Recombinant Pseudomonas putida Elongation factor Ts (tsf)

What is Elongation Factor Ts (tsf) in Pseudomonas putida and what is its primary function?

Elongation Factor Ts (tsf) is a protein involved in the elongation phase of protein synthesis in Pseudomonas putida. It functions as a nucleotide exchange factor that catalyzes the regeneration of active EF-Tu·GTP from inactive EF-Tu·GDP. The tsf gene in P. putida has been identified with GenBank accession number CAK16908 . It plays a crucial role in maintaining efficient translation by ensuring continuous availability of active EF-Tu molecules.

The significance of tsf extends beyond basic translation, as studies have demonstrated that this protein contributes to organic solvent tolerance (OST) in P. putida. Recombinant expression of tsf can increase OST by 0.25 units as measured by OD660 in experimental systems . This indicates that tsf has multifunctional properties that may be exploited in various research applications focused on stress tolerance mechanisms.

How is the tsf gene typically cloned from Pseudomonas putida?

Cloning the tsf gene from P. putida involves a systematic molecular biology approach. Based on established protocols, researchers typically begin by isolating chromosomal DNA from P. putida strains such as P. putida JUCS, which serves as the template for PCR amplification . The gene is then amplified using specific primers designed to incorporate appropriate restriction enzyme sites for subsequent directional cloning.

The following primers have been successfully used for PCR amplification of the tsf gene:

In this design, the forward primer includes a BamHI restriction site (GGATCC) while the reverse primer incorporates a HindIII site (AAGCTT), facilitating directional cloning into expression vectors such as pQE-80L . Following PCR amplification, both the PCR product and the chosen vector are digested with the appropriate restriction enzymes, ligated, and transformed into a suitable host such as E. coli JM109 using the heat shock method. Verification of recombinant plasmids typically involves double-enzyme restriction analysis and confirmation of protein expression via SDS-PAGE following IPTG induction (1 mM concentration) .

What relationship exists between Elongation Factor Ts (tsf) and other elongation factors in Pseudomonas?

Elongation Factor Ts functions in close coordination with Elongation Factor Tu (EF-Tu) in P. putida. While tsf serves as the guanine nucleotide exchange factor, EF-Tu functions as the carrier of aminoacyl-tRNAs to the ribosome during translation elongation . Research has demonstrated that these elongation factors are subject to distinct regulatory mechanisms and post-translational modifications.

Notably, EF-Tu has been identified as a substrate for Pseudomonas prolyl-hydroxylase domain-containing protein (PPHD), which catalyzes trans-prolyl-4-hydroxylation at Pro54 in the switch I loop of EF-Tu . This modification has been confirmed through mass spectrometric studies and amino acid analyses. In contrast, tsf has not been reported to undergo similar modifications, suggesting differential regulation of these functionally related proteins. The interaction between tsf and EF-Tu is critical for maintaining translation efficiency, particularly under stress conditions, indicating a regulatory network that coordinates the activity of these elongation factors in response to environmental changes.

How does recombinant tsf expression contribute to organic solvent tolerance mechanisms in P. putida?

The contribution of tsf to organic solvent tolerance (OST) in P. putida represents a significant area of research interest. Experimental studies have demonstrated that expression of the tsf gene contributes to enhanced OST, resulting in a measurable increase of 0.25 in OD660 in recombinant systems . This finding suggests that tsf plays a role beyond protein synthesis in cellular adaptation to toxic compounds.

The mechanism likely involves integration with the broader stress response network in P. putida. Analysis of P. putida DOT-T1E, a strain known for its exceptional tolerance to toluene and other toxic hydrocarbons, has identified several proteins involved in solvent tolerance, including stress-related proteins and factors involved in energy metabolism . While the exact mechanism of tsf contribution to OST remains to be fully elucidated, it potentially functions by:

• Maintaining translation efficiency under solvent stress conditions

• Participating in the broader cellular stress response network

• Contributing to metabolic adaptations required for energy-intensive solvent tolerance mechanisms

Further research using knockout mutants and comparative proteomic analysis would provide deeper insights into the specific role of tsf in the complex mechanisms of solvent tolerance in P. putida.

What methodological approaches are most effective for studying the function of recombinant tsf in heterologous expression systems?

Investigating recombinant tsf function in heterologous systems requires a multi-faceted methodological approach. Based on established protocols, the following methods have proven effective:

• Expression vector selection and optimization: The cis-repressed pQE-80L vector has been successfully used for recombinant tsf expression . This system allows for tight regulation of expression and produces sufficient protein yields for functional studies. Expression should be verified using SDS-PAGE following IPTG induction.

• Functional complementation assays: To assess the biological activity of recombinant tsf, researchers can employ complementation assays in which the heterologously expressed protein rescues phenotypic defects in strains lacking functional tsf. This approach can reveal whether the recombinant protein maintains its native functionality.

• Stress tolerance assessment: Given the role of tsf in organic solvent tolerance, exposing recombinant strains to increasing concentrations of organic solvents (such as toluene) and measuring growth parameters provides valuable functional data . The OD660 measurement has been established as a reliable indicator of OST in these systems.

• Protein-protein interaction studies: Co-immunoprecipitation or bacterial two-hybrid systems can be employed to investigate interactions between recombinant tsf and its molecular partners, particularly EF-Tu, providing insights into functional mechanisms.

• Comparative proteomic analysis: This approach can identify changes in the proteome resulting from tsf expression, revealing potential downstream targets or pathways affected by tsf activity .

These methodologies, when combined, provide a comprehensive framework for investigating the functional properties of recombinant tsf in heterologous expression systems.

How can researchers assess the impact of post-translational modifications on elongation factor function in Pseudomonas species?

Post-translational modifications (PTMs) of elongation factors significantly influence their function in Pseudomonas species. While EF-Tu is known to undergo prolyl hydroxylation at Pro54 in the switch I loop , the PTM profile of tsf is less characterized. To assess the impact of these modifications, researchers should employ a multi-analytical approach:

• Mass spectrometric analysis: High-resolution mass spectrometry has successfully identified the +16-Da mass shift indicative of prolyl hydroxylation in EF-Tu . This technique can be applied to purified recombinant tsf to identify potential modifications. Both MALDI-TOF MS and LC-MS/MS approaches have proven effective for identifying PTMs in elongation factors.

• Amino acid analysis: Following protein hydrolysis, amino acid analysis can confirm the nature of modifications. This method has been used to verify trans-prolyl-4-hydroxylation of EF-Tu in P. putida .

• Site-directed mutagenesis: Generating variants with mutations at potential modification sites allows for functional assessment of specific PTMs. By comparing the activity of wild-type and mutant proteins, researchers can determine the functional significance of identified modifications.

• Structural studies: Analysis of protein complexes, such as the PPHD:EF-Tu complex , provides valuable insights into conformational changes resulting from PTMs. Similar approaches could be applied to tsf to understand how modifications affect its structure and function.

• In vivo modification studies: Comparative analysis of proteins isolated from wild-type strains versus modification enzyme knockout mutants (such as PPHD insertional mutants) can reveal the occurrence and significance of PTMs under physiological conditions .

These approaches collectively provide a comprehensive strategy for characterizing the PTM landscape of elongation factors and understanding their functional implications.

What challenges exist in studying the role of tsf in Pseudomonas genetic regulatory networks?

Investigating the role of tsf within P. putida genetic regulatory networks presents several significant challenges:

• Functional redundancy: Translation factors often display partial functional redundancy, complicating the interpretation of knockout phenotypes. This necessitates careful design of conditional mutants or partial depletion systems to study tsf function without lethal consequences.

• Integration with multiple stress response pathways: Evidence suggests that tsf contributes to organic solvent tolerance , but determining its precise role within the complex network of stress response mechanisms remains challenging. Two-dimensional gel analyses of proteins extracted from P. putida under stress conditions have revealed that numerous proteins are differentially expressed , making it difficult to isolate tsf-specific effects.

• Temporal dynamics of expression: The dynamics of tsf expression in response to environmental stimuli may involve complex feedback mechanisms. Existing research on regulatory networks in P. putida, such as the TOL network with its negative and positive feedback loops , suggests that gene expression timing can dramatically alter circuit function. Similar dynamics may affect tsf regulation but remain largely unexplored.

• Technical limitations in detecting protein-protein interactions: Identifying all interaction partners of tsf requires sensitive detection methods. While mass spectrometry has successfully identified EF-Tu as a binding partner for related proteins , comprehensive interaction mapping for tsf remains challenging.

• Strain-specific variations: Different P. putida strains may exhibit variations in tsf sequence and regulation. For instance, studies on organic solvent tolerance have employed specific strains such as P. putida JUCS and P. putida DOT-T1E , potentially limiting the generalizability of findings.

Addressing these challenges requires integrated approaches combining genetics, proteomics, and systems biology to unravel the complex role of tsf in P. putida regulatory networks.

How can synthetic biology approaches be used to engineer enhanced tsf functionality in Pseudomonas putida?

Synthetic biology offers powerful approaches for engineering enhanced tsf functionality in P. putida, particularly for applications requiring increased stress tolerance. The following strategies have shown promise:

• Promoter engineering: Modifying the regulatory elements controlling tsf expression can significantly alter its functionality. Research on the TOL network in P. putida has demonstrated that altering the connectivity of transcription factors through different promoters can dramatically change signal specificity and output strength . Similar approaches could be applied to tsf, placing it under the control of promoters with different input/output transfer functions to optimize expression for specific conditions.

• Feedback loop modification: Creating synthetic feedback loops can enhance signal specificity. Studies on the XylR regulatory system in P. putida have shown that replacing natural negative feedback with positive feedback loops results in higher promoter activity and increased signal-to-background ratio . Similar principles could be applied to tsf regulation to enhance its expression under specific stress conditions.

• Domain engineering: Fusion of tsf with sensing domains or protein tags can create bifunctional proteins with enhanced or novel capabilities. This approach could be particularly useful for creating biosensors based on tsf functionality.

• Chromosomal integration strategies: Methods for random chromosomal insertions using transposons such as mini-Tn5 have been developed for P. putida . These techniques enable sampling of different genomic locations to identify optimal integration sites for enhanced tsf expression. The approach has been successfully used to generate libraries of approximately 200 distinct P. putida clones with different insertion sites .

• Codon optimization: Optimizing the codon usage of the tsf gene for expression in P. putida can significantly improve protein yields. This approach has been employed for heterologous gene expression in P. putida and could be applied to enhance tsf expression.

These synthetic biology strategies, informed by the understanding of P. putida regulatory networks, provide a framework for engineering enhanced tsf functionality for various biotechnological applications.

What are the optimal conditions for purifying recombinant P. putida tsf protein for functional studies?

Purification of recombinant P. putida tsf requires careful optimization to maintain protein functionality. Based on established protocols for related proteins, the following methodology is recommended:

• Expression system selection: The pQE-80L vector system with a 6×His tag has proven effective for tsf expression . This system facilitates purification while minimizing interference with protein function. Expression in E. coli JM109 provides good yields with minimal formation of inclusion bodies.

• Induction conditions: Optimal induction is achieved with 1 mM IPTG when cultures reach mid-logarithmic phase (OD600 of 0.6-0.8) . Lower induction temperatures (16-20°C) often improve the solubility of recombinant tsf, though longer induction times may be required.

• Cell lysis: Gentle lysis methods using lysozyme treatment (1 mg/ml, 30 minutes on ice) followed by sonication (6 × 10 seconds with 10-second cooling intervals) in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10 mM imidazole preserves protein structure and function.

• Affinity chromatography: Ni-NTA affinity chromatography represents the primary purification step, with binding in the presence of 10 mM imidazole to reduce non-specific binding, followed by washing with 20-30 mM imidazole and elution with 250 mM imidazole.

• Secondary purification: Size exclusion chromatography using Superdex 75 or 200 columns effectively removes aggregates and ensures homogeneity of the purified protein.

• Buffer optimization: Final buffer composition significantly affects tsf stability and activity. A buffer containing 20 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2, and 5% glycerol has been found suitable for maintaining elongation factor activity.

• Quality control: SDS-PAGE, Western blotting, and mass spectrometry should be employed to verify purity and integrity of the recombinant protein before functional studies.

These optimized conditions ensure high-quality recombinant tsf suitable for downstream functional and structural analyses.

How can researchers effectively assess the interaction between recombinant tsf and EF-Tu in experimental systems?

Investigating the interaction between recombinant tsf and EF-Tu requires specialized techniques that capture both physical associations and functional consequences. The following methodological approach is recommended:

• Co-purification assays: Tandem affinity purification using differentially tagged tsf and EF-Tu can effectively isolate the protein complex. This approach has been successfully used to identify EF-Tu as a binding partner for related proteins in P. putida .

• Surface plasmon resonance (SPR): SPR allows for real-time monitoring of protein-protein interactions and determination of binding kinetics. For tsf-EF-Tu interactions, immobilizing one partner on the sensor chip and flowing the other protein at various concentrations provides quantitative binding parameters.

• Isothermal titration calorimetry (ITC): ITC measures the thermodynamic parameters of binding, providing insights into the energetics of tsf-EF-Tu interactions under various conditions, including the influence of nucleotides (GDP/GTP) on complex formation.

• Nucleotide exchange assays: Since a primary function of tsf is to catalyze nucleotide exchange on EF-Tu, measuring the rate of GDP dissociation or GTP binding to EF-Tu in the presence of varying concentrations of tsf provides functional evidence of interaction. This can be accomplished using fluorescently labeled nucleotides or radioactive tracers.

• Structural studies: The structure of the EF-Tu:EF-Ts complex reveals important conformational changes that occur upon binding. Similar approaches, including crystallography or cryo-electron microscopy, could be applied to the P. putida proteins. Previous studies have successfully determined structures of related complexes, such as the PPHD:EF-Tu complex .

• In vivo FRET assays: Fusing fluorescent proteins to tsf and EF-Tu allows for monitoring their interaction in living cells using Förster resonance energy transfer (FRET). This approach provides insights into the spatial and temporal dynamics of the interaction under physiological conditions.

These complementary approaches provide a comprehensive assessment of both the physical and functional aspects of the tsf-EF-Tu interaction, critical for understanding the role of these proteins in translation and stress response.