Recombinant Bdellovibrio bacteriovorus Proline--tRNA ligase (proS)

What is the biological function of Proline--tRNA ligase in Bdellovibrio bacteriovorus?

Proline--tRNA ligase (ProRS) in B. bacteriovorus, like in other bacteria, catalyzes the attachment of proline to its cognate tRNA in a two-step process critical for protein translation. This enzyme specifically recognizes and activates proline using ATP to form prolyl-AMP, then transfers the amino acid to the appropriate tRNA molecule. The process is essential for maintaining accuracy during protein synthesis in this predatory bacterium . ProRS activity is particularly important in B. bacteriovorus due to its unique predatory lifecycle, which requires precise protein synthesis during both attack phase and intraperiplasmic growth within prey bacteria .

How does the structure of B. bacteriovorus ProRS compare to that of non-predatory bacteria?

B. bacteriovorus ProRS belongs to the class II aminoacyl-tRNA synthetase family. Similar to other prokaryotic ProRS enzymes, it likely contains a large insertion domain (INS) between conserved motifs 2 and 3, which spans approximately 180 amino acids . This insertion domain plays a critical role in the editing mechanism that ensures translation fidelity. Comparative structural analysis suggests that the editing activity resides in a domain functionally and structurally distinct from the aminoacylation active site, allowing the enzyme to maintain high specificity during protein synthesis . The unique predatory lifestyle of B. bacteriovorus may have influenced subtle structural adaptations in ProRS that optimize its function during the rapid metabolic transitions required during predation cycles.

What techniques are typically used for recombinant expression of B. bacteriovorus ProRS?

Recombinant expression of B. bacteriovorus ProRS typically employs E. coli-based expression systems with careful optimization to ensure proper folding and functionality. The methodology involves:

• Gene amplification from B. bacteriovorus HD100 genomic DNA using PCR with high-fidelity polymerase

• Cloning into expression vectors containing appropriate affinity tags (His6, GST, etc.)

• Transformation into E. coli expression strains optimized for recombinant protein production

• Induction of expression under controlled temperature conditions (typically 16-25°C to enhance solubility)

• Cell lysis and protein purification using affinity chromatography followed by size exclusion and/or ion exchange chromatography

Expression conditions must be carefully calibrated to avoid inclusion body formation, which is common with recombinant aminoacyl-tRNA synthetases. Experimental validation of enzyme activity involves measuring aminoacylation efficiency using radioactive amino acids or fluorescent assays that monitor ATP consumption .

How does the editing mechanism of B. bacteriovorus ProRS contribute to predatory fitness?

B. bacteriovorus ProRS likely possesses both pre- and post-transfer editing mechanisms similar to those documented in E. coli ProRS . This editing function prevents the misactivation and mischarging of alanine, which is smaller than proline and can be mistakenly incorporated during protein synthesis. The prokaryotic insertion domain (INS) contains conserved residues that specifically contribute to this editing function .

For B. bacteriovorus, maintaining translational fidelity through effective ProRS editing would be particularly critical during:

• Attack phase - when the predator must synthesize specific proteins for prey recognition, attachment, and penetration

• Intraperiplasmic growth - when rapid, accurate protein synthesis is essential for utilizing prey resources

• Bdelloplast formation - when precise regulation of cell division proteins determines reproductive success

The editing efficiency may directly impact predatory success rates by ensuring proper synthesis of proteins needed for the complex predatory lifecycle. Future research comparing editing domain mutations with predatory efficiency measurements could provide valuable insights into this relationship .

What experimental approaches can resolve contradictory data regarding ProRS substrate specificity in B. bacteriovorus?

When facing contradictory data regarding substrate specificity of B. bacteriovorus ProRS, researchers should implement a multi-faceted approach:

• In vitro biochemical assays: Perform detailed kinetic analyses (kcat/KM determinations) with purified recombinant enzyme using:

  • ATP-PPi exchange assays to measure amino acid activation

  • tRNA aminoacylation assays with varying amino acid concentrations

  • Pre-steady-state kinetics to distinguish binding from catalytic steps

• Structural validation: Employ X-ray crystallography or cryo-EM to compare substrate-bound and apo-enzyme structures, with particular focus on the insertion domain implicated in editing functions .

• Mutagenesis studies: Conduct alignment-guided alanine scanning mutagenesis targeting conserved residues in the insertion domain, similar to the approach used for E. coli ProRS . Test each mutant for both aminoacylation and editing activities.

• In vivo approaches:

  • Create B. bacteriovorus strains with ProRS editing domain mutations

  • Assess predatory efficiency against various prey bacteria

  • Evaluate protein translation fidelity using reporter systems

• Computational analysis: Employ molecular dynamics simulations to analyze substrate interactions with the active site and editing domain.

By integrating data from these complementary approaches, researchers can resolve contradictions and develop a cohesive model of substrate specificity.

How can researchers effectively study the role of ProRS in B. bacteriovorus predatory cycle using gene manipulation techniques?

Studying ProRS function in B. bacteriovorus predation requires sophisticated genetic approaches due to the essential nature of this enzyme and the complex predatory lifecycle:

• Conditional expression systems: Implement anhydrotetracycline-inducible promoters similar to those used for testing P_tet constructs in other bacterial systems , allowing for controlled expression of wild-type or mutant ProRS variants.

• Silent deletion strategy: Create precise in-frame deletions of specific ProRS domains (particularly editing domains) using suicide vectors like pK18mobsacB, similar to the approach used for pilA gene deletion in B. bacteriovorus . This approach avoids introducing antibiotic resistance cassettes that might interfere with predation assays.

• Domain swapping experiments: Replace the editing domain of B. bacteriovorus ProRS with homologous domains from non-predatory bacteria to evaluate functional conservation.

• Real-time tracking: Label ProRS with fluorescent tags to track its localization during different stages of the predatory cycle.

• Quantitative predation assays: Measure predation efficiency using techniques like:

  • Prey optical density measurements over time (similar to Fig. 1A in source )

  • Endpoint prey enumeration to quantify population reduction percentages

  • Electron microscopy to visualize predator-prey interactions at different stages

These approaches would reveal how ProRS function, particularly its editing activity, influences the predatory efficiency of B. bacteriovorus.

What are the optimal conditions for assaying B. bacteriovorus ProRS activity in vitro?

Optimal conditions for assaying recombinant B. bacteriovorus ProRS activity include:

The assay should incorporate controls that distinguish aminoacylation from editing functions, particularly when investigating the insertion domain's role in maintaining translational fidelity . Temperature stability should be carefully monitored, as B. bacteriovorus enzymes may have adaptations related to survival in different environmental conditions during predatory and free-living states .

How can researchers differentiate between the roles of ProRS and other factors in B. bacteriovorus predatory efficiency?

Differentiating the specific contribution of ProRS from other factors influencing B. bacteriovorus predation requires systematic experimental design:

• Genetic complementation analysis:

  • Generate ProRS variants with mutations in: (a) aminoacylation domain only, (b) editing domain only, (c) both domains

  • Complement these into appropriate genetic backgrounds

  • Compare predatory efficiencies using established assays like those used for pilA mutant validation

• Combined genetic-biochemical approach:

  • Isolate B. bacteriovorus at different stages of predation

  • Measure ProRS aminoacylation and editing activities biochemically

  • Correlate enzyme activities with predatory efficiency metrics

• Controlled comparative studies:

  • Compare wild-type B. bacteriovorus with strains containing editing-deficient ProRS

  • Test predation on multiple prey species (similar to the E. coli vs. Salmonella comparison in source )

  • Assess predation under varying conditions (temperature, osmolarity) that might influence ProRS function

• Utilize non-predatory controls:

  • Similar to how non-predatory pilA mutants were used to distinguish predatory effects from mere presence of bacterial cells

  • Compare ProRS mutants with other non-predatory mutants affecting different aspects of the predatory cycle

This multi-faceted approach would distinguish ProRS-specific effects from other factors influencing predation, enabling more precise understanding of this enzyme's role in B. bacteriovorus biology.

What considerations should be made when designing experiments to study potential therapeutic applications of B. bacteriovorus ProRS inhibitors?

When investigating B. bacteriovorus ProRS as a potential therapeutic target, researchers should consider:

• Selectivity determination:

  • Compare inhibition profiles between B. bacteriovorus ProRS and human cytoplasmic/mitochondrial ProRS

  • Test effects on other bacterial ProRS enzymes, particularly from gut microbiome species

  • Design assays measuring IC₅₀ values across multiple species to create selectivity indices

• In vivo testing framework:

  • Utilize animal infection models similar to those described for testing B. bacteriovorus as a biological therapeutic

  • Develop appropriate controls to distinguish ProRS inhibition from other antimicrobial mechanisms

  • Monitor both pathogen reduction (e.g., Salmonella enumeration) and host response (cecal abnormalities)

• Potential combination therapies:

  • Test ProRS inhibitors in combination with intact B. bacteriovorus predatory cells

  • Evaluate potential synergistic effects with conventional antibiotics

  • Assess effects on predator-prey dynamics when both populations are present

• Safety profiling:

  • Evaluate effects on commensal bacteria using microbiome analysis

  • Assess potential for resistance development through serial passage experiments

  • Measure immunological responses to ensure lack of adverse inflammation

• Delivery considerations:

  • Develop formulations accounting for gut conditions (pH, enzymes)

  • Consider antacid co-administration (like CaCO₃ used with B. bacteriovorus cells)

  • Evaluate stability under physiologically relevant conditions

These approaches would enable responsible development of B. bacteriovorus ProRS inhibitors as potential therapeutics with appropriate selectivity and safety profiles.

What are common challenges in expressing recombinant B. bacteriovorus ProRS and how can they be overcome?

Researchers often encounter several challenges when expressing recombinant B. bacteriovorus ProRS:

These challenges reflect the unique biology of B. bacteriovorus as a predatory bacterium with potential differences in codon usage, protein folding mechanisms, and cofactor requirements compared to common expression hosts .

How can researchers address inconsistent results when studying the editing function of B. bacteriovorus ProRS?

When facing inconsistent results in studies of B. bacteriovorus ProRS editing function, researchers should implement the following systematic troubleshooting approach:

• Reagent and enzyme quality control:

  • Verify enzyme homogeneity using SDS-PAGE and analytical size exclusion chromatography

  • Confirm ATP quality and absence of contaminating ATPases

  • Use freshly prepared reagents and control for temperature fluctuations during assays

• Assay validation:

  • Implement multiple complementary assays to measure editing activity:

    • Thin-layer chromatography to monitor AMP formation (pre-transfer editing)

    • Acid gel electrophoresis to measure deacylation (post-transfer editing)

    • Misactivation of alanine using ATP-PPi exchange assays

  • Include E. coli ProRS as a positive control with established editing activity

• Environmental variables:

  • Systematically vary ionic conditions to mimic different B. bacteriovorus habitats

  • Test activity at different pH values reflecting various stages of predation

  • Consider the impact of molecular crowding agents to better mimic cellular conditions

• Substrate considerations:

  • Test both homologous and heterologous tRNA substrates

  • Verify tRNA aminoacylation status using acid urea gel electrophoresis

  • Evaluate the impact of tRNA modifications on editing efficiency

• Data analysis refinement:

  • Implement statistical approaches designed for enzyme kinetics with high variability

  • Use technical replicates (n≥3) and multiple biological replicates

  • Apply appropriate normalization strategies when comparing different protein preparations

This systematic approach enables identification of variables contributing to inconsistent results, leading to more reliable measurements of B. bacteriovorus ProRS editing function.

What methodological approaches enable accurate assessment of B. bacteriovorus ProRS contributions to predatory capability?

To accurately assess how ProRS contributes to B. bacteriovorus predatory capabilities, researchers should employ methodological approaches that bridge molecular enzymology with predation phenotypes:

• Quantitative predation assays:

  • Develop standardized prey reduction assays using optical density measurements

  • Implement end-point prey enumeration to quantify predation efficiency

  • Use fluorescently labeled prey to track predation dynamics in real-time

  • Compare predation on different prey species with varying tRNA^Pro characteristics

• Correlation analysis between enzyme properties and predatory success:

  • Measure ProRS aminoacylation and editing activities from B. bacteriovorus isolated at different predation stages

  • Correlate biochemical parameters (kcat/KM, editing efficiency) with predatory metrics

  • Develop mathematical models relating ProRS function to predatory fitness

• Genetic complementation with structure-guided variants:

  • Generate B. bacteriovorus strains expressing ProRS variants with specific alterations in catalytic or editing domains

  • Compare predatory capabilities of these strains under various conditions

  • Use site-directed mutagenesis to create variants with altered specificity or efficiency

• Competitive fitness assays:

  • Conduct head-to-head competition experiments between strains with different ProRS variants

  • Measure relative fitness using fluorescent labeling or genetic markers

  • Evaluate performance under stress conditions (nutrient limitation, temperature fluctuation)

• Multi-omics integration:

  • Combine transcriptomics and proteomics to measure global effects of ProRS alterations

  • Use metabolomics to detect changes in amino acid pools affecting ProRS function

  • Implement RNA-Seq analysis similar to methods described in source

These methodological approaches enable researchers to establish causal relationships between ProRS function and predatory capabilities, moving beyond correlative observations.

What are promising avenues for exploring the therapeutic potential of recombinant B. bacteriovorus ProRS?

Several promising research directions could advance our understanding of B. bacteriovorus ProRS as a therapeutic target:

• Structure-based drug design:

  • Determine high-resolution crystal structures of B. bacteriovorus ProRS

  • Identify unique structural features distinguishing it from human ProRS

  • Develop small molecule inhibitors targeting bacterial-specific pockets

  • Use computational screening to accelerate discovery of selective inhibitors

• ProRS inhibition to modulate predatory behavior:

  • Investigate whether partial ProRS inhibition could enhance predatory activity against specific pathogens

  • Develop compounds that selectively enhance editing functions to improve translational fidelity during predation

  • Explore potential for engineering B. bacteriovorus with modified ProRS to improve therapeutic applications

• Combination therapies:

  • Test synergistic effects between ProRS inhibitors and conventional antibiotics

  • Evaluate how ProRS modulation affects B. bacteriovorus predation on antibiotic-resistant pathogens

  • Develop dual-targeting approaches affecting both predator and prey bacterial populations

• Biofilm applications:

  • Assess how ProRS function influences B. bacteriovorus ability to penetrate and disrupt biofilms

  • Develop ProRS modulators that enhance anti-biofilm activity

  • Explore potential for targeting ProRS in biofilm-forming pathogens while preserving B. bacteriovorus predatory function

• Immunological interactions:

  • Investigate how B. bacteriovorus ProRS or its inhibition affects host immune recognition

  • Explore potential for engineering B. bacteriovorus with modified ProRS to reduce inflammatory responses

  • Evaluate implications for therapeutic applications in inflammatory bowel diseases

These directions build upon the established potential of B. bacteriovorus as a "living antibiotic" while focusing specifically on the role of ProRS in this unique predatory bacterium.

How might research on B. bacteriovorus ProRS contribute to understanding evolutionary adaptation in predatory bacteria?

Research on B. bacteriovorus ProRS offers unique insights into evolutionary adaptation in predatory bacteria:

• Comparative genomics and structural biology:

  • Compare ProRS sequences and structures across predatory and non-predatory bacteria

  • Identify selection pressures unique to the predatory lifestyle

  • Analyze insertion domains for evidence of specialized functions beyond those found in E. coli

• Functional adaptation studies:

  • Investigate whether B. bacteriovorus ProRS has evolved enhanced editing capabilities to maintain translational fidelity during rapid predatory growth

  • Examine kinetic parameters across predatory and non-predatory species

  • Test cross-species complementation to identify predator-specific functions

• Coevolutionary dynamics:

  • Explore whether B. bacteriovorus ProRS shows adaptations related to specific prey bacteria

  • Investigate potential molecular arms races between predator ProRS and prey defense mechanisms

  • Study how bacterial predation relates to translational fidelity across ecological niches

• Environmental adaptation markers:

  • Compare ProRS from B. bacteriovorus strains isolated from different environments

  • Assess temperature stability profiles related to different ecological niches

  • Evaluate how ProRS function varies under different conditions (similar to experiments testing survival at different temperatures)

• Horizontal gene transfer assessment:

  • Analyze ProRS and its insertion domain for evidence of horizontal gene transfer

  • Investigate whether predatory bacteria exchange tRNA synthetase components

  • Evaluate potential genetic exchange between predators and prey affecting translation machinery

This research could establish ProRS as a model system for understanding molecular adaptations enabling bacterial predation, complementing existing knowledge about predatory mechanisms .

What are the key considerations for researchers newly entering the field of B. bacteriovorus ProRS research?

Researchers newly entering B. bacteriovorus ProRS research should consider several key aspects:

• Interdisciplinary approach requirement:

  • Combine expertise in enzymology, microbiology, and predator-prey dynamics

  • Develop collaborations spanning structural biology to in vivo modeling

  • Establish methodological approaches that bridge molecular mechanisms with ecological function

• Technical challenges:

  • Anticipate difficulties in recombinant expression and purification

  • Prepare for the complexity of working with a predatory bacterium

  • Develop specialized assays that account for B. bacteriovorus biology

• Conceptual framework:

  • Consider ProRS function within the broader context of predatory lifecycle

  • Recognize the potential therapeutic applications while maintaining scientific rigor

  • Appreciate evolutionary implications of studying translation components in predatory bacteria

• Research infrastructure:

  • Establish both biochemical and microbiological capacities

  • Develop expertise in predation assays similar to those described in source

  • Consider computational resources for structural analysis and modeling

• Ethical and regulatory considerations:

  • Address biosafety considerations for working with predatory bacteria

  • Consider implications for potential therapeutic development

  • Engage with broader discussions about biological control agents