Recombinant Yersinia pseudotuberculosis serotype O:3 Elongation factor Ts (tsf)

What is the role of Elongation Factor Ts (tsf) in Yersinia pseudotuberculosis protein synthesis?

Elongation Factor Ts (EF-Ts) plays a crucial role in bacterial protein synthesis by functioning as a guanine nucleotide exchange factor for Elongation Factor Tu (EF-Tu). In Y. pseudotuberculosis, EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu, regenerating active EF-Tu-GTP complexes that deliver aminoacyl-tRNAs to the ribosome during the elongation phase of translation.

While studying EF-Ts function, it's important to consider its interaction with the bacterial translation machinery in the context of Y. pseudotuberculosis growth dynamics. Research shows that Y. pseudotuberculosis undergoes growth arrest during expression of virulence factors like the type-III secretion system (T3SS), which is associated with altered ribosomal protein expression . This growth modulation likely affects translation factors including EF-Ts, suggesting a potential interplay between virulence mechanisms and protein synthesis machinery.

How does Y. pseudotuberculosis serotype O:3 differ from other serotypes in terms of pathogenesis and protein expression?

When designing experiments to study EF-Ts in serotype O:3, researchers should account for these distinguishing virulence factors. For instance, CNFy toxin production might affect metabolism and protein synthesis requirements, thereby influencing EF-Ts expression or function. Comparative studies between serotypes can reveal whether EF-Ts exhibits structural or functional adaptations specific to O:3 strains.

What conservation patterns exist for the tsf gene across Yersinia species?

The tsf gene shows high conservation across Yersinia species, reflecting its essential role in bacterial protein synthesis. Comparative genomic analyses similar to those performed for IscR binding sites (which showed over 93% conservation between Y. pseudotuberculosis and Y. pestis ) suggest that core metabolic genes like tsf maintain strong sequence conservation.

To analyze tsf conservation:

• Perform multiple sequence alignment of tsf nucleotide and amino acid sequences from different Yersinia species and strains

• Calculate sequence identity percentages and identify conserved domains

• Construct phylogenetic trees to visualize evolutionary relationships

• Map any variable regions to the protein structure to assess functional implications

Despite high conservation in coding regions, regulatory elements of tsf may show greater variation, potentially reflecting adaptation to different ecological niches and host environments.

What are the optimal expression systems for producing recombinant Y. pseudotuberculosis EF-Ts?

The optimal expression system for recombinant Y. pseudotuberculosis EF-Ts depends on research objectives, but E. coli-based systems typically offer the best balance of yield and functionality. When designing an expression strategy, consider:

• Vector selection: pET vectors with T7 promoters provide high-level inducible expression

• Host strain considerations:

  • BL21(DE3) for standard expression

  • Rosetta strains if Y. pseudotuberculosis codon usage differs significantly from E. coli

  • Arctic Express strains for enhanced protein folding at lower temperatures

• Expression conditions optimization:

For studying interactions with Y. pseudotuberculosis-specific partners, consider co-expression strategies incorporating bacterial chaperones or binding partners. The methodological approaches used for Yop-TEM translational fusions provide a framework that can be adapted for EF-Ts expression, particularly regarding promoter selection and verification of protein production.

What purification strategy yields the highest activity for recombinant Y. pseudotuberculosis EF-Ts?

A multi-step purification strategy is recommended to obtain highly active recombinant EF-Ts from Y. pseudotuberculosis:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tagged EF-Ts

  • Buffer optimization critical: typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5-10% glycerol

  • Include 1-5 mM β-mercaptoethanol to maintain reduced cysteines

  • Elute with imidazole gradient (50-300 mM)

• Intermediate purification: Ion exchange chromatography

  • Based on theoretical pI calculation for Y. pseudotuberculosis EF-Ts

  • Typically anion exchange (Q-Sepharose) at pH 8.0

• Polishing step: Size exclusion chromatography

  • Removes aggregates and provides buffer exchange

  • Superdex 75 or 200 columns appropriate for EF-Ts

• Activity preservation:

  • Include 1-2 mM DTT in final buffer

  • Add 10% glycerol for freeze storage

  • Flash-freeze in liquid nitrogen and store at -80°C

Assessment of purity should include SDS-PAGE and western blotting similar to methods used for verification of Yop-TEM fusion proteins . Activity assays should measure GDP/GTP exchange rates with cognate Y. pseudotuberculosis EF-Tu if available, or with E. coli EF-Tu as a surrogate.

How can researchers ensure proper folding of recombinant Y. pseudotuberculosis EF-Ts?

Ensuring proper folding of recombinant Y. pseudotuberculosis EF-Ts requires attention to both expression conditions and post-purification analysis:

• Expression optimization:

  • Lower temperatures (16-20°C) slow protein synthesis, allowing more time for folding

  • Co-expression with chaperones (GroEL/ES, DnaK/J systems) facilitates folding

  • Use of E. coli strains with enhanced disulfide bond formation capabilities if relevant

• Folding assessment methods:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure content

  • Fluorescence spectroscopy to assess tertiary structure through tryptophan fluorescence

  • Thermal shift assays to determine stability and proper folding

  • Limited proteolysis to probe for well-structured domains

• Functional verification:

  • Nucleotide exchange assay with EF-Tu (measuring GDP release or GTP binding)

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify binding to EF-Tu

Monitoring protein behavior during expression using methods similar to those employed for tracking Y. pseudotuberculosis T3SS effects on growth can provide insights into potential toxicity or folding issues of recombinant EF-Ts. Correlations between growth patterns and protein yield can guide optimization strategies.

What methods can effectively measure the nucleotide exchange activity of Y. pseudotuberculosis EF-Ts?

Several complementary approaches can accurately measure the nucleotide exchange activity of Y. pseudotuberculosis EF-Ts:

• Direct measurement of nucleotide exchange rates:

  • Fluorescent nucleotide analogs (mant-GDP/GTP) to monitor binding/release kinetics

  • Radiolabeled nucleotides ([³H]GDP/[³⁵S]GTPγS) for filter-binding assays

  • Stopped-flow kinetics to determine rate constants for association/dissociation

• Indirect activity measurements:

  • Coupled enzymatic assays linking GDP/GTP exchange to NADH oxidation

  • Measurement of inorganic phosphate release using colorimetric assays

  • Thermal shift assays to monitor nucleotide-dependent stability changes

• Experimental design considerations:

When interpreting results, consider that Y. pseudotuberculosis growth conditions significantly affect protein synthesis machinery . Therefore, recombinant EF-Ts activity should be assessed under conditions mimicking different bacterial growth states, including those representing T3SS expression.

How does the interaction between EF-Ts and EF-Tu differ in Y. pseudotuberculosis compared to model organisms?

• Structural comparison approaches:

  • Homology modeling of Y. pseudotuberculosis EF-Ts and EF-Tu based on solved structures

  • Identification of non-conserved residues at the interaction interface

  • Molecular dynamics simulations to predict functional consequences of sequence variations

• Binding kinetics analysis:

  • Surface plasmon resonance to determine association/dissociation rates

  • Isothermal titration calorimetry for thermodynamic parameters

  • Analytical ultracentrifugation to characterize complex formation

• Mutagenesis studies:

  • Site-directed mutagenesis of Y. pseudotuberculosis-specific residues

  • Reciprocal mutations in EF-Ts from model organisms

  • Activity assays to correlate structural differences with functional consequences

The methodology established for tracking translocation of Yersinia effector proteins offers principles that can be adapted to study EF-Ts:EF-Tu interaction dynamics, particularly regarding the creation of fusion proteins for detection and visualization of these interactions in bacterial systems.

How does iron limitation affect EF-Ts expression and function in Y. pseudotuberculosis?

Iron limitation significantly impacts Y. pseudotuberculosis physiology and virulence gene expression, with potential consequences for EF-Ts expression and function:

• Expression analysis under iron limitation:

  • qRT-PCR to measure tsf transcript levels

  • Western blotting to quantify EF-Ts protein

  • Reporter constructs (similar to the S10-GFP reporter ) to visualize tsf expression patterns

• Iron-dependent functional changes:

  • Nucleotide exchange assays under varying iron concentrations

  • Assessment of EF-Ts stability using thermal denaturation

  • Potential post-translational modifications in low-iron conditions

Iron starvation triggers IscR-mediated regulation of multiple pathways in Y. pseudotuberculosis , and translation machinery components may be affected as part of this response. While EF-Ts itself may not contain iron-sulfur clusters, its expression and activity could be indirectly regulated through iron-responsive pathways, especially considering the observed relationship between T3SS expression (which is IscR-regulated) and altered ribosomal protein expression .

What is the relationship between Y. pseudotuberculosis Type III Secretion System expression and EF-Ts function?

The relationship between Y. pseudotuberculosis T3SS expression and EF-Ts function represents an important intersection between virulence and core cellular processes:

• Growth modulation effects:
  Recent research demonstrates that T3SS expression in Y. pseudotuberculosis induces growth arrest and alters ribosomal protein expression . Since EF-Ts is integral to the protein synthesis machinery, its function is likely affected during T3SS-induced growth modulation. Specifically, the decreased expression of ribosomal proteins like S10 during T3SS activity suggests coordinated downregulation of translation machinery, potentially including EF-Ts.

• Experimental approaches to study this relationship:

  • Transcriptomic analysis comparing tsf expression levels with and without T3SS induction

  • Reporter fusion constructs to visualize EF-Ts expression patterns during T3SS activity

  • Measurement of nucleotide exchange rates in cell extracts from T3SS-expressing bacteria

  • Assessment of translation efficiency during T3SS expression

• Potential regulatory mechanisms:

  • Direct regulation through shared transcription factors

  • Indirect effects via altered metabolic state during virulence expression

  • Resource allocation between virulence expression and translation machinery

The methods developed for detecting Yop translocation could be adapted to investigate whether EF-Ts function is altered in cells that have been injected with T3SS effectors, providing insights into host-pathogen interactions at the translational level.

How does antibiotic tolerance in Y. pseudotuberculosis relate to EF-Ts expression and activity?

Y. pseudotuberculosis exhibits altered antibiotic susceptibility during virulence factor expression, with potential connections to EF-Ts function:

• Observed antibiotic tolerance phenomena:
  High levels of T3SS expression in Y. pseudotuberculosis promote decreased susceptibility to gentamicin, a ribosome-targeting antibiotic . Since gentamicin targets the ribosome, and EF-Ts is integral to the translation machinery, alterations in EF-Ts expression or activity could contribute to this antibiotic tolerance mechanism.

• Experimental approaches to investigate EF-Ts involvement:

  • Quantification of EF-Ts levels in antibiotic-tolerant vs. susceptible populations

  • Creation of EF-Ts overexpression and depletion strains to test antibiotic susceptibility

  • Site-directed mutagenesis of EF-Ts to identify residues important for antibiotic tolerance

  • Ribosome binding assays with and without EF-Ts under antibiotic pressure

• Potential mechanisms connecting EF-Ts to antibiotic tolerance:

  • Altered translation kinetics affecting antibiotic binding to ribosomes

  • Structural changes in the translation machinery

  • Compensatory responses to ribosome-targeting antibiotics

Research shows that bacterial cells surviving gentamicin treatment have heightened T3SS expression and lower S10 levels , suggesting a coordinated response that may include alterations in EF-Ts function or abundance.

What role might EF-Ts play during Y. pseudotuberculosis infection of mesenteric lymph nodes?

EF-Ts may serve specialized functions during Y. pseudotuberculosis infection of mesenteric lymph nodes (MLNs), a key site of pathogenesis:

• Infection microenvironment considerations:
  Y. pseudotuberculosis forms extracellular microcolonies within pyogranulomas in MLNs . This unique growth environment likely imposes specific demands on the bacterial translation machinery, including EF-Ts. The bacteria encounter neutrophils, inflammatory monocytes, and lymphocytes , creating stress conditions that may require adaptive responses in translation.

• Investigation approaches:

  • In vivo expression analysis of tsf during different stages of MLN infection

  • Comparison of tsf expression between bacteria in different microenvironments within MLNs

  • Creation of tsf reporter strains to visualize expression patterns during infection

  • Competitive infection experiments with tsf mutants vs. wild-type bacteria

• Potential specialized functions:

  • Adaptation of translation to nutrient limitation within pyogranulomas

  • Response to host immune factors targeting bacterial protein synthesis

  • Coordination with virulence factor expression during tissue colonization

Methods for tracking Yop translocation in infected tissues provide a framework that could be adapted to monitor EF-Ts activity or expression patterns during MLN infection, potentially revealing spatial and temporal dynamics of translation regulation during pathogenesis.

How can researchers determine the three-dimensional structure of Y. pseudotuberculosis EF-Ts and its complexes?

Determining the three-dimensional structure of Y. pseudotuberculosis EF-Ts requires a multi-technique approach:

• X-ray crystallography workflow:

  • High-purity protein preparation (>95% homogeneity)

  • Crystallization screening using commercial kits and specialized conditions

  • Data collection at synchrotron radiation facilities

  • Structure determination using molecular replacement with E. coli EF-Ts as a search model

  • Refinement and validation following crystallographic standards

• Cryo-electron microscopy approach:

  • Particularly valuable for EF-Ts:EF-Tu complexes

  • Sample preparation optimized for homogeneity and concentration

  • High-resolution data collection on modern cryo-EM instruments

  • Image processing and 3D reconstruction

  • Flexible fitting of domains if resolution is limited

• Complementary structural techniques:

  • Nuclear magnetic resonance (NMR) for dynamics studies

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Hydrogen-deuterium exchange mass spectrometry for protein-protein interfaces

These structural studies should consider potential conformational changes that might occur during T3SS expression or antibiotic stress, as Y. pseudotuberculosis shows significant physiological adaptations during these conditions .

What systems biology approaches can reveal the network of interactions involving EF-Ts in Y. pseudotuberculosis?

Systems biology approaches can provide comprehensive insights into EF-Ts function within the broader network of Y. pseudotuberculosis cellular processes:

• Protein-protein interaction mapping:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Bacterial two-hybrid screening

  • Proximity labeling methods (BioID, APEX)

  • Crosslinking mass spectrometry for transient interactions

• Multi-omics integration:

  • Correlation of transcriptomics, proteomics, and metabolomics data

  • Network analysis to identify pathways connected to EF-Ts function

  • Flux analysis to determine impact of EF-Ts perturbation on metabolic pathways

• Computational modeling:

  • Protein-protein docking to predict novel interaction partners

  • Genome-scale metabolic modeling incorporating translation

  • Agent-based models of bacterial population dynamics with varying EF-Ts activity

The observed connections between T3SS expression, ribosomal protein levels, and antibiotic tolerance suggest that EF-Ts functions within a complex adaptive network. Systems approaches can reveal how this translation factor contributes to the integration of virulence, metabolism, and stress responses in Y. pseudotuberculosis.

How do post-translational modifications affect EF-Ts function in Y. pseudotuberculosis during different growth phases?

Post-translational modifications (PTMs) of EF-Ts may serve as regulatory mechanisms in Y. pseudotuberculosis adapting to different growth phases and environmental conditions:

• Identification of PTMs:

  • Mass spectrometry-based proteomics focusing on EF-Ts

  • Enrichment strategies for phosphorylation, acetylation, and other modifications

  • Comparison of PTM profiles across growth phases and stress conditions

  • Site-specific antibodies for western blotting validation

• Functional consequences of PTMs:

  • Site-directed mutagenesis of modified residues

  • Activity assays comparing wild-type and PTM-mimicking variants

  • Structural analysis of how PTMs affect EF-Ts:EF-Tu interaction

  • In vivo studies with PTM-deficient mutants

• Regulation of PTMs:

  • Identification of enzymes responsible for adding/removing modifications

  • Environmental triggers affecting PTM patterns

  • Cross-talk with virulence regulation networks

Given that Y. pseudotuberculosis undergoes significant physiological adaptation during virulence expression , PTMs of translation factors like EF-Ts may serve as rapid response mechanisms to adjust protein synthesis capacity during transitions between growth and virulence states.