerging high-throughput methodologies offer promising avenues for advancing EF-P research:

• Proteome-wide interaction studies: Techniques like protein microarrays using recombinant EF-P as bait could identify the complete set of proteins that interact with EF-P under different conditions.

• CRISPR screens: Genome-wide CRISPR screens in C. violaceum could identify genes that interact synthetically with efp, revealing functional connections.

• Deep mutational scanning: Systematic creation and functional testing of all possible single amino acid variants of EF-P to create a comprehensive map of structure-function relationships.

• Transcriptome-wide analysis of translation: Ribosome profiling across diverse growth conditions could comprehensively map the mRNAs whose translation is most dependent on EF-P.

• Comparative genomics and systems biology: Integration of EF-P functional data with genomic, transcriptomic, and proteomic datasets across multiple bacterial species could reveal conserved and species-specific aspects of EF-P function.

These approaches could significantly expand our understanding of EF-P beyond its canonical role in facilitating translation of polyproline sequences, potentially revealing unexplored functions in bacterial physiology and pathogenesis.

What is the relationship between EF-P function and quorum sensing in C. violaceum?

C. violaceum is a model organism for studying quorum sensing due to its production of the purple pigment violacein, which is regulated by quorum sensing mechanisms . The potential relationship between EF-P and quorum sensing represents an intriguing research question that can be approached through:

• EF-P knockout studies: Analyze how EF-P deletion affects violacein production and other quorum sensing-regulated phenotypes.

• Translational analysis of quorum sensing genes: Determine whether key quorum sensing regulators contain polyproline motifs that might make their translation particularly dependent on EF-P.

• Stress-response integration: Investigate whether environmental stresses that alter EF-P levels also impact quorum sensing, potentially revealing a mechanism by which bacteria coordinate population behavior with stress responses.

• Co-expression network analysis: Perform transcript and protein co-expression analysis to identify potential regulatory relationships between EF-P and quorum sensing pathways.

Understanding this relationship could provide insights into how translation regulation interfaces with bacterial communication systems, potentially revealing new targets for anti-virulence strategies that don't directly kill bacteria but disrupt their coordinated behaviors.