Recombinant Salmonella newport Elongation factor Ts (tsf)

What is Elongation factor Ts (EF-Ts) and how does it function in bacterial translation?

Elongation factor Ts (EF-Ts) is a protein that functions during translational elongation in protein synthesis. EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu, catalyzing the release of GDP from EF-Tu and allowing the formation of an EF-Tu-GTP complex that can bind aminoacyl-tRNA . This regeneration of active EF-Tu is critical for continuous protein synthesis.

The protein synthesis elongation process in bacteria is remarkably efficient, proceeding at a rate of 15-20 amino acids per second. EF-Ts plays an essential role in maintaining this speed while ensuring translation accuracy . In the bacterial elongation factor system, EF-Ts interacts specifically with EF-Tu, whereas in eukaryotes, the homologous factor eEF-1B (βγ) performs a similar function with eEF-1A.

How is the tsf gene structured and conserved in Salmonella Newport compared to other Salmonella serotypes?

The tsf gene in Salmonella Newport, like other bacterial species, is highly conserved due to its essential function in protein synthesis. While the search results don't specifically describe the structure of the tsf gene in Salmonella Newport, we can infer that as a core housekeeping gene, it likely maintains high sequence similarity across Salmonella serotypes.

Comparative analysis between different Salmonella serotypes shows that core genomic elements tend to be highly conserved, while virulence factors often show greater variation. Research on Salmonella virulence genotyping has demonstrated that genes associated with Salmonella Pathogenicity Islands (SPIs) are present in 95-100% of both environmental and clinical isolates . Though tsf is not directly mentioned in these comparative studies, as a core gene for protein synthesis, it likely follows similar conservation patterns as other housekeeping genes.

What expression systems are most effective for producing recombinant Salmonella Newport EF-Ts?

For recombinant expression of Salmonella Newport EF-Ts, several expression systems can be employed, with E. coli being the most common host organism. The methodological approach typically involves:

• PCR amplification of the tsf gene from Salmonella Newport genomic DNA

• Cloning into an appropriate expression vector (pET, pBAD, or pGEX systems)

• Transformation into an E. coli expression strain (BL21(DE3), Rosetta, or Arctic Express)

• Induction of protein expression under optimized conditions

The choice of expression system should consider factors such as protein solubility, yield requirements, and downstream applications. For structural studies, expression systems that allow isotopic labeling (15N, 13C) may be preferred. For functional studies, systems that preserve native protein folding and minimize aggregation are essential.

What methods are recommended for purifying recombinant Salmonella Newport EF-Ts?

Purification of recombinant EF-Ts typically employs a multi-step chromatography approach:

• Initial capture: Affinity chromatography using His-tag (IMAC), GST-tag, or MBP-tag depending on the expression construct

• Intermediate purification: Ion exchange chromatography to separate proteins based on charge differences

• Polishing: Size exclusion chromatography to achieve high purity and remove aggregates

For optimal results, researchers should consider:

• Maintaining reducing conditions throughout purification to prevent disulfide bond formation

• Using protease inhibitors to prevent degradation

• Optimizing buffer conditions (pH, salt concentration) to maintain protein stability

• Conducting purification at 4°C to minimize protein degradation

How might antimicrobial resistance in Salmonella Newport affect studies of translation factors including EF-Ts?

Salmonella Newport MDR-AmpC strains have demonstrated resistance to multiple antimicrobials, including extended-spectrum cephalosporins . This resistance is primarily mediated by the plasmid-borne blaCMY gene, which was found in all Newport MDR-AmpC isolates studied . While antimicrobial resistance genes primarily affect drug susceptibility, they may indirectly influence protein synthesis machinery.

When studying recombinant EF-Ts from resistant strains, researchers should consider:

What experimental approaches are most effective for studying interactions between EF-Ts and other translation factors in Salmonella Newport?

To study the interactions between EF-Ts and other translation factors in Salmonella Newport, several methodological approaches can be employed:

• Co-immunoprecipitation (Co-IP): Using antibodies against EF-Ts to pull down interaction partners

• Surface Plasmon Resonance (SPR): Measuring real-time binding kinetics between EF-Ts and EF-Tu

• Isothermal Titration Calorimetry (ITC): Determining thermodynamic parameters of protein-protein interactions

• Crosslinking mass spectrometry: Identifying interaction interfaces between proteins

• Yeast two-hybrid or bacterial two-hybrid systems: Screening for potential interaction partners

For functional characterization, in vitro translation assays using purified components can assess how EF-Ts influences translation efficiency and accuracy. These experiments should include controls with EF-Ts from susceptible Salmonella Newport strains to identify any functional differences associated with drug resistance.

How do genetic variations in the tsf gene correlate with Salmonella Newport virulence and antimicrobial resistance profiles?

While direct correlations between tsf gene variations and Salmonella Newport virulence have not been specifically documented in the search results, we can analyze this question by considering the relationship between core genes and virulence factors.

Salmonella Pathogenicity Islands (SPIs) are key determinants of virulence in Salmonella species. Studies have shown that SPI-associated genes are present in 95-100% of both environmental and clinical Salmonella isolates . In contrast, virulence genes like shdA and sopE were found to be lacking in a significant number of Salmonella Newport isolates compared to Salmonella Typhi .

For researchers investigating potential correlations between tsf gene variations and virulence, the following methodological approaches are recommended:

• Sequence comparative analysis of tsf genes from multiple isolates with defined virulence profiles

• Phylogenetic analysis to identify potential correlations between tsf variants and virulence markers

• Experimental verification using gene replacement or complementation studies

• Phenotypic characterization of strains with tsf variants in infection models

What methodological considerations are important when designing experiments to study the impact of recombinant EF-Ts on Salmonella Newport protein synthesis?

When designing experiments to study the impact of recombinant EF-Ts on Salmonella Newport protein synthesis, researchers should consider:

• Protein activity verification: Ensure that recombinant EF-Ts maintains its native activity after purification

• Concentration effects: Titrate EF-Ts concentrations to identify optimal levels for protein synthesis

• Interaction with other factors: Include purified EF-Tu and other translation components in the experimental design

• In vitro translation systems: Use reconstituted translation systems with defined components

• Ribosome preparation: Consider using ribosomes from the same Salmonella Newport strain for homologous system studies

The experimental design should include appropriate controls:

• Wild-type EF-Ts versus mutant variants

• Comparison with EF-Ts from drug-susceptible Salmonella Newport strains

• Controls with EF-Ts from E. coli or other bacteria to assess serotype-specific effects

How do horizontal gene transfer and recombination events affect the evolution of translation factors in Salmonella Newport?

Horizontal gene transfer (HGT) plays a significant role in Salmonella evolution, particularly for virulence and antimicrobial resistance genes. Research has shown that Salmonella can acquire mutations in mutS that result in "relaxation of the internal barriers that normally restrict homeologous recombination following the horizontal acquisition of foreign DNA" .

Research methodologies to study HGT effects on translation factors should include:

• Comparative genomic analysis of tsf genes across diverse Salmonella isolates

• Phylogenetic incongruence testing to identify potential recombination events

• Calculation of dN/dS ratios to assess selective pressures on the tsf gene

• Analysis of flanking regions for mobile genetic elements or recombination hotspots

How can recombinant Salmonella Newport EF-Ts be utilized in antimicrobial drug discovery programs?

Recombinant Salmonella Newport EF-Ts can serve as a valuable target in antimicrobial drug discovery, particularly for developing drugs against multi-drug resistant strains. The methodological approach would include:

• High-throughput screening: Developing assays to identify compounds that inhibit EF-Ts-EF-Tu interactions

• Structure-based drug design: Using crystal structures of EF-Ts to design specific inhibitors

• Fragment-based screening: Identifying small molecule fragments that bind to functional pockets in EF-Ts

• Differential targeting: Exploiting structural differences between bacterial and human elongation factors

The utility of targeting translation factors stems from their essential role in bacterial survival. Since EF-Ts is necessary for efficient protein synthesis, inhibitors could potentially be effective against multidrug-resistant strains like Newport MDR-AmpC, which has shown resistance to at least nine antimicrobials .

What analytical techniques are most informative for characterizing the structure-function relationship of Salmonella Newport EF-Ts?

A comprehensive structural and functional characterization of Salmonella Newport EF-Ts requires multiple complementary techniques:

• X-ray crystallography: Determining high-resolution 3D structure

• NMR spectroscopy: Analyzing protein dynamics and ligand interactions in solution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Mapping conformational changes upon binding

• Cryo-electron microscopy: Visualizing EF-Ts in complex with ribosomes or other translation factors

• Site-directed mutagenesis: Identifying key residues for function through systematic mutation

Functional characterization can be performed using:

• GDP exchange assays measuring EF-Tu recycling

• In vitro translation assays assessing elongation rates

• Thermal shift assays evaluating protein stability

• Binding assays quantifying interaction with other translation components

The integration of structural data with functional assays provides the most comprehensive understanding of how EF-Ts contributes to protein synthesis in Salmonella Newport.

How can comparative genomics approaches be applied to study EF-Ts variation across Salmonella Newport isolates with different virulence profiles?

Comparative genomics offers powerful methods for understanding how EF-Ts varies across Salmonella Newport isolates and how these variations might relate to virulence. A methodological framework would include:

• Whole genome sequencing: Generate genome data from diverse S. Newport isolates with characterized virulence

• Multiple sequence alignment: Align tsf genes and flanking regions to identify variations

• Phylogenetic analysis: Construct gene trees to understand evolutionary relationships

• SNP analysis: Identify single nucleotide polymorphisms that correlate with virulence traits

• Recombination detection: Apply methods like GARD or RDP to detect recombination events

Studies on Salmonella Newport have used pulsed-field gel electrophoresis (PFGE) to characterize strain relationships, revealing that some patterns are indistinguishable among isolates from humans and animals . Similar approaches could be applied to study tsf gene variations.

Research has already demonstrated that certain virulence genes show different prevalence between Salmonella Newport and other serotypes like Salmonella Typhi . A similar analysis focused specifically on the tsf gene could reveal whether translation factors show comparable patterns of variation.

What are the main challenges in expressing and purifying functional recombinant EF-Ts from multidrug-resistant Salmonella Newport strains?

Expressing and purifying functional recombinant EF-Ts from multidrug-resistant Salmonella Newport strains presents several technical challenges:

• Genetic stability: MDR strains often contain mobile genetic elements that can affect genomic stability

• Codon usage optimization: Differences in codon usage between S. Newport and expression hosts

• Protein solubility: Ensuring proper folding of the recombinant protein

• Contaminant co-purification: Plasmid-encoded proteins may co-purify with the target

• Activity verification: Confirming that the purified protein maintains native functionality

Methodological solutions include:

• Expression system optimization: Testing different vectors, promoters, and fusion tags

• Codon optimization: Adapting the coding sequence for the expression host

• Solubility enhancement: Using solubility tags (MBP, SUMO) or expressing at lower temperatures

• Rigorous purification: Implementing multi-step chromatography to ensure high purity

• Activity assays: Developing robust assays to verify functional integrity

Studies on multidrug-resistant Salmonella Newport have shown that these strains contain multiple resistance elements, including class 1 integrons with resistance genes like aadA and dhfr . The presence of these elements could potentially affect genomic stability when cloning genes from these strains.

How can researchers address issues of protein instability or misfolding when working with recombinant Salmonella Newport EF-Ts?

Protein instability and misfolding are common challenges when working with recombinant proteins. For Salmonella Newport EF-Ts, researchers can implement the following strategies:

• Buffer optimization: Systematically test different buffer compositions (pH, salt, additives)

• Expression temperature: Lower temperatures often improve protein folding

• Co-expression with chaperones: Express molecular chaperones to assist proper folding

• Fusion partners: Use solubility-enhancing fusion tags like MBP, SUMO, or TRX

• Refolding protocols: Develop denaturation and refolding procedures if inclusion bodies form

Experimental approach to optimize stability:

• Conduct thermal shift assays to identify stabilizing buffer conditions

• Perform limited proteolysis to identify and remove flexible regions prone to degradation

• Add stabilizing agents such as glycerol, sucrose, or specific ions

• Test protein stability under various storage conditions (temperature, freeze-thaw cycles)

When designing purification strategies, researchers should consider that Salmonella Newport has been found to possess multiple virulence factors and resistance elements . These characteristics might influence the expression and stability of recombinant proteins derived from these strains.

How is the study of Elongation factor Ts contributing to our understanding of Salmonella Newport pathogenesis?

While the direct connection between EF-Ts and Salmonella Newport pathogenesis isn't explicitly addressed in the search results, we can analyze how translation factors might contribute to bacterial virulence:

• Growth rate influence: EF-Ts affects protein synthesis efficiency, which can impact bacterial growth rates during infection

• Stress response regulation: Translation factors can play roles in bacterial adaptation to host environments

• Interaction with host factors: Bacterial translation machinery may interact with host defense mechanisms

• Target for antimicrobial development: As an essential factor, EF-Ts presents a potential drug target

Research has shown that Salmonella Newport MDR-AmpC strains have been associated with severe infections and deaths in both animals and humans . The rapid emergence of these resistant strains highlights the need to better understand core bacterial processes, including protein synthesis, that might be targeted for novel therapeutic approaches.

Future research directions might include:

• Investigating whether EF-Ts expression levels change during infection

• Determining if EF-Ts from virulent strains has distinct functional characteristics

• Exploring potential moonlighting functions of EF-Ts beyond its canonical role in translation

What new technologies are emerging for studying the role of translation factors in bacterial antibiotic resistance?

Emerging technologies for studying translation factors in the context of antibiotic resistance include:

• Cryo-electron microscopy: Providing high-resolution structures of translation complexes

• Ribosome profiling: Monitoring translation efficiency and regulation genome-wide

• CRISPR-Cas9 gene editing: Creating precise mutations in translation factor genes

• Single-molecule techniques: Visualizing translation dynamics in real-time

• Proteomics approaches: Identifying post-translational modifications and interaction networks

These technologies can help researchers understand how translation factors like EF-Ts might be affected by or contribute to antibiotic resistance mechanisms. Studies have shown that Salmonella Newport MDR-AmpC isolates have resistance to multiple antimicrobials, including extended-spectrum cephalosporins . Understanding how protein synthesis machinery functions in these resistant strains could reveal new therapeutic approaches.