Recombinant Phenylobacterium zucineum Elongation factor Ts (tsf)

What is Elongation factor Ts (tsf) and what is its function in bacterial protein synthesis?

Elongation factor Ts (EF-Ts) is a protein encoded by the tsf gene that functions as a guanine nucleotide exchange factor for elongation factor Tu (EF-Tu) during bacterial protein synthesis. EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu, thereby recycling EF-Tu for subsequent rounds of aminoacyl-tRNA delivery to the ribosome. This exchange reaction is crucial for maintaining the efficiency of protein translation.

In organisms like Chlamydia trachomatis, the tsf gene encodes a 282-amino-acid polypeptide with a calculated molecular weight of 30,824 Da . The protein's primary function is to increase the rate of GDP exchange with both its own species' EF-Tu and potentially EF-Tu from other bacterial species, as demonstrated by cross-species activity studies .

How is the tsf gene typically organized in bacterial genomes, and what is known about its genomic context in Phenylobacterium zucineum?

The tsf gene is typically found in a conserved gene cluster in many bacterial species. In Chlamydia trachomatis, genomic analysis revealed that the tsf gene is located in a cluster similar to the rpsB-tsf-pyrH(smbA)-frr region of Escherichia coli . While specific information about the genomic organization of the tsf gene in Phenylobacterium zucineum is not directly available from the search results, comparative genomic analysis indicates that P. zucineum is phylogenetically closest to Caulobacter crescentus .

The complete genome of Phenylobacterium zucineum consists of a circular chromosome (3,996,255 bp) and a circular plasmid (382,976 bp), encoding 3,861 putative proteins . Given the conservation of certain genomic features between P. zucineum and C. crescentus, particularly the cell cycle master regulator CtrA and its regulatory network, it is reasonable to hypothesize that the tsf gene might also be found in a conserved genomic context in P. zucineum.

What expression systems are most efficient for producing recombinant Phenylobacterium zucineum Elongation factor Ts?

Based on approaches used for similar proteins, several expression systems can be considered for recombinant P. zucineum EF-Ts production:

For basic research purposes, E. coli expression systems are likely most suitable, as demonstrated by successful expression of chlamydial tsf gene in E. coli as both a nonfusion protein and as a 6x His-tagged fusion protein . The latter approach facilitates purification while maintaining protein function.

What are the optimal conditions for purifying recombinant Phenylobacterium zucineum Elongation factor Ts to ensure biological activity?

While specific purification protocols for P. zucineum EF-Ts are not detailed in the search results, insights can be drawn from successful approaches with similar proteins:

• Affinity chromatography: His-tagged recombinant EF-Ts can be purified using nickel affinity chromatography, which has been shown to yield functionally active protein for other bacterial EF-Ts proteins .

• Buffer optimization: Phosphate or Tris-based buffers (pH 7.5-8.0) containing 100-300 mM NaCl are typically suitable for maintaining stability during purification.

• Reducing agents: Including reducing agents such as DTT or β-mercaptoethanol (1-5 mM) helps prevent oxidation of cysteine residues.

• Activity preservation: Adding glycerol (10-20%) to storage buffers helps maintain protein stability and activity during freeze-thaw cycles.

• Quality control: Purified protein should be assessed for proper folding using circular dichroism and for activity using GDP exchange assays with EF-Tu.

The functional integrity of purified recombinant EF-Ts can be verified by its ability to catalyze GDP exchange with EF-Tu, as demonstrated in studies with chlamydial EF-Ts .

How does the structure and function of Phenylobacterium zucineum Elongation factor Ts compare with that of model organisms?

While specific structural information for P. zucineum EF-Ts is not available in the search results, comparative analysis can provide insights:

• Sequence homology: Based on patterns observed with other bacterial EF-Ts proteins, P. zucineum EF-Ts likely shares conserved domains with model organisms. For reference, chlamydial EF-Ts shows 34% identity and an additional 14% similarity with E. coli EF-Ts .

• Functional conservation: Despite sequence divergence, EF-Ts proteins generally maintain their GDP exchange function across species. Notably, chlamydial EF-Ts demonstrated activity comparable to E. coli EF-Ts in exchange reactions with E. coli EF-Tu, suggesting functional conservation despite structural differences .

• Species-specific adaptations: Given P. zucineum's unique lifestyle as a facultative intracellular bacterium that maintains stable associations with host cells without affecting their growth , its EF-Ts might exhibit adaptations related to this lifestyle.

A comprehensive structural comparison would require experimental determination of the P. zucineum EF-Ts structure using X-ray crystallography or cryo-electron microscopy.

What role might Phenylobacterium zucineum Elongation factor Ts play in the organism's unique intracellular lifestyle?

P. zucineum is notable for maintaining a stable association with human cells without affecting their growth or morphology , suggesting specialized adaptations for intracellular survival:

• Protein synthesis regulation: EF-Ts might be adapted to function under the unique intracellular conditions of the host cell, potentially with modified kinetics or stability.

• Host-pathogen interactions: As a key component of the bacterial translational machinery, EF-Ts could be subject to regulation during adaptation to the intracellular environment.

• Stress response: Intracellular bacteria often face nutritional and oxidative stresses; EF-Ts might play a role in modulating protein synthesis rates during these stress conditions.

• Potential interaction with host factors: P. zucineum's benign relationship with host cells might involve specialized interactions between its translational machinery and host cytoplasmic components.

Research methodologies to explore these hypotheses could include:

• Comparative transcriptomics of P. zucineum grown in various conditions

• Protein-protein interaction studies between P. zucineum EF-Ts and host cell components

• Mutational analysis of EF-Ts to identify residues critical for intracellular survival

How might structural differences in Phenylobacterium zucineum Elongation factor Ts be exploited for developing targeted antimicrobials?

The development of targeted antimicrobials based on P. zucineum EF-Ts would leverage several factors:

• Essential function: Translation factors like EF-Ts are essential for bacterial survival, making them attractive drug targets.

• Structural differences from host factors: Bacterial EF-Ts differs significantly from eukaryotic translation factors, providing a basis for selectivity.

• Specialized research approaches:

  • Structure-based drug design targeting unique pockets in P. zucineum EF-Ts

  • High-throughput screening for inhibitors of the EF-Ts:EF-Tu interaction

  • Peptide mimetics that disrupt the function of EF-Ts

• Potential applications:

  • Development of narrow-spectrum antibiotics against P. zucineum

  • Creation of research tools to study the role of EF-Ts in bacterial physiology

  • Insight into translation factor inhibition as a broader antimicrobial strategy

While P. zucineum itself may not be a primary pathogen, understanding its unique EF-Ts could provide templates for targeting related proteins in pathogenic species.

What is the relationship between Phenylobacterium zucineum Elongation factor Ts and the organism's antimicrobial compounds?

P. zucineum has been associated with the production of antimicrobial compounds, including zucinodin, a lassopeptide identified in this bacterium . While direct relationships between EF-Ts and antimicrobial production are not established in the search results, several hypotheses can be considered:

• Translational regulation: As a translation factor, EF-Ts could influence the expression of genes involved in antimicrobial compound biosynthesis.

• Metabolic coordination: Protein synthesis and secondary metabolite production compete for cellular resources; EF-Ts activity may indirectly influence the allocation of resources to antimicrobial production.

• Stress response coordination: Both translation regulation and antimicrobial production can be stress responses; they might be coordinately regulated under certain conditions.

Research to explore these connections might include:

• Transcriptomic analysis correlating tsf expression with antimicrobial biosynthetic gene clusters

• Metabolomic profiling of wild-type versus tsf mutant strains

• Investigation of regulatory networks connecting translation efficiency and secondary metabolism

What protocol modifications are necessary when expressing Phenylobacterium zucineum Elongation factor Ts in heterologous hosts?

Expressing P. zucineum EF-Ts in heterologous hosts requires several considerations:

• Codon optimization: P. zucineum likely has different codon usage compared to common expression hosts. Optimizing the coding sequence for the target expression system can significantly improve yield.

• Expression vector selection:

  • For E. coli expression: pET series vectors with T7 promoter systems offer tight regulation and high expression

  • For baculovirus expression: pFastBac vectors enable efficient transfer to insect cells

  • For mammalian expression: pcDNA or pCMV vectors provide strong constitutive expression

• Fusion tags and solubility:

  • N-terminal or C-terminal 6×His tags facilitate purification while typically maintaining function

  • Solubility-enhancing tags (SUMO, MBP, GST) may improve folding and solubility

  • Inclusion of cleavage sites allows tag removal if necessary for functional studies

• Expression conditions:

  • Lower temperatures (16-25°C) often improve soluble protein yield

  • Induction strength modulation (IPTG concentration for E. coli systems)

  • Extended expression times with lower inducer concentrations

• Host strain selection:

  • BL21(DE3) derivatives for basic expression

  • Origami or SHuffle strains if disulfide bonds are present

  • Rosetta strains if rare codons are abundant

These protocols can be adjusted based on initial expression trials and protein characterization results.

How can isotope labeling of recombinant Phenylobacterium zucineum Elongation factor Ts facilitate structural studies?

Isotope labeling is essential for advanced structural studies of proteins using NMR spectroscopy and can facilitate other structural techniques:

• NMR structural studies:

  • Uniform 15N labeling: Grow expression host in minimal media with 15NH4Cl as sole nitrogen source

  • Uniform 13C/15N double labeling: Use 13C-glucose and 15NH4Cl for complete backbone assignment

  • Selective amino acid labeling: Incorporate specific labeled amino acids for focused studies

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Deuterium labeling can reveal solvent-accessible regions and conformational dynamics

  • Provides information about protein-protein interaction surfaces

• Neutron diffraction:

  • Deuterium labeling enhances contrast in neutron diffraction studies

  • Can provide unique information about hydrogen bonding networks

• Protocol considerations:

  • M9 minimal media is typically used for isotope labeling in E. coli

  • Higher inoculum densities and longer growth periods are often necessary

  • Protein yields are typically lower in minimal media

  • SILAC approaches can be used for mammalian expression systems

• Applications to P. zucineum EF-Ts:

  • Structural comparison with EF-Ts from model organisms

  • Mapping of interaction surfaces with EF-Tu

  • Identification of potential unique structural features related to P. zucineum's lifestyle

What are the optimal conditions for assessing the nucleotide exchange activity of Phenylobacterium zucineum Elongation factor Ts in vitro?

Assessing nucleotide exchange activity requires careful experimental design:

• Basic assay components:

  • Purified recombinant P. zucineum EF-Ts

  • Purified EF-Tu (either from P. zucineum or model organisms)

  • Fluorescently labeled GDP/GTP or radiolabeled nucleotides

  • Appropriate buffer system (typically HEPES or Tris, pH 7.5-8.0)

• Measurement approaches:

  • Real-time fluorescence monitoring using mant-GDP

  • Filter binding assays with radiolabeled nucleotides

  • FRET-based assays for protein-protein interactions

• Kinetic parameters to determine:

  • kcat for the exchange reaction

  • Km for EF-Tu:GDP complex

  • Effects of temperature, pH, and salt concentration

• Comparative analysis:

  • Activity with P. zucineum's own EF-Tu vs. EF-Tu from other species

  • Comparison with well-characterized EF-Ts proteins (e.g., from E. coli)

Based on research with chlamydial EF-Ts, P. zucineum EF-Ts might show activity with both its own EF-Tu and EF-Tu from other bacterial species , which would be an important characteristic to verify experimentally.

How can computational approaches be used to predict structure and function of Phenylobacterium zucineum Elongation factor Ts?

Computational approaches offer valuable insights when experimental structural data is limited:

• Homology modeling:

  • Using crystal structures of EF-Ts from model organisms as templates

  • Refinement through molecular dynamics simulations

  • Validation using energy minimization and stereochemical quality checks

• Protein-protein interaction prediction:

  • Docking studies with EF-Tu to predict interaction interfaces

  • Molecular dynamics simulations to assess stability of predicted complexes

  • Determination of crucial residues for site-directed mutagenesis

• Phylogenetic analysis:

  • Evolutionary conservation mapping to identify functionally important residues

  • Comparison with EF-Ts proteins from bacteria with similar lifestyles

  • Identification of uniquely conserved motifs in P. zucineum EF-Ts

• Molecular dynamics simulations:

  • Assessment of conformational flexibility

  • Investigation of nucleotide binding/release mechanisms

  • Prediction of effects of pH, temperature, and other environmental factors

• Integration with experimental data:

  • Refinement of models based on limited experimental data

  • Design of targeted experiments to validate computational predictions

  • Iterative improvement of structural models

These computational approaches can guide experimental design and provide testable hypotheses about P. zucineum EF-Ts structure and function.

How can recombinant Phenylobacterium zucineum Elongation factor Ts be used to study bacterial evolution and adaptation?

Recombinant P. zucineum EF-Ts provides a valuable tool for evolutionary studies:

• Comparative biochemistry:

  • Cross-species activity assays to determine functional conservation

  • Thermostability comparisons to assess environmental adaptations

  • Structure-function relationships across bacterial lineages

• Molecular evolution analysis:

  • Identification of positively selected residues in the P. zucineum tsf gene

  • Correlation of sequence changes with ecological niches

  • Horizontal gene transfer analysis of translation factors

• Experimental evolution approaches:

  • Expression of P. zucineum EF-Ts in heterologous hosts under selective pressure

  • Directed evolution to identify potential functional adaptations

  • Competition assays between wild-type and modified EF-Ts variants

• Intracellular adaptation studies:

  • Investigation of how P. zucineum's unique intracellular lifestyle has shaped EF-Ts function

  • Comparison with EF-Ts from obligate intracellular pathogens

  • Correlation of EF-Ts properties with host range and host-microbe interactions

These approaches can illuminate how essential translation factors evolve during bacterial adaptation to specialized ecological niches.

What techniques can resolve contradictory findings regarding biochemical properties of Phenylobacterium zucineum Elongation factor Ts?

When faced with contradictory results regarding P. zucineum EF-Ts properties, several methodological approaches can help resolve discrepancies:

• Standardization of recombinant protein preparation:

  • Consistent expression systems and purification protocols

  • Detailed characterization of protein purity and folding state

  • Batch-to-batch consistency verification

• Multiple complementary assay systems:

  • Different nucleotide exchange activity measurement techniques

  • Various buffer conditions and experimental setups

  • Independent laboratory verification of key findings

• Advanced biophysical characterization:

  • Circular dichroism to confirm secondary structure

  • Thermal shift assays to assess protein stability

  • Size exclusion chromatography with multi-angle light scattering for oligomerization state

• Molecular biology verification:

  • Site-directed mutagenesis of key residues

  • Chimeric proteins combining domains from different species

  • In vivo complementation studies

• Systematic review of methodological differences:

  • Creation of a standardized protocol based on best practices

  • Meta-analysis of existing data to identify patterns in contradictory results

  • Design of decisive experiments targeting specific contradictions

By applying these rigorous approaches, researchers can develop consensus on the biochemical properties of P. zucineum EF-Ts and resolve apparent contradictions in the literature.

What are the most promising research applications for Phenylobacterium zucineum Elongation factor Ts in microbial biotechnology?

The unique properties of P. zucineum and its EF-Ts suggest several promising research directions:

• Protein engineering applications:

  • Development of EF-Ts variants with enhanced nucleotide exchange activity

  • Creation of chimeric translation factors with novel properties

  • Design of EF-Ts-based biosensors for bacterial metabolism

• Host-microbe interaction studies:

  • Investigation of P. zucineum's non-pathogenic intracellular lifestyle

  • Development of bacterial delivery vectors based on P. zucineum properties

  • Understanding mechanisms of stable intracellular colonization

• Antimicrobial development:

  • Structure-based design of translation inhibitors

  • Exploration of P. zucineum's antimicrobial compounds like zucinodin

  • Identification of natural products targeting bacterial translation

• Synthetic biology tools:

  • Optimization of translation systems for heterologous protein expression

  • Development of minimal translation systems for in vitro applications

  • Creation of orthogonal translation machinery

These research directions leverage P. zucineum's unique biological properties while addressing important challenges in biotechnology and medicine.

How can structural studies of Phenylobacterium zucineum Elongation factor Ts inform drug discovery efforts?

Structural characterization of P. zucineum EF-Ts can significantly advance drug discovery through multiple approaches:

• Structure-based drug design:

  • Identification of druggable pockets unique to bacterial EF-Ts

  • Virtual screening of compound libraries against the EF-Ts structure

  • Fragment-based drug discovery targeting the EF-Ts:EF-Tu interface

• Comparative structural analysis:

  • Identification of conserved features across bacterial EF-Ts proteins

  • Mapping of species-specific structural elements

  • Analysis of conformational changes during nucleotide exchange

• Mechanistic insights:

  • Understanding of dynamic protein-protein interactions

  • Elucidation of rate-limiting steps in the exchange reaction

  • Identification of allosteric regulation sites

• Translation to related pathogens:

  • Application of structural insights to EF-Ts from pathogenic species

  • Development of broad-spectrum translation inhibitors

  • Target validation using structural biology approaches

• Integration with other data:

  • Correlation of structure with antimicrobial resistance mechanisms

  • Combination with genomic and transcriptomic data

  • Machine learning approaches to predict drug interactions

These structural biology approaches can provide crucial insights for rational drug design targeting bacterial translation machinery.