Recombinant Stenotrophomonas maltophilia Elongation factor Ts (tsf)

What is Stenotrophomonas maltophilia and why is elongation factor Ts significant?

Stenotrophomonas maltophilia is an opportunistic pathogen causing nosocomial and community-acquired infections, particularly affecting immunocompromised individuals, cancer patients, and those with cystic fibrosis. It is associated with significant mortality rates ranging from 14-69% in bacteremia cases . S. maltophilia is environmentally ubiquitous, found in aqueous habitats including plant rhizospheres, animals, foods, and water sources .

Elongation factor Ts (tsf) is a critical component of the bacterial protein synthesis machinery. While specific information on S. maltophilia tsf is limited, research has demonstrated that bacterial tsf proteins play essential roles in protein translation by facilitating the regeneration of active EF-Tu~GTP complexes. Recently, tsf has gained additional research interest as fusion experiments have shown its potential in protein display applications, where recombinant proteins fused to tsf can be successfully displayed on bacterial floc structures .

How does S. maltophilia tsf compare structurally to elongation factors in other bacterial species?

S. maltophilia tsf likely shares conserved domains with other bacterial elongation factors while possessing species-specific variations. The protein likely contains N-terminal and C-terminal domains connected by a flexible linker region, similar to other bacterial EF-Ts proteins. The N-terminal domain typically interacts with EF-Tu, while the C-terminal domain often contributes to stability.

To compare S. maltophilia tsf with other species, researchers should:

• Perform multiple sequence alignments using tools like MUSCLE or Clustal Omega

• Analyze conserved motifs using MEME Suite or similar tools

• Generate homology models using Swiss-Model or Phyre2 if crystal structures are unavailable

• Validate models through molecular dynamics simulations

Functional analysis through complementation studies in heterologous systems can further reveal species-specific characteristics of S. maltophilia tsf compared to model organisms like E. coli.

What genetic characteristics define the tsf gene in S. maltophilia?

The tsf gene in S. maltophilia is likely part of the str operon, which typically contains genes involved in ribosomal protein synthesis and translation. While specific information about the S. maltophilia tsf gene organization is limited in the search results, researchers should examine:

• Promoter elements and regulatory regions controlling tsf expression

• Codon usage bias patterns that might affect heterologous expression

• Genetic linkage to other translation-related genes

• Strain-to-strain variability across different S. maltophilia genogroups

S. maltophilia exhibits significant genetic heterogeneity across different strains and genogroups, which may extend to the tsf gene . Researchers must consider this variability when designing experiments, as different clinical and environmental isolates may contain variations in the tsf sequence and expression patterns.

What expression systems are most effective for producing recombinant S. maltophilia tsf?

For recombinant expression of S. maltophilia tsf, several systems can be considered, each with specific advantages:

• E. coli expression systems: The pET vector system with BL21(DE3) or its derivatives offers high protein yields and inducible expression. For S. maltophilia proteins, codon optimization may be necessary due to differences in codon usage between the species.

• Cell-free expression systems: These can be advantageous for potentially toxic proteins, offering rapid expression without concerns about protein toxicity to host cells.

• Yeast expression systems: For proteins requiring eukaryotic post-translational modifications or when bacterial expression is problematic.

• Native expression: Using S. maltophilia itself as an expression host might preserve native folding and modifications, though this approach presents challenges due to the organism's pathogenicity and limited genetic tools.

Based on available research, an E. coli system using a fusion approach has been successful for tsf-related constructs. In one study, researchers successfully expressed a fusion protein consisting of Tsf and green fluorescence protein (GFP) on E. coli flocs, with the fusion protein comprising approximately 15% (w/w) of total floc protein .

How can researchers optimize purification protocols for recombinant S. maltophilia tsf?

Purification of recombinant S. maltophilia tsf requires a methodical approach:

• Affinity tags selection:

  • His6-tag for IMAC purification is common and generally effective

  • GST-tags can improve solubility but add significant size

  • FLAG or Strep-II tags for applications requiring higher purity

• Optimization of lysis conditions:

  • Test multiple buffer systems (HEPES, Tris, Phosphate) at pH 7.0-8.0

  • Include protease inhibitors to prevent degradation

  • Consider detergents for membrane-associated fractions

• Chromatography sequence:

  • Initial capture: Affinity chromatography (IMAC for His-tagged constructs)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography for highest purity

• Quality control:

  • SDS-PAGE and Western blotting to confirm identity

  • Mass spectrometry for precise molecular weight determination

  • Circular dichroism to assess secondary structure integrity

For scale-up considerations, on-column refolding protocols may be necessary if inclusion bodies form. Additionally, researchers should determine optimal storage conditions (buffer composition, pH, additives like glycerol) to maintain long-term stability.

What strategies can address solubility challenges with recombinant S. maltophilia tsf?

Bacterial proteins, including those from S. maltophilia, often face solubility challenges during recombinant expression. Several strategies can address these issues:

• Expression condition optimization:

  • Lower induction temperature (16-25°C)

  • Reduce inducer concentration

  • Use enriched media formulations

  • Test different growth phases for induction

• Fusion partners to enhance solubility:

  • MBP (Maltose-Binding Protein)

  • SUMO tag

  • Thioredoxin

  • NusA

• Co-expression approaches:

  • Chaperones (GroEL/GroES, DnaK/DnaJ)

  • Partner proteins from the translation complex

• Buffer optimization during purification:

  • Mild detergents (0.05-0.1% Triton X-100)

  • Stabilizing osmolytes (glycerol, sucrose)

  • Binding partners or substrates

The fusion protein approach has shown success with tsf, as demonstrated in the study where Tsf was successfully fused with GFP and expressed on E. coli flocs . This suggests that fusion strategies may be particularly effective for recombinant S. maltophilia tsf expression.

How can recombinant S. maltophilia tsf be utilized in protein display systems?

Research indicates that tsf can be effectively used for protein display applications. The methodology involves:

• Design of fusion constructs:

  • Create genetic fusions between tsf and the target protein

  • Include flexible linkers (e.g., (GGGGS)n) to prevent steric hindrance

  • Consider both N- and C-terminal fusions to determine optimal orientation

• Expression optimization:

  • In one study, a fusion protein consisting of Tsf and GFP expressed on E. coli flocs reached approximately 15% (w/w) of total floc protein

  • This suggests significant capacity for loading recombinant proteins

• Application protocols:

  • Induce flocculation by overexpressing the fusion protein

  • Harvest and process flocs according to downstream applications

  • Quantify display efficiency using fluorescent proteins or antibody detection

• Potential applications:

  • Immobilized enzyme systems

  • Biosensors

  • Bioadsorbents for environmental applications

  • Vaccine development through antigen display

The advantage of the tsf-based display system includes the self-produced flocculation, which provides a naturally immobilized platform without requiring additional carrier materials .

S. maltophilia exhibits intrinsic multidrug resistance through various mechanisms . Recombinant tsf can serve as a tool for studying these resistance mechanisms:

• Protein synthesis inhibitor research:

  • Aminoglycoside resistance in S. maltophilia involves ribosomal protection mechanisms

  • Recombinant tsf can be used to study interactions with the translation machinery components affected by antibiotics

  • In vitro translation assays with purified components including recombinant tsf can measure the impact of resistance mutations

• Stress response studies:

  • Examining tsf expression levels under antibiotic stress

  • Determining if tsf mutations or modifications contribute to translational adaptation during antibiotic exposure

• Protein-protein interaction studies:

  • Pull-down assays with recombinant tsf to identify binding partners in resistant strains

  • Comparative interactomics between susceptible and resistant isolates

• Structural biology approaches:

  • Crystal structures of S. maltophilia tsf alone and in complex with antibiotics

  • Molecular dynamics simulations to predict resistance-associated conformational changes

This research could provide insights into S. maltophilia's intrinsic resistance mechanisms, which include aminoglycoside-modifying enzymes, efflux pumps, and protected ribosomal components .

How does the structure-function relationship of S. maltophilia tsf inform therapeutic targeting?

Understanding the structure-function relationship of S. maltophilia tsf can reveal potential targets for therapeutic intervention:

• Critical functional domains:

  • The N-terminal domain typically interacts with EF-Tu

  • The C-terminal domain contributes to stability and may have species-specific features

  • The linker region flexibility affects catalytic efficiency

• Methodological approaches:

  • Site-directed mutagenesis to identify essential residues

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics

  • Cross-linking studies to capture transient interactions

  • CRISPR interference to modulate tsf expression levels in vivo

• Potential therapeutic strategies:

  • Small molecule inhibitors targeting tsf-specific pockets

  • Peptide mimetics disrupting tsf-EF-Tu interactions

  • RNA-based approaches to modulate tsf expression

• Validation methods:

  • In vitro translation assays with purified components

  • Cell-based reporter systems measuring translation efficiency

  • Animal infection models evaluating compound efficacy

Since protein synthesis is essential for bacterial survival and pathogenesis, targeting S. maltophilia tsf could represent a novel approach against this multidrug-resistant pathogen that shows resistance to aminoglycosides, β-lactams, and quinolones .

What techniques can elucidate interactions between tsf and other components of the S. maltophilia translation machinery?

Studying protein-protein interactions involving S. maltophilia tsf requires multiple complementary approaches:

• In vitro binding assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Microscale Thermophoresis (MST) for interactions in complex solutions

• Structural studies:

  • X-ray crystallography of tsf-EF-Tu complexes

  • Cryo-EM to visualize larger assemblies within the translation machinery

  • NMR spectroscopy for dynamic interaction studies

• In vivo approaches:

  • Bacterial two-hybrid systems

  • Fluorescence Resonance Energy Transfer (FRET)

  • Proximity-dependent biotin labeling (BioID)

  • Co-immunoprecipitation with antibodies against tsf

• Systems biology methods:

  • Ribosome profiling to assess translation effects

  • Transcriptomics to identify co-regulated genes

  • Network analysis to place tsf in the broader translation machinery context

These methods can reveal how S. maltophilia tsf interacts with other translation factors and whether these interactions differ from model organisms, potentially contributing to the pathogen's adaptability in various environments.

How can researchers assess the impact of tsf mutations on S. maltophilia fitness and virulence?

Evaluating the effects of tsf mutations on S. maltophilia requires a comprehensive approach:

• Genetic manipulation strategies:

  • Site-directed mutagenesis of recombinant tsf

  • Chromosomal modification using CRISPR-Cas9 or recombineering

  • Complementation studies with mutated tsf variants

• Phenotypic assays:

  • Growth rate measurements under various conditions

  • Biofilm formation quantification using crystal violet staining

  • Antibiotic susceptibility testing (especially protein synthesis inhibitors)

  • Motility assays (swimming, swarming, twitching)

• Virulence assessment:

  • Cell culture infection models measuring:

    • Adhesion efficiency

    • Invasion rates

    • Intracellular survival

    • Cytotoxicity

  • Animal infection models evaluating:

    • Colonization efficiency

    • Tissue damage

    • Inflammatory response

    • Survival rates

• Molecular analysis:

  • Proteomics to identify changes in global protein expression

  • Analysis of specific virulence factor production

  • Assessment of stress response pathways activation

These approaches would provide insights into whether tsf functions beyond translation contribute to S. maltophilia pathogenicity, similar to moonlighting functions observed in other bacterial translation factors.

What controls should be included when studying recombinant S. maltophilia tsf activity?

Rigorous experimental design for S. maltophilia tsf studies should include:

• Positive controls:

  • Well-characterized tsf from model organisms (E. coli)

  • Native (non-recombinant) S. maltophilia tsf when available

  • Commercially available translation factors

• Negative controls:

  • Inactive tsf variants (site-directed mutants)

  • Denatured protein samples

  • Buffer-only conditions

• Specificity controls:

  • Unrelated proteins of similar size/structure

  • Other translation factors to demonstrate specificity

  • Species-specific variants to assess evolutionary conservation

• System validation:

  • In vitro translation system functionality verification

  • Dose-dependent responses to establish quantitative relationships

  • Time-course experiments to capture kinetic parameters

When working with fusion constructs like Tsf-GFP , additional controls should include the tag/fusion partner alone and alternative fusion orientations to distinguish between effects of tsf and effects of the fusion partner.

How can researchers validate the functional activity of purified recombinant S. maltophilia tsf?

Functional validation of recombinant S. maltophilia tsf should include:

• In vitro nucleotide exchange assays:

  • Measure EF-Tu·GDP to EF-Tu·GTP conversion rates

  • Monitor nucleotide exchange using fluorescent GDP/GTP analogs

  • Compare kinetic parameters with those of established tsf proteins

• Translation reconstitution assays:

  • In vitro translation systems with purified components

  • Poly(U)-directed poly(Phe) synthesis

  • Reporter protein synthesis (luciferase, GFP)

• Structural verification:

  • Circular dichroism to confirm secondary structure

  • Thermal shift assays to assess stability

  • Limited proteolysis to verify domain organization

• Binding validation:

  • EF-Tu binding assays (SPR, ITC, pull-down)

  • Ribosome association studies

  • Competition assays with known binding partners

For tsf fusion proteins used in display systems, additional validation should include surface accessibility assays and confirmation that the fusion does not compromise the function of either partner protein .

What are common pitfalls in experimental design when working with S. maltophilia proteins?

Researchers should be aware of several challenges specific to S. maltophilia proteins:

• Genetic heterogeneity issues:

  • S. maltophilia shows significant strain-to-strain variability

  • Different genogroups may express proteins with sequence variations

  • Solution: Sequence multiple clinical and environmental isolates before designing constructs

• Expression optimization challenges:

  • Codon usage bias differences between S. maltophilia and expression hosts

  • Potential toxicity of S. maltophilia proteins in heterologous systems

  • Solution: Use codon-optimized sequences and inducible, tightly controlled expression systems

• Purification complications:

  • Potential for contamination with host proteins

  • Formation of inclusion bodies

  • Solutions:

    • Multiple orthogonal purification steps

    • Rigorous endotoxin removal for proteins destined for immunological studies

    • Optimization of solubilization conditions

• Functional assay limitations:

  • Model systems may not recapitulate S. maltophilia's unique biology

  • Environmental conditions affecting protein function

  • Solution: Validate findings in native-like conditions and consider S. maltophilia-specific factors

When working specifically with tsf-based display systems, researchers should consider the potential for steric hindrance affecting displayed protein functionality and implement controls to assess accessibility and activity .