Recombinant Bdellovibrio bacteriovorus Elongation factor P (efp)

What expression systems are most effective for producing recombinant B. bacteriovorus efp?

Two primary expression systems have been documented for recombinant B. bacteriovorus efp:

• Baculovirus expression system: This system has been used successfully to express full-length recombinant B. bacteriovorus efp (residues 1-188) with high purity (>85% by SDS-PAGE) .

• Mammalian cell expression: Alternative systems using mammalian cells have also been implemented for expression of the full-length protein .

When designing your expression strategy, consider:

• Including appropriate affinity tags that can be determined during the manufacturing process

• Expressing the full protein (residues 1-188) to maintain native functionality

• Using expression conditions optimized for bacterial proteins (lower temperature expression at 18-25°C may improve folding)

What are the optimal storage and reconstitution conditions for recombinant B. bacteriovorus efp?

For optimal stability and activity of recombinant B. bacteriovorus efp:

• Storage: The shelf life depends on multiple factors, including buffer composition and temperature. Generally:

  • Liquid form: 6 months at -20°C/-80°C

  • Lyophilized form: 12 months at -20°C/-80°C

• Reconstitution protocol:

  • Centrifuge vial briefly before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (recommended 50%)

  • Aliquot for long-term storage at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

How can researchers utilize recombinant B. bacteriovorus efp to study the unique translation mechanisms in predatory bacteria?

Recombinant B. bacteriovorus efp can serve as a valuable tool for investigating:

• Predatory lifecycle-specific translation: Using the recombinant protein in in vitro translation assays can help determine if efp is particularly important for synthesizing proteins involved in prey invasion and utilization.

• Comparative studies: Reconstituting translation systems with efp from B. bacteriovorus versus non-predatory bacteria can reveal mechanistic differences in translation control.

• Ribosome binding assays: Experiments comparing the affinity of B. bacteriovorus efp for ribosomes during different stages of the predatory lifecycle may reveal regulatory mechanisms .

Particularly valuable would be studying whether efp plays a role in the rapid protein synthesis required during the transition from attack phase to growth phase, when B. bacteriovorus must quickly adapt its proteome after invading prey .

How does efp expression change during the B. bacteriovorus predatory lifecycle?

Based on proteome analysis of B. bacteriovorus lifecycle stages, researchers can track expression patterns of translation factors like efp. A methodological approach would include:

• Synchronize cultures of B. bacteriovorus using established protocols with prey bacteria (e.g., E. coli ML35)

• Sample at defined timepoints representing different lifecycle stages:

  • Attack phase (free-swimming predators)

  • Early invasion (1 hour post-mixing)

  • Growth phase (2 hours post-mixing)

  • Late growth/septation (3 hours post-mixing)

• Extract RNA for semi-quantitative RT-PCR or total protein for proteomic analysis

• Normalize expression levels to constitutively expressed reference genes (e.g., Bd2400)

This approach allows researchers to determine if efp is differentially regulated during the predatory lifecycle, providing insights into its role in the rapid adaptation required during predation .

What controls should be included when studying recombinant B. bacteriovorus efp function in translation assays?

When designing experiments to study B. bacteriovorus efp function:

• Positive controls:

  • Include well-characterized efp from model organisms (e.g., E. coli)

  • Test translation of known polyproline-containing substrates

• Negative controls:

  • Translation reactions without efp

  • Translation of mRNAs lacking polyproline motifs

  • Denatured or inactive efp variants

• Experimental variables to control:

  • Buffer composition (particularly ion concentrations like Mg²⁺ and K⁺)

  • Temperature (optimal for B. bacteriovorus is 30-35°C)

  • pH (optimal range 7.0-8.0)

When conducting translation assays, consider the potential impact of the tags used in recombinant protein production, as these may affect function .

How can researchers validate that recombinant B. bacteriovorus efp is functionally active?

A multi-step validation approach is recommended:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Size exclusion chromatography to verify monomeric state

• Functional assays:

  • In vitro translation assays using polyproline-containing reporter constructs

  • Ribosome binding studies to confirm interaction with bacterial ribosomes

  • Complementation assays in efp-deficient bacterial strains

• Activity comparison:

  • Side-by-side comparison with native B. bacteriovorus efp (if available)

  • Comparison with efp from well-characterized bacterial species

How should researchers interpret contradictory results when studying B. bacteriovorus efp function?

When faced with contradictory results regarding B. bacteriovorus efp function:

• Consider lifecycle context: B. bacteriovorus has distinct lifecycle phases (attack phase vs. intraperiplasmic growth phase) that may affect efp function and requirements.

• Examine strain differences: Different B. bacteriovorus strains (e.g., HD100 vs. host-independent variants) may show different efp expression patterns or requirements.

• Assess experimental conditions: Temperature, pH, and ion concentrations significantly affect B. bacteriovorus metabolism and may influence efp function .

• Statistical approach: When analyzing contradictory data, conduct:

  • Outlier analysis to identify potential experimental anomalies

  • Multi-factorial analysis to identify interaction effects between variables

  • Meta-analysis if multiple datasets are available

• Validate with multiple techniques: If contradictory results persist, use complementary experimental approaches (e.g., both in vitro and in vivo studies) .

What advanced techniques can be used to study B. bacteriovorus efp interactions with the translational machinery?

Advanced techniques for studying efp interactions include:

• Cryo-electron microscopy (cryo-EM): Can visualize efp-ribosome complexes at near-atomic resolution, particularly valuable given the recent advances in imaging the B. bacteriovorus lifecycle at nanometer-scale resolution .

• Ribosome profiling: Can identify specific mRNAs whose translation is most dependent on efp in B. bacteriovorus.

• Crosslinking mass spectrometry: Can map the interaction interfaces between efp and other components of the translational machinery.

• Fluorescence microscopy with tagged efp: Can track the localization of efp during different stages of the predatory lifecycle, similar to approaches used for other B. bacteriovorus proteins .

• Genetic approaches:

  • CRISPR interference to modulate efp expression levels

  • Site-directed mutagenesis to create efp variants with altered function

  • Riboswitch-based regulation of efp expression using established tools for B. bacteriovorus gene expression control .

How can studies of efp contribute to understanding B. bacteriovorus as a potential "living antibiotic"?

Elongation factor P research may contribute to the development of B. bacteriovorus as a living antibiotic in several ways:

• Understanding growth regulation: Since efp is critical for translation of specific proteins, it may be involved in regulating the transition between attack phase and growth phase in the predatory lifecycle.

• Optimizing recombinant strain development: Knowledge of efp function could inform genetic engineering strategies to enhance predatory activity or target specificity.

• Metabolic insights: Studies of translation factors like efp can reveal how B. bacteriovorus efficiently converts prey resources into its own biomass, which is essential for its therapeutic potential .

• Predation efficiency markers: Expression levels of efp and other translation factors could serve as indicators of predatory fitness when evaluating engineered strains .

Research into translation factors could ultimately help address practical challenges in developing B. bacteriovorus for therapeutic applications, particularly in optimizing growth conditions and predatory efficiency .

What methodological approaches can be used to study the impact of efp on B. bacteriovorus predation efficiency?

To investigate how efp affects predation efficiency:

• Gene expression modulation:

  • Create strains with controllable efp expression using established riboswitch technology for B. bacteriovorus

  • Monitor predation kinetics using standardized assays:

    • Optical density measurements of prey cultures

    • Plaque formation assays

    • Fluorescence microscopy of labeled predator and prey

• Predation assay protocol:

  • Prepare synchronized cultures of B. bacteriovorus (10⁴ PFU/mL)

  • Add to E. coli or other Gram-negative prey (10⁸ CFU/mL)

  • Monitor at 24-hour intervals for:

    • Reduction in prey optical density

    • Increase in predator numbers

    • Morphological changes using microscopy

• Comparative analysis:

  • Compare wild-type vs. efp-modulated strains

  • Assess predation under varied conditions (temperature, pH, oxygen availability)

  • Analyze data using predation efficiency metrics:

    Strain	Prey Clearance Rate	Progeny Yield	Invasion Time
    Wild-type	Baseline	Baseline	Baseline
    efp-enhanced	Measure % change	Measure % change	Measure % change
    efp-reduced	Measure % change	Measure % change	Measure % change

• Molecular analysis:

  • Protein synthesis rate measurements during predation

  • Identification of key polyproline-containing proteins expressed during invasion