Recombinant Francisella tularensis subsp. mediasiatica Ribosome-recycling factor (frr)

What is the biological function of Ribosome-recycling factor (frr) in Francisella tularensis?

Ribosome-recycling factor (frr) in Francisella tularensis is responsible for the critical process of releasing ribosomes from messenger RNA at the termination of protein biosynthesis. The protein functions to increase the efficiency of translation by recycling ribosomes from one completed round of translation to subsequent rounds. This process is essential for maintaining proper protein synthesis rates and ensuring bacterial survival under various growth conditions . In the context of intracellular pathogens like F. tularensis, efficient protein synthesis is crucial for adapting to changing host environments and expressing virulence factors at appropriate times during infection.

How does F. tularensis subsp. mediasiatica differ from other Francisella subspecies?

Francisella tularensis subsp. mediasiatica is one of four recognized subspecies of F. tularensis, which also include subsp. tularensis, holarctica, and novicida. The mediasiatica subspecies is distinguished by:

• Geographical distribution: Originally thought to be limited to Central Asia, recent findings have identified a natural focus of F. tularensis subsp. mediasiatica in the Altai region of Russia, more than 1500 km eastward from previously known foci in Kazakhstan and Turkmenistan .

• Genetic structure: Current research divides mediasiatica into three phylogeographic groups (M.I, M.II, and M.III) based on MLVA (multiple-locus variable number tandem repeat analysis) profiles and geographical locations .

• Virulence profile: The virulence of subsp. mediasiatica in a vaccinated mouse model is intermediate between the highly virulent subsp. tularensis and the less virulent subsp. holarctica .

• Population genetics: Genomic analyses show that the distance from the Most Recent Common Ancestor (MRCA) to contemporary strains is approximately 2400 SNPs for mediasiatica, compared to 1130-1290 SNPs for tularensis and 2000-2400 for holarctica .

What expression systems are most effective for producing recombinant F. tularensis frr protein?

For recombinant expression of F. tularensis frr, several methodological approaches have proven successful:

• E. coli expression systems: The BL21(DE3) strain with pET vector systems has been widely used for expression of Francisella proteins. For frr specifically, codon optimization may be necessary due to differences in codon usage between Francisella and E. coli.

• Expression conditions: Optimal expression is typically achieved by induction with 0.5-1.0 mM IPTG at 18-25°C for 16-18 hours, as lower temperatures reduce inclusion body formation for this protein.

• Solubility enhancement: Addition of 5-10% glycerol and 1-5 mM βME (beta-mercaptoethanol) to lysis buffers has been shown to improve solubility of Francisella proteins.

• Safety considerations: When working with proteins from select agents like F. tularensis, researchers must ensure proper biosafety measures and potentially use attenuated strains like the avirulent 60(B)57 strain of subsp. mediasiatica for initial studies .

What purification strategies yield highest purity and activity of recombinant frr?

To obtain high-purity, active frr protein from F. tularensis subsp. mediasiatica, a multi-step purification protocol is recommended:

• Affinity chromatography: His-tagged frr can be purified using Ni-NTA columns with gradual imidazole elution (50-250 mM).

• Ion exchange chromatography: Given the predicted pI of frr, anion exchange (Q-Sepharose) at pH 8.0 serves as an effective secondary purification step.

• Size exclusion chromatography: Final polishing via gel filtration (Superdex 75) in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM DTT.

• Quality control: Protein purity should be assessed by SDS-PAGE (with expected band at approximately 20.6 kDa ), and functional activity confirmed through ribosome-binding assays.

• Storage stability: The purified protein maintains optimal activity when stored at -80°C in buffer containing 10% glycerol, with minimal freeze-thaw cycles.

How can researchers accurately assess the functional activity of recombinant frr?

Functional assessment of recombinant frr requires assays that evaluate its ribosome-recycling capacity:

• In vitro translation termination assay: Using purified ribosomes, mRNA with stop codons, and necessary translation factors to measure frr's ability to release ribosomes from mRNA.

• Polysome profiling: Comparing polysome profiles in the presence and absence of functional frr to quantify its impact on ribosome recycling.

• Complementation assays: Testing whether the recombinant frr can rescue growth defects in frr-depleted bacterial strains.

• Binding affinity measurements: Using techniques such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) to determine binding constants between frr and ribosomal components.

• Activity comparisons: Comparing activity of F. tularensis subsp. mediasiatica frr with orthologs from other subspecies to identify functional differences that might relate to pathogenicity.

How does frr contribute to F. tularensis pathogenicity and intracellular survival?

The contribution of frr to F. tularensis pathogenicity involves multiple aspects of bacterial physiology:

What structural and functional differences exist between frr proteins across Francisella subspecies?

Analysis of frr proteins across Francisella subspecies reveals several important differences:

These differences may contribute to the varying virulence profiles observed across subspecies, though direct experimental validation is needed to confirm the functional impact of these variations.

How might frr serve as a potential therapeutic target against F. tularensis infections?

The essential nature of frr for bacterial viability makes it a potential therapeutic target against F. tularensis:

• Target validation: As frr is critical for bacterial protein synthesis and has sufficient structural differences from mammalian translation factors, it represents a potentially selective antimicrobial target.

• Small molecule inhibitor design: In silico screening and rational drug design approaches could identify compounds that specifically interfere with F. tularensis frr-ribosome interactions.

• Peptide inhibitors: Short peptides mimicking ribosomal binding regions that compete with frr function could serve as therapeutic leads.

• Combination therapy strategies: Sub-inhibitory concentrations of frr inhibitors might sensitize F. tularensis to existing antibiotics, providing synergistic therapeutic effects.

• Resistance considerations: The highly conserved nature of frr across bacterial species suggests a high fitness cost for resistance mutations, potentially reducing the rapid emergence of resistance.

What are common challenges in expressing and purifying active F. tularensis frr protein?

Researchers frequently encounter several challenges when working with F. tularensis frr:

• Protein solubility issues:

  • Problem: Formation of inclusion bodies during expression

  • Solution: Express at lower temperatures (16-18°C), use solubility-enhancing tags (SUMO, MBP), or employ detergent screening

• Proteolytic degradation:

  • Problem: Partial degradation during purification

  • Solution: Include protease inhibitor cocktails, perform purification at 4°C, and minimize purification time

• Activity loss during purification:

  • Problem: Purified protein lacks functional activity

  • Solution: Include stabilizing agents (glycerol, reducing agents), avoid harsh elution conditions, and verify proper folding by circular dichroism

• Contamination with E. coli ribosomes:

  • Problem: Co-purification of host ribosomes due to frr's natural affinity

  • Solution: Include high-salt washing steps (500 mM NaCl) during affinity purification and additional ion exchange chromatography

• Biosafety considerations:

  • Problem: Working with proteins from select agents

  • Solution: Use attenuated strains or rely on recombinant expression in approved host systems

How can researchers optimize frr functional assays to obtain reproducible results?

To enhance reproducibility in frr functional assays, researchers should consider:

• Ribosome quality control:

  • Ensure ribosomes used in assays are freshly prepared and quantified by both A260 measurements and activity controls

  • Verify ribosome integrity by sucrose gradient analysis

• Reaction conditions optimization:

  • Systematically test buffer components (Mg2+ concentration is particularly critical, typically 5-10 mM)

  • Optimize temperature (typically 30-37°C) and pH (usually 7.4-7.8)

• Proper controls:

  • Include positive controls (known active frr from E. coli)

  • Use negative controls (heat-inactivated frr)

  • Implement system controls lacking specific components

• Data normalization:

  • Express results relative to internal standards

  • Account for batch-to-batch variation in ribosome preparations

• Statistical robustness:

  • Perform all experiments in at least triplicate

  • Apply appropriate statistical tests to determine significance of results

What techniques can address data inconsistencies when comparing frr activity across different F. tularensis subspecies?

When comparing frr activity across F. tularensis subspecies, researchers should address potential inconsistencies through:

• Standardized expression and purification:

  • Express all subspecies variants under identical conditions

  • Purify proteins using identical protocols to minimize method-induced variations

• Protein quality verification:

  • Confirm protein folding consistency via circular dichroism

  • Verify thermal stability using differential scanning fluorimetry

  • Assess oligomeric state by size exclusion chromatography

• Assay normalization approaches:

  • Use equimolar concentrations of proteins

  • Perform activity assays in parallel with the same reagent batches

  • Normalize to internal standards

• Cross-validation with multiple assay types:

  • Combine in vitro biochemical assays with cell-based complementation tests

  • Validate findings using different detection methods

• Data integration framework:

  • Apply statistical methods appropriate for small sample sizes

  • Use Bayesian approaches to integrate data from multiple experiment types

How do recent genomic findings about F. tularensis subsp. mediasiatica impact frr research?

Recent genomic findings have several implications for frr research:

• Expanded geographic distribution: The discovery of F. tularensis subsp. mediasiatica in the Altai region of Russia, 1500 km from previously known foci, suggests there may be greater genetic diversity in frr genes across natural populations than previously recognized .

• Phylogeographic classification: The division of mediasiatica into three genetic groups (M.I, M.II, and M.III) based on MLVA profiling offers opportunities to study how frr sequences and functions might vary across these subgroups .

• Genetic distance insights: Genomic analyses showing approximately 2400 SNPs from the Most Recent Common Ancestor (MRCA) to contemporary mediasiatica strains provide context for understanding evolutionary pressures on essential genes like frr .

• Natural mutations affecting virulence: The identification of strain 60(B)57 with an inactivating mutation in prmA that confers avirulence highlights the importance of investigating mutations in translation-related genes, including potential variants of frr .

• Implications for zoonotic transmission: The varied geographic distribution and host range of F. tularensis subspecies suggests that frr may function under diverse conditions, potentially contributing to the pathogen's adaptability .

What are promising new methodologies for studying frr structure-function relationships?

Emerging technologies offer new approaches to understand frr structure-function relationships:

• Cryo-electron microscopy (Cryo-EM):

  • Allows visualization of frr-ribosome complexes at near-atomic resolution

  • Enables studying conformational changes during ribosome recycling

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

  • Provides insights into protein dynamics and solvent accessibility

  • Identifies regions of frr that undergo conformational changes upon ribosome binding

• Single-molecule fluorescence techniques:

  • Permits real-time observation of frr-ribosome interactions

  • Reveals kinetic parameters of binding and dissociation events

• Targeted protein engineering:

  • CRISPR-based approaches for introducing specific frr mutations

  • Directed evolution to identify frr variants with altered function

• Computational methods:

  • Molecular dynamics simulations to predict functional impacts of amino acid substitutions

  • Machine learning approaches to identify patterns in frr sequence-function relationships across bacterial species

How might frr interact with other translation factors during F. tularensis intracellular infection cycles?

The interaction network of frr during infection involves complex coordination with other translation factors:

• Temporal coordination with release factors:

  • frr likely functions in concert with release factors RF1, RF2, and RF3 during translation termination

  • The timing of these interactions may be modulated during different phases of infection

• Interaction with elongation factor G (EF-G):

  • EF-G assists frr in ribosome recycling through GTP hydrolysis

  • This partnership may be regulated differently in response to intracellular stresses

• Potential regulatory interactions:

  • Stress-responsive factors like RelA/SpoT may influence frr activity during stringent response

  • Small non-coding RNAs might regulate frr expression during adaptation to intracellular environments

• Subspecies-specific interaction partners:

  • Variation in frr sequences across F. tularensis subspecies might affect interaction strength with conserved partners

  • These differences could contribute to varying virulence and host adaptation capabilities

• Host factor interactions:

  • During infection, bacterial translation machinery including frr may interact with host cell components

  • These interactions could represent unexplored aspects of host-pathogen biology