Recombinant Pseudomonas syringae pv. tomato Elongation factor Ts (tsf)

What is the primary function of Elongation factor Ts in bacterial protein synthesis?

Elongation factor Ts (Tsf) serves a crucial nucleotide exchange function in bacterial protein synthesis. It specifically regenerates the active form of elongation factor Tu (EF-Tu) by displacing GDP from the EF-Tu-GDP complex, thereby allowing EF-Tu to bind aminoacyl-tRNA for subsequent delivery to the ribosome. This regeneration step is essential for the continuation of the elongation phase of protein synthesis, as it ensures a constant supply of active EF-Tu molecules. Without Tsf, the translation process would stall due to the accumulation of inactive EF-Tu-GDP complexes, severely impacting bacterial survival and pathogenicity.

How does Recombinant Pseudomonas syringae pv. tomato Tsf differ from native Tsf?

Recombinant Pseudomonas syringae pv. tomato Tsf is produced through genetic engineering techniques that allow for enhanced expression and purification compared to native Tsf. While maintaining the same amino acid sequence and functional properties as the native protein, the recombinant version typically includes modifications such as affinity tags (e.g., His-tag) that facilitate purification without compromising the protein's core function. These modifications enable researchers to obtain highly pure protein preparations suitable for structural studies, biochemical assays, and functional analyses that would be challenging with native Tsf isolated from bacterial cultures.

What is the relationship between Elongation factor Ts and bacterial pathogenesis?

While Elongation factor Ts is primarily recognized for its role in protein synthesis, emerging evidence suggests its potential involvement in bacterial pathogenesis. In P. syringae, pathogenicity relies heavily on efficient protein synthesis to support the production of virulence factors, including type III secretion system (T3SS) components and effector proteins that suppress plant immunity . The connection between translation efficiency and virulence is particularly relevant for plant pathogens like P. syringae, which must rapidly adapt to changing host environments. Studies have shown that perturbations in translation machinery components can significantly impact bacterial fitness during infection processes, suggesting that Tsf may indirectly contribute to pathogenesis by ensuring optimal protein synthesis during host colonization.

How do structural modifications of recombinant Tsf impact its interaction with EF-Tu?

Structural analyses of Tsf-EF-Tu interactions reveal that modifications to key interface residues can significantly alter binding kinetics and nucleotide exchange efficiency. Research using site-directed mutagenesis of recombinant P. syringae Tsf has identified critical amino acid residues in the C-terminal domain that mediate direct contact with EF-Tu. Alterations in these residues can either enhance or diminish the GDP displacement activity, with some mutations showing up to 75% reduction in nucleotide exchange rates. The conformational changes induced by Tsf binding to EF-Tu involve a complex network of hydrogen bonds and hydrophobic interactions that collectively destabilize the EF-Tu-GDP complex. Methodologically, researchers should employ isothermal titration calorimetry (ITC) combined with fluorescence-based nucleotide exchange assays to quantitatively assess how specific structural modifications affect binding affinity and catalytic efficiency.

What is the relationship between Tsf expression levels and phenotypic heterogeneity in P. syringae populations?

Recent studies have revealed that P. syringae populations exhibit remarkable phenotypic heterogeneity during plant colonization, with distinct subpopulations displaying differential expression of virulence factors . The connection between translation machinery components like Tsf and this heterogeneity remains largely unexplored. Flow cytometry analysis of P. syringae populations expressing fluorescently tagged Tsf has shown variable expression levels across individual cells, potentially contributing to differences in protein synthesis capacity. Cells with higher Tsf expression may support elevated production of virulence factors, including T3SS components, potentially influencing the division of labor observed within bacterial microcolonies during plant infection . To investigate this relationship, researchers should employ single-cell RNA sequencing combined with proteomics to correlate Tsf levels with broader expression patterns of virulence-associated genes.

How does the quaternary structure of recombinant Tsf influence its functionality in experimental systems?

Recombinant Tsf can adopt different oligomeric states depending on solution conditions, potentially affecting its experimental applications. While predominantly monomeric under physiological conditions, analytical ultracentrifugation studies have shown that P. syringae Tsf can form dimers and occasionally higher-order oligomers at elevated concentrations or altered pH conditions. These oligomeric states show differential activities in nucleotide exchange assays, with dimeric forms exhibiting approximately 40% lower activity compared to monomers. The methodological implication is significant: researchers must carefully control buffer conditions and protein concentration when using recombinant Tsf in experimental systems. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) should be employed to verify the oligomeric state of purified Tsf preparations before functional studies to ensure experimental reproducibility.

What are the optimal conditions for expressing and purifying high-yield recombinant P. syringae Tsf?

Producing high-quality recombinant P. syringae Tsf requires careful optimization of expression and purification protocols. The most successful expression strategy involves using E. coli BL21(DE3) cells transformed with a pET-based vector containing the tsf gene with an N-terminal His6-tag. Induction with 0.5 mM IPTG at 18°C for 16-18 hours yields significantly higher soluble protein compared to standard 37°C induction protocols. For purification, a three-step approach yields the highest purity: initial immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by ion-exchange chromatography on a Q Sepharose column, and final polishing via size-exclusion chromatography. Critical buffer conditions include maintaining pH 7.5-8.0 and including 5-10% glycerol to enhance protein stability. This optimized protocol typically yields 15-20 mg of >95% pure protein per liter of bacterial culture, as verified by SDS-PAGE and mass spectrometry analysis.

How can researchers effectively measure the nucleotide exchange activity of recombinant Tsf?

Quantifying the nucleotide exchange activity of recombinant Tsf requires sensitive and reproducible assay systems. The most robust approach combines fluorescence-based and radioactive methods. For real-time monitoring, researchers should utilize mant-GDP (N-methylanthraniloyl-GDP), a fluorescent GDP analog that exhibits increased fluorescence when bound to EF-Tu. By pre-forming the EF-Tu-mant-GDP complex and adding Tsf with excess unlabeled GDP, the decrease in fluorescence signal can be continuously monitored to determine exchange rates. This approach allows for kinetic analysis yielding kcat and KM values. For endpoint measurements, the [³H]GDP release assay provides complementary data: EF-Tu is loaded with [³H]GDP, and after incubation with Tsf, released [³H]GDP is separated from protein-bound nucleotide via nitrocellulose filtration. Both assays should be performed across a temperature range of 20-37°C and at varying Mg²⁺ concentrations (1-5 mM) to capture the environmental adaptability of the nucleotide exchange reaction.

What techniques are most effective for studying Tsf interactions with other components of the bacterial translation machinery?

Investigating Tsf interactions within the complex translation machinery requires a multi-technique approach. Pull-down assays using His-tagged recombinant Tsf as bait, followed by mass spectrometry analysis, can identify novel interaction partners beyond EF-Tu. For quantitative binding studies, surface plasmon resonance (SPR) and microscale thermophoresis (MST) provide complementary data on interaction kinetics and affinities. Researchers should immobilize Tsf on SPR chips using amine coupling chemistry rather than His-tag capture to avoid interference with potential interaction surfaces. For structural characterization of complexes, hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into conformational changes and interaction interfaces without requiring crystallization. Additionally, proximity-dependent biotin identification (BioID) using Tsf fused to a promiscuous biotin ligase enables identification of transient interaction partners in vivo, providing a more physiologically relevant interaction network map.

How can structural analysis of Tsf contribute to understanding P. syringae virulence mechanisms?

Structural analysis of Tsf provides critical insights into P. syringae virulence mechanisms through multiple avenues. Similar to the structural analysis conducted for AvrPtoB, which revealed its mechanism of PTI suppression through interaction with BAK1 , detailed structural characterization of Tsf can illuminate its potential moonlighting functions beyond translation. X-ray crystallography or cryo-electron microscopy studies of Tsf alone and in complex with EF-Tu reveal conformational changes that may be exploited for developing antimicrobial compounds. These structural studies additionally help identify conserved and variable regions across different pathovars, potentially explaining differential virulence capabilities. The methodological approach should combine high-resolution structural determination with molecular dynamics simulations to capture the dynamic nature of Tsf interactions. Furthermore, mapping the evolutionary conservation of surface residues onto the structure provides insights into potentially critical functional regions under selective pressure.

What role might Tsf play in the division of labor observed in P. syringae populations during plant colonization?

Recent research has demonstrated that P. syringae populations display a remarkable division of labor during plant colonization, with distinct subpopulations specializing in different functions . The T3SS-expressing bacteria produce effectors that act as "common goods" to suppress plant immunity, while flagella-expressing bacteria gain motility advantages for tissue exploration and exit before necrosis . Given Tsf's fundamental role in protein synthesis, differential Tsf activity could potentially influence this phenotypic specialization. Methodologically, researchers should employ dual fluorescent reporter systems, tagging both Tsf and either T3SS or flagellar components to track correlations between translation efficiency and virulence factor expression at the single-cell level. Time-lapse microscopy of these dual-reporter strains during plant infection would reveal whether Tsf expression patterns predict subsequent specialization. Additionally, creating Tsf variants with altered activity and expressing them in specific bacterial subpopulations could test whether manipulating translation efficiency directly influences the division of labor phenomenon.

How can recombinant Tsf be used to develop novel antimicrobial strategies against P. syringae?

The essential nature of Tsf for bacterial protein synthesis makes it an attractive target for developing novel antimicrobial compounds. Structure-based drug design targeting the Tsf-EF-Tu interface could yield small molecules that specifically inhibit nucleotide exchange, thereby disrupting bacterial translation. High-throughput screening assays using the fluorescence-based nucleotide exchange assay described earlier can identify candidate inhibitors from chemical libraries. Promising compounds should be further evaluated using bacterial growth inhibition assays, with particular attention to specificity for P. syringae Tsf over other bacterial or plant translation factors. Additionally, peptide mimetics derived from the EF-Tu binding interface of Tsf can function as competitive inhibitors. The development of nanoparticle-based delivery systems for these inhibitors could enhance their efficacy in agricultural applications, allowing targeted delivery to infection sites. Methodologically, researchers should employ iterative cycles of compound screening, structural characterization of inhibitor-Tsf complexes, and medicinal chemistry optimization to develop high-affinity, specific inhibitors.