Recombinant Burkholderia multivorans Elongation factor Ts (tsf)

What is Elongation factor Ts (EF-Ts) and what is its role in Burkholderia multivorans?

Elongation factor Ts (EF-Ts) is a critical protein involved in the bacterial translation process. In Burkholderia multivorans, EF-Ts functions as a guanine nucleotide exchange factor that catalyzes the release of GDP from the elongation factor Tu (EF-Tu), allowing subsequent binding of GTP and continuation of the translation cycle. This process is essential for protein synthesis in these bacteria, as it enables the recycling of EF-Tu during translation elongation . EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of approximately 6 × 10^4 and 3 × 10^3, respectively, significantly accelerating the nucleotide exchange process beyond what would occur spontaneously .

How is Burkholderia multivorans Elongation factor Ts identified in experimental settings?

Burkholderia multivorans Elongation factor Ts can be identified through proteomic approaches, most notably two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In comparative studies, cultured supernatants of B. multivorans are processed through 2-DE, and spots are analyzed for differential expression compared to other Burkholderia species (such as B. cenocepacia) . When analyzing secretory proteins, researchers must verify that detected proteins are truly from the secretome rather than cell lysis by measuring markers like LDH release. Significant protein spots (with fold changes ≥1.5 and P values ≤0.05) can then be selected for protein identification by mass spectrometry, where EF-Ts shows characteristic peptide mass fingerprints that enable its precise identification within the B. multivorans proteome .

What is the taxonomic significance of Elongation factor Ts in the Burkholderia cepacia complex?

Elongation factor Ts has emerged as an important biomarker for taxonomic differentiation within the Burkholderia cepacia complex (Bcc). The Bcc consists of nine closely related species that present diagnostic challenges due to their phenotypic similarities . EF-Ts is one of the discriminatory proteins identified between Burkholderia multivorans and Burkholderia cenocepacia, making it valuable for species-level identification within this complex . Given the clinical importance of distinguishing between these species in cystic fibrosis patients, EF-Ts serves as a potential diagnostic marker that can complement multilocus sequence typing (MLST) methods for accurate Burkholderia species identification . The distinctive expression patterns of EF-Ts across different Burkholderia species provide researchers with a protein-based approach to complement nucleic acid-based identification methods.

What are the optimal conditions for expressing recombinant Burkholderia multivorans Elongation factor Ts?

The optimal expression of recombinant B. multivorans Elongation factor Ts requires careful consideration of expression systems and conditions. Based on protocols for similar Burkholderia species proteins, two primary expression systems have demonstrated success: E. coli-based expression and baculovirus-insect cell systems . For E. coli expression, BL21(DE3) or similar strains with reduced protease activity typically yield better results. Expression should be induced at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG, followed by cultivation at 18-25°C for 16-18 hours to enhance soluble protein production and reduce inclusion body formation. For baculovirus systems, Sf9 or Hi5 insect cells provide a eukaryotic environment that may improve protein folding. Post-expression, purification via affinity chromatography (typically using His-tag or GST-tag systems) followed by ion-exchange chromatography yields high-purity protein. Final preparations should be maintained in buffer containing 20-50 mM Tris-HCl (pH 7.5), 100-200 mM NaCl, 1 mM DTT, and 10% glycerol .

What purification strategies yield the highest purity and activity for recombinant B. multivorans EF-Ts?

A multi-step purification approach produces the highest purity and activity for recombinant B. multivorans EF-Ts. The optimal protocol typically involves:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged protein)

• Intermediate purification via ion-exchange chromatography (typically Q-Sepharose)

• Final polishing step using size-exclusion chromatography

The table below outlines a recommended purification workflow with expected outcomes:

Throughout purification, it's essential to maintain the temperature at 4°C and include protease inhibitors in the initial lysis buffer. Final protein quality should be assessed by SDS-PAGE (target purity >85%) and activity assays measuring nucleotide exchange capacity . To maintain activity, purified EF-Ts should be stored with 10-50% glycerol at -20°C for short-term storage or -80°C for long-term preservation, avoiding repeated freeze-thaw cycles .

How should researchers assess the functional activity of purified B. multivorans EF-Ts?

Assessing the functional activity of purified B. multivorans EF-Ts requires examination of its nucleotide exchange capability. The most robust approach employs stopped-flow fluorescence techniques measuring the acceleration of GDP/GTP exchange on EF-Tu . A comprehensive functional assessment should include:

• GDP Dissociation Assay: Measure the rate of mant-GDP (fluorescently labeled GDP) dissociation from EF-Tu in the presence and absence of EF-Ts. The rate enhancement (typically 10^3-10^4 fold) provides a quantitative measure of EF-Ts activity .

• Binary Complex Formation: Assess the formation of the EF-Tu·EF-Ts binary complex using techniques such as native gel electrophoresis, analytical ultracentrifugation, or surface plasmon resonance to determine binding kinetics.

• Ternary Complex Analysis: Examine the ability of EF-Ts to facilitate formation of the EF-Tu·GTP·aminoacyl-tRNA ternary complex, which represents the physiologically relevant endpoint of the exchange reaction.

For accurate activity comparisons between different EF-Ts preparations, standardized reaction conditions (temperature, pH, Mg^2+ concentration) must be maintained, as these factors significantly influence nucleotide exchange rates. An active EF-Ts preparation should demonstrate GDP dissociation enhancement factors comparable to those reported for E. coli EF-Ts (approximately 6 × 10^4 for GDP) .

How can structural studies of B. multivorans EF-Ts inform antibiotic development strategies?

Structural studies of B. multivorans EF-Ts provide crucial insights for antibiotic development through multiple mechanisms. EF-Ts represents an attractive antimicrobial target due to its essential role in bacterial protein synthesis and its structural differences from eukaryotic counterparts. High-resolution structural characterization through X-ray crystallography or cryo-electron microscopy enables identification of unique binding pockets that could be targeted by small-molecule inhibitors. Particular focus should be placed on the EF-Tu interaction interfaces, as disrupting this protein-protein interaction would inhibit bacterial translation .

Molecular dynamics simulations can further identify transient binding pockets and allosteric sites that might not be evident in static structures. Structure-based virtual screening approaches then allow for the in silico identification of potential inhibitors before experimental validation. Importantly, comparative structural analysis between Burkholderia EF-Ts and human mitochondrial EF-Ts (which shares the same function but has distinct structural features) can guide the development of selective inhibitors that minimize off-target effects . Since Burkholderia species are intrinsically resistant to many antibiotics, targeting the essential EF-Ts protein presents a promising strategy for developing novel therapeutics against these challenging pathogens.

What are the key differences between B. multivorans EF-Ts and EF-Ts proteins from other bacterial species?

B. multivorans EF-Ts exhibits notable structural and functional differences from EF-Ts proteins of other bacterial species, particularly in domain organization and key interaction residues. While the core function of nucleotide exchange on EF-Tu is conserved, sequence analysis reveals species-specific variations that influence protein-protein interactions and kinetic parameters .

Analysis of primary sequence data shows that B. multivorans EF-Ts maintains the characteristic N-terminal domain, core domain, and C-terminal domain structure found in other bacterial EF-Ts proteins, but with distinctive variations in key interaction interfaces. These variations likely contribute to the specificity of EF-Tu·EF-Ts interactions within Burkholderia species. Kinetic studies of nucleotide exchange mechanisms reveal that the rate enhancement of GDP/GTP dissociation from EF-Tu varies significantly between species, with Burkholderia EF-Ts potentially showing different catalytic efficiency compared to well-characterized systems like E. coli .

Furthermore, B. multivorans EF-Ts serves as a potential species-specific biomarker within the Burkholderia cepacia complex, highlighting its unique characteristics compared to other closely related bacterial species . These distinctive features make B. multivorans EF-Ts valuable both for taxonomic identification and as a potential target for species-specific therapeutic interventions.

How does post-translational modification affect the function of B. multivorans EF-Ts?

Post-translational modifications (PTMs) of B. multivorans EF-Ts can significantly influence its nucleotide exchange activity, protein-protein interactions, and cellular localization. While comprehensive PTM mapping of B. multivorans EF-Ts has not been fully characterized, insights from related systems suggest several potential mechanisms of regulation. Phosphorylation at serine, threonine, or tyrosine residues can alter the electrostatic properties of key interaction domains, potentially modulating the affinity for EF-Tu or the rate of nucleotide exchange catalysis .

Proteomic studies using two-dimensional gel electrophoresis have revealed multiple protein spots corresponding to EF-Ts, suggesting the presence of different proteoforms potentially arising from PTMs . These modifications may serve as regulatory mechanisms allowing bacteria to fine-tune translation rates in response to environmental stresses or nutrient availability. Mass spectrometry-based approaches combining phosphoproteomics, glycoproteomics, and acetylomics offer the most comprehensive strategy for identifying and quantifying PTMs on native B. multivorans EF-Ts.

Researchers investigating PTM effects should employ site-directed mutagenesis to create phosphomimetic (e.g., Ser→Asp) or phosphodeficient (e.g., Ser→Ala) variants, followed by functional nucleotide exchange assays to assess the impact on catalytic activity. Additionally, examining PTM patterns under different growth conditions or stress responses may reveal regulatory mechanisms that could be exploited for therapeutic intervention.

How does B. multivorans EF-Ts interact with other components of the bacterial translation machinery?

B. multivorans EF-Ts participates in an intricate network of interactions within the bacterial translation machinery, primarily centered on its relationship with EF-Tu. The EF-Tu·EF-Ts interaction serves as the cornerstone of translation elongation regulation, with EF-Ts accelerating the GDP/GTP exchange on EF-Tu by factors of 10^3-10^4 . This dramatic rate enhancement is achieved through structural rearrangements that disrupt the Mg^2+ binding site in EF-Tu, though this mechanism alone doesn't fully explain the catalytic efficiency of EF-Ts .

Beyond its canonical interaction with EF-Tu, emerging evidence suggests EF-Ts may engage in moonlighting functions through interactions with additional translation factors or regulatory proteins. The EF-Ts interaction network likely extends to components including:

• Ribosomes: Potential direct interactions during the elongation cycle

• Aminoacyl-tRNA synthetases: Possible coordination of tRNA charging and utilization

• Regulatory factors: Interaction with stress-response proteins under particular growth conditions

These extended interactions may vary between bacterial species, potentially contributing to the unique characteristics of B. multivorans translation regulation. Researchers investigating these interaction networks should employ techniques such as pull-down assays, cross-linking mass spectrometry, or proximity labeling approaches to capture the complete interactome of B. multivorans EF-Ts under various physiological conditions .

What role does EF-Ts play in Burkholderia multivorans virulence and pathogenicity?

The contribution of EF-Ts to B. multivorans virulence and pathogenicity operates through both direct and indirect mechanisms. As a fundamental component of the translation machinery, EF-Ts ensures efficient protein synthesis necessary for bacterial growth and adaptation within host environments . This core function underscores its indirect contribution to virulence through enabling the translation of virulence factors and stress-response proteins essential for host colonization and immune evasion.

More directly, EF-Ts has been identified among the secreted proteins of B. multivorans, suggesting potential extracellular functions beyond its canonical role in translation . Secreted bacterial translation factors can sometimes serve as moonlighting proteins with immunomodulatory properties or contribute to host-pathogen interactions. Furthermore, EF-Ts has emerged as a distinctive biomarker for differentiating between Burkholderia species in clinical samples, particularly in the context of cystic fibrosis infections where accurate species identification informs treatment decisions .

Research strategies to explore the virulence-related functions of EF-Ts should include:

• Conditional knockdown systems to assess the impact of EF-Ts depletion on virulence factor expression

• Animal infection models comparing wild-type and EF-Ts-modulated strains

• Immunological studies examining potential interactions between secreted EF-Ts and host immune components

Understanding these connections between EF-Ts and virulence could reveal novel therapeutic strategies targeting this essential bacterial protein .

How does the genomic context of the tsf gene vary across Burkholderia species and what are the evolutionary implications?

The genomic context of the tsf gene encoding Elongation factor Ts shows both conservation and variation across Burkholderia species, providing insights into evolutionary relationships and functional adaptations. In many bacteria, including Burkholderia species, the tsf gene typically resides within the str operon, which contains genes encoding several ribosomal and translation-related proteins . This operon organization reflects the functional coordination of translation components.

Comparative genomic analysis across the Burkholderia cepacia complex reveals subtle variations in the sequence and regulatory elements of the tsf gene that contribute to species differentiation . Multilocus sequence typing approaches incorporating tsf along with other housekeeping genes have proven valuable for resolving the complex taxonomy of Burkholderia species . The table below summarizes key genomic characteristics of the tsf gene across select Burkholderia species:

Evolutionary analysis suggests that while the core function of EF-Ts is highly conserved due to selective pressure maintaining translation accuracy, species-specific variations have accumulated primarily in regions less critical for nucleotide exchange activity . These variations likely contribute to fine-tuning translation efficiency under different environmental conditions encountered by various Burkholderia species, reflecting their diverse ecological niches ranging from soil to the human respiratory tract.

What are common challenges in expressing and purifying recombinant B. multivorans EF-Ts and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant B. multivorans EF-Ts, each requiring specific troubleshooting approaches:

• Poor solubility: B. multivorans proteins often have high GC content, potentially leading to codon usage issues and protein misfolding. This can be addressed by:

  • Using codon-optimized synthetic genes matching the expression host

  • Expressing at lower temperatures (16-20°C) with reduced inducer concentration

  • Adding solubility-enhancing fusion tags (SUMO, Thioredoxin, or MBP) rather than just His-tags

  • Including solubility enhancers (0.5-1% Triton X-100, 5-10% glycerol) in lysis buffers

• Proteolytic degradation: EF-Ts can be susceptible to proteolysis during expression and purification. Countermeasures include:

  • Using protease-deficient expression strains

  • Adding a comprehensive protease inhibitor cocktail to all buffers

  • Maintaining samples at 4°C throughout purification

  • Minimizing processing time between purification steps

• Loss of activity: EF-Ts may lose nucleotide exchange activity during purification or storage due to oxidation or denaturation. Solutions include:

  • Adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to all buffers

  • Including 10-50% glycerol in storage buffers

  • Avoiding repeated freeze-thaw cycles by preparing small aliquots

  • Validating activity immediately after purification and periodically during storage

Implementing these strategies can significantly improve the yield and quality of recombinant B. multivorans EF-Ts preparations for subsequent functional and structural studies.

How can researchers verify the specificity of interactions between B. multivorans EF-Ts and EF-Tu?

Verifying the specificity of B. multivorans EF-Ts and EF-Tu interactions requires a multi-technique approach to establish both physical binding and functional consequences. Researchers should implement the following complementary methods:

• Biochemical Interaction Assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics (ka, kd) and affinity (KD)

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

  • Pull-down assays with controlled negative controls (e.g., unrelated proteins) to confirm specificity

• Functional Verification:

  • Nucleotide exchange assays measuring GDP/GTP dissociation rates from EF-Tu with wild-type EF-Ts versus mutant variants

  • Competition assays where homologous EF-Ts proteins from related species compete for binding to B. multivorans EF-Tu

• Structural Evidence:

  • Hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

  • Cross-linking coupled with mass spectrometry to identify proximity relationships between specific residues

• Specificity Controls:

  • Testing EF-Ts activity with EF-Tu proteins from increasingly distant bacterial species to establish taxonomic specificity

  • Site-directed mutagenesis of predicted interface residues to demonstrate sequence-specific interactions

Taken together, these approaches provide robust evidence for genuine and specific EF-Ts:EF-Tu interactions while ruling out non-specific binding artifacts that could confound interpretation of experimental results .

What analytical techniques are most effective for detecting structural changes in EF-Ts during nucleotide exchange?

Multiple complementary analytical techniques can effectively detect the structural changes in EF-Ts during the nucleotide exchange process. The most informative approaches include:

By integrating data from these complementary techniques, researchers can construct a comprehensive model of the dynamic structural changes that enable EF-Ts to catalyze nucleotide exchange on EF-Tu with such remarkable efficiency.