Recombinant Pseudomonas putida Elongation factor Ts (tsf)

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

Elongation factor Ts (tsf) is a protein that plays a critical role in the protein translation process in Pseudomonas putida. Its primary function is to catalyze the regeneration of the EF-Tu-GDP complex during protein synthesis . More specifically, tsf is involved in the protein translation elongation phase, where it facilitates the exchange of GDP for GTP on EF-Tu, enabling EF-Tu to bind to aminoacyl-tRNA and continue the translation cycle.

Beyond its canonical role in translation, research has demonstrated that tsf contributes significantly to organic solvent tolerance mechanisms in P. putida, making it relevant for biotechnological applications in harsh chemical environments .

What cloning and expression strategies have been successful for studying P. putida tsf gene?

Research has demonstrated effective methodologies for cloning and expressing the P. putida tsf gene:

• Template Selection: Chromosomal DNA from P. putida strains serves as an appropriate template for PCR amplification of the tsf gene (GenBank accession number: CAK16908) .

• Vector Selection and Cloning: The cis-repressed pQE-80L vector has been successfully used for expression studies. The procedure involves restriction enzyme digestion of both the PCR product and vector, followed by ligation to create recombinant plasmids .

• Transformation and Verification: Transformation into E. coli JM109 using heat shock methods, followed by verification through double-enzyme cleavage to confirm successful cloning .

• Expression Conditions: Induction with 1 mM IPTG has been effective for protein expression, with subsequent SDS-PAGE analysis confirming the presence of the recombinant protein .

This methodology has enabled researchers to study the functional properties of tsf, particularly its role in organic solvent tolerance mechanisms.

How can researchers evaluate the effect of tsf expression on organic solvent tolerance?

Based on published methodologies, the following experimental approaches have proven effective for evaluating tsf's impact on organic solvent tolerance:

• Liquid Culture Growth Assays:

  • Transform E. coli JM109 with the recombinant plasmid containing the tsf gene or empty vector control

  • Culture cells at 37°C until reaching early exponential phase (OD₆₆₀ of approximately 0.2)

  • Add organic solvent (e.g., cyclohexane at 4% v/v) and continue incubation

  • Monitor growth by measuring optical density at regular intervals

  • Compare growth curves between tsf-expressing strains and controls

• Colony Formation Efficiency Tests:

  • Prepare serial dilutions of bacterial cultures (10⁷ to 10³ cells)

  • Spot dilutions on agar plates

  • Overlay with an organic solvent such as decalin

  • Incubate and assess colony formation efficiency

  • Compare results between tsf-expressing strains and controls

When evaluated using these methods, E. coli expressing P. putida tsf showed increased growth in the presence of 4% cyclohexane compared to control strains, with an OD₆₆₀ increase of 0.25, demonstrating the contribution of tsf to organic solvent tolerance .

How does tsf expression compare to other proteins involved in P. putida's organic solvent tolerance mechanisms?

Comparative analysis of proteins involved in P. putida's organic solvent tolerance reveals a hierarchy of effectiveness:

Two-dimensional gel electrophoresis of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane identified five high-abundance protein spots with over 60% intensity discrepancies under different solvent conditions. These included arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase (mmsB), protein chain elongation factor EF-Ts (tsf), and isochorismatase superfamily hydrolase (PSEEN0851) .

While tsf demonstrates a significant contribution to organic solvent tolerance, the data indicates that mmsB exhibits the most prominent effect among the three genes that were functionally characterized .

What molecular mechanisms may explain tsf's contribution to organic solvent tolerance?

The molecular mechanisms underlying tsf's contribution to organic solvent tolerance remain incompletely characterized, but several hypotheses can be proposed based on experimental evidence:

The observation that E. coli expressing tsf shows increased solvent tolerance provides strong evidence for its functional role in this process, though the precise molecular interactions remain to be fully elucidated .

What are the optimal conditions for production and storage of recombinant P. putida Elongation factor Ts?

Based on commercially available recombinant P. putida Elongation factor Ts specifications, the following conditions are recommended:

• Expression System: E. coli has been successfully employed as a host for recombinant tsf production .

• Purification: Purification protocols should aim for >85% purity as assessed by SDS-PAGE .

• Reconstitution: The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage Considerations:

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

  • Store at -20°C for regular use, or -80°C for extended storage

  • Avoid repeated freeze-thaw cycles

  • Working aliquots may be stored at 4°C for up to one week

• Shelf Life:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

These parameters ensure optimal stability and activity of the recombinant protein for research applications.

How might tsf be integrated into metabolic engineering strategies for biomanufacturing in organic solvent environments?

The role of tsf in organic solvent tolerance presents several opportunities for metabolic engineering applications:

• Enhanced Host Strain Development: Co-expression of tsf with other solvent tolerance genes (such as mmsB and PSEEN0851) could create robust production hosts capable of functioning in biphasic systems where organic solvents are used for product extraction .

• Metabolic Pathway Stabilization: In P. putida strain designs for lignin-derived feedstocks like p-coumarate, stabilizing protein synthesis machinery through tsf expression could help maintain metabolic pathway function under stressful conditions .

• Genome-Scale Design Integration: Tsf could be incorporated into genome-scale metabolic models (GSMMs) used to identify gene deletion sets for growth coupling and improved strain performance .

• Cross-Feeding Strategy Enhancement: Based on strategies employed for fumarase hydratase in P. putida, controlled expression of tsf could help overcome limitations in metabolic designs requiring multiple gene edits .

• Bioconversion Process Optimization: For processes converting aromatic compounds in organic solvent environments, tsf expression could help stabilize the translation machinery, maintaining the expression of key catalytic enzymes.

A methodical approach would involve integrating tsf expression with other tolerance mechanisms while carefully optimizing expression levels to balance the benefits of solvent tolerance against potential metabolic burdens.

What techniques are available for structural characterization of P. putida tsf and its interactions with organic solvents?

Several advanced techniques can be employed to characterize the structure of P. putida tsf and its interactions with organic solvents:

• X-ray Crystallography: To determine the three-dimensional structure of tsf at atomic resolution, potentially revealing solvent-interacting domains.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For studying the dynamic interactions between tsf and organic solvents in solution, particularly useful for identifying flexible regions that may be involved in solvent adaptation.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To identify regions of tsf that show altered solvent accessibility in the presence of organic solvents, providing insights into conformational changes.

• Molecular Dynamics Simulations: To model the behavior of tsf in the presence of various organic solvents, predicting potential interaction sites and conformational changes.

• Site-Directed Mutagenesis Combined with Functional Assays: Systematic mutation of potential solvent-interacting residues followed by organic solvent tolerance testing to validate the functional importance of specific amino acids.

These approaches, used in combination, would provide comprehensive insights into the structural basis of tsf's contribution to organic solvent tolerance, potentially enabling rational design of enhanced variants.

How can research on P. putida tsf contribute to our understanding of stringent response and stress adaptation in bacteria?

Research on P. putida tsf can provide valuable insights into broader stress adaptation mechanisms, particularly in relation to the stringent response:

• Integration with ppGpp-Mediated Regulation: Studies have highlighted the pivotal role of ppGpp-mediated stringent response in orchestrating metabolic and transcriptional regulation when P. putida cells are exposed to glucose or ammonia starvation . Investigation of how tsf expression and function are influenced by ppGpp levels could reveal connections between translation regulation and the stringent response.

• Transcriptional Coordination: Gene expression analyses have shown that rpoS (RNA polymerase sigma S factor) and relA (ppGpp synthase) are upregulated in P. putida under stress conditions, alongside changes in spoT (ppGpp hydrolase) expression . Understanding how tsf fits into this regulatory network could illuminate the coordination between translation machinery and transcriptional responses.

• Metabolic Adaptation Mechanisms: P. putida accumulates storage compounds like 3-hydroxydecanoic acid (C10) and 3-hydroxydodecanoic acid (C12) under limiting conditions . Research could explore whether tsf-mediated translation regulation influences the metabolic shifts that lead to these adaptations.

• Cross-Stress Protection: Investigating whether tsf-mediated solvent tolerance provides cross-protection against other stresses (nutrient limitation, oxidative stress) would contribute to understanding integrated stress response networks in bacteria.

This research direction has potential implications for industrial biotechnology, particularly for developing robust microbial cell factories capable of functioning under multiple stress conditions.