Recombinant Bdellovibrio bacteriovorus Threonine--tRNA ligase (thrS), partial

What is Bdellovibrio bacteriovorus Threonine--tRNA ligase and what is its biological function?

Bdellovibrio bacteriovorus Threonine--tRNA ligase (thrS), also known as Threonyl-tRNA synthetase (ThrRS), is a class II aminoacyl-tRNA synthetase enzyme with EC number 6.1.1.3. This essential enzyme catalyzes the attachment of threonine to its cognate tRNA (tRNA^Thr) through a two-step aminoacylation reaction necessary for protein synthesis. The enzyme first binds threonine and ATP to form threonyl adenylate (Thr-AMP) with the release of pyrophosphate, then transfers the threonine moiety to the 3'OH site of tRNA^Thr . The enzyme is homodimeric, with a molecular weight of approximately 150 kDa in bacteria . In B. bacteriovorus HD100, this enzyme plays a critical role in the predator's unique lifecycle, supporting protein synthesis during both the free-living attack phase and the intraperiplasmic growth phase within prey bacteria .

How does B. bacteriovorus thrS differ structurally and functionally from thrS in other bacterial species?

While the core catalytic mechanism remains conserved across species, B. bacteriovorus thrS contains structural adaptations that reflect the specialized metabolism of this predatory bacterium. The enzyme maintains the characteristic class II aminoacyl-tRNA synthetase architecture, with N-terminal domains and a catalytic core, but may feature specific adaptations related to the predatory lifestyle . These differences potentially influence substrate recognition, catalytic efficiency, and regulation during the transition between predatory phases.

Key structural distinctions include:

These structural differences may contribute to the enzyme's function during the predatory lifecycle, particularly during the transition from attack phase to growth phase inside prey bacteria .

What are the optimal storage and handling conditions for recombinant B. bacteriovorus thrS?

Optimal storage and handling conditions for recombinant B. bacteriovorus thrS depend on preparation format and research requirements:

Storage conditions:

• Liquid formulations: Store at -20°C to -80°C with a typical shelf life of 6 months

• Lyophilized formulations: Store at -20°C to -80°C with an extended shelf life of 12 months

• Working aliquots: Store at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is commonly recommended) for long-term storage

• Aliquot to minimize freeze-thaw cycles

Important handling considerations:

• Avoid repeated freeze-thaw cycles as they can compromise enzyme activity

• For experimental work, maintain the enzyme at appropriate temperature (typically 4°C) during assay preparation

• Consider buffer composition for specific applications, especially regarding divalent metal ion requirements for activity

What expression systems have been validated for producing recombinant B. bacteriovorus thrS?

Two primary expression systems have been validated for producing recombinant B. bacteriovorus thrS:

E. coli expression system:

• Most commonly used for bacterial protein expression

• Yields functional recombinant protein with >85% purity (SDS-PAGE)

• Suitable for structural studies and biochemical characterization

• Expression typically employs T7-based expression vectors

• Requires optimization of induction conditions and purification strategies

Yeast expression system:

• Alternative system reported for expression of B. bacteriovorus proteins

• May provide different post-translational modifications

• Reported to produce functional protein with >85% purity (SDS-PAGE)

• Can be advantageous for proteins that are toxic in bacterial systems

Recent advances in B. bacteriovorus genetic engineering have expanded options for heterologous expression, including:

• Development of Golden Standard (GS) cloning techniques specifically adapted for B. bacteriovorus HD100

• Characterization of constitutive and inducible promoters for controlled expression

• Implementation of Tn7 transposon-based chromosomal integration methods

How can I verify the enzymatic activity of recombinant B. bacteriovorus thrS in vitro?

Verification of recombinant B. bacteriovorus thrS enzymatic activity involves several complementary approaches:

ATP-PPi exchange assay:

• Measure the formation of [32P]ATP from [32P]PPi and AMP in the presence of threonine

• Reaction components: thrS enzyme, threonine, ATP, [32P]PPi, and appropriate buffer (typically containing Mg2+)

• Quantify radiolabeled ATP formation as a measure of adenylation activity

Aminoacylation assay:

• Monitor the attachment of [14C]threonine or [3H]threonine to tRNA^Thr

• Reaction components: thrS enzyme, labeled threonine, ATP, purified tRNA^Thr, and buffer

• Precipitate charged tRNA with TCA, filter, and quantify radioactivity

• Alternatively, use a more modern thin-layer chromatography (TLC) approach to separate charged and uncharged tRNAs

Coupled spectrophotometric assay:

• Monitor ATP hydrolysis during aminoacylation using coupled enzymes (pyruvate kinase and lactate dehydrogenase)

• Follow NADH oxidation spectrophotometrically at 340 nm

• Calculate enzyme activity based on the rate of NADH consumption

Activity validation should include appropriate controls:

• Negative control: reaction mixture without threonine

• Positive control: commercially available E. coli thrS (if available)

• Substrate specificity control: test with non-cognate amino acids

How can recombinant B. bacteriovorus thrS be applied in studies of bacterial predation mechanisms?

Recombinant B. bacteriovorus thrS serves as a valuable tool for investigating predation mechanisms through several research strategies:

Lifecycle stage-specific protein synthesis:

• Use thrS activity assays to measure protein synthesis capacity during different predatory lifecycle stages

• Correlate thrS activity with transcriptomic data to understand translational regulation during predation

• Develop thrS-based reporter constructs to visualize protein synthesis dynamics during prey invasion and consumption

Prey-predator interaction studies:

• Examine how B. bacteriovorus maintains translation fidelity during different predatory phases

• Investigate whether prey-derived amino acids or nucleotides directly influence thrS activity

• Use thrS as a marker for predator metabolic activity when co-cultured with prey bacteria

Host-independent growth investigation:

• Compare thrS expression and activity between host-dependent (HD) and host-independent (HI) B. bacteriovorus strains

• Determine if alterations in thrS function contribute to the HI phenotype

• Study how translation machinery adapts between predatory and saprophytic lifestyles

These applications can be facilitated through recently developed genetic tools for B. bacteriovorus, including:

• Inducible expression systems like PJ^ExD/EliR

• Constitutive high-expression promoters like P^BG37

• Chromosomal integration using Tn7 transposon technology

What role might B. bacteriovorus thrS play in the bacterium's potential applications as a "living antibiotic"?

B. bacteriovorus thrS may influence several aspects of the bacterium's potential as a "living antibiotic":

Predatory efficiency and metabolic activity:

• thrS activity directly impacts protein synthesis rates needed for efficient predation

• Optimization of thrS expression could potentially enhance predatory capabilities against pathogens

• Understanding thrS regulation may reveal strategies to maintain predatory fitness in therapeutic settings

Host range determination:

• Translation machinery adaptability may influence the range of bacterial prey that can be effectively utilized

• thrS function could be critical when B. bacteriovorus transitions between diverse prey environments

• Potential correlation between thrS activity and predation efficiency against specific pathogens

Survival in therapeutic contexts:

• thrS functionality under physiological conditions (temperature, pH, serum proteins) relevant to therapeutic applications

• Potential for engineering thrS variants with enhanced stability for increased predator persistence in therapeutic settings

• Role in predator recovery and reproduction after completing predation cycles in vivo

Research data supporting therapeutic potential:

What are the challenges in developing genetic manipulation approaches for studying B. bacteriovorus thrS function?

Developing genetic tools to study B. bacteriovorus thrS function presents several significant challenges:

Essential gene manipulation:

• thrS is likely essential for predator viability, complicating knockout studies

• Requires conditional expression systems or partial loss-of-function approaches

• Host-independent (HI) derivatives may be needed to study genes essential for predatory growth

Limited genetic toolbox:

• Despite recent advances, B. bacteriovorus genetic manipulation remains challenging

• Available approaches include:

  • Conjugation-based DNA transfer from E. coli (most common method)

  • Transposon mutagenesis in HI isolates

  • Antisense RNA for gene downregulation

  • Synthetic riboswitches for regulated expression

Technical challenges:

• Predatory lifestyle complicates isolation and maintenance of pure cultures

• Host-dependent (HD) growth requires co-culture with prey bacteria

• Difficulty in selecting transformants due to obligate predatory lifestyle

• Limited promoter options for controlled gene expression

Recent methodological advances that can be applied to thrS studies:

• Marker-free deletion techniques using suicide plasmids containing sacB for counter-selection (pSSK10, pK18mobsacB)

• The Golden Standard (GS) hierarchical assembly cloning technique adapted for B. bacteriovorus HD100

• Tn7 transposon-based chromosomal integration systems

• Characterized constitutive promoters (e.g., P^BG37) and inducible systems (PJ^ExD/EliR)

How might structural analyses of B. bacteriovorus thrS inform the development of novel antimicrobial strategies?

Structural analysis of B. bacteriovorus thrS could contribute to antimicrobial development through several approaches:

Selective inhibitor design:

• Identification of structural differences between B. bacteriovorus thrS and pathogen thrS enzymes

• Design of inhibitors that selectively target pathogen thrS while preserving B. bacteriovorus enzyme function

• Development of combination therapies using predatory bacteria and synthetase inhibitors with complementary target spectra

Predation enhancement strategies:

• Structural insights into regulatory domains that control thrS activity during predatory lifecycle

• Engineering thrS variants with optimized catalytic efficiency for enhanced predator performance

• Identification of structural adaptations that support protein synthesis during the transition from attack phase to growth phase

Novel antimicrobial peptide discovery:

• Investigation of potential moonlighting functions of thrS fragments

• Many aminoacyl-tRNA synthetases harbor domains with secondary functions or give rise to bioactive peptides

• Structural characterization may reveal peptide regions with direct antimicrobial activity

Structural features of interest:

What are the recommended purification strategies for recombinant B. bacteriovorus thrS?

Purification of recombinant B. bacteriovorus thrS can be achieved through the following optimized approach:

Expression and initial preparation:

• Express with appropriate affinity tag (His-tag is commonly used)

• Harvest cells and disrupt by sonication or French press

• Clarify lysate by centrifugation (30,000 × g, 30 minutes, 4°C)

• Filter supernatant through 0.45 μm membrane

Multi-step purification strategy:

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Wash with increasing imidazole concentrations (10-50 mM)

  • Elute with high imidazole buffer (250-300 mM)

• Ion exchange chromatography:

  • Apply to anion exchange column (Q Sepharose) equilibrated with low salt buffer

  • Elute with linear NaCl gradient (0-500 mM)

  • Collect and pool fractions containing thrS (monitor by SDS-PAGE and activity)

• Size exclusion chromatography:

  • Apply concentrated sample to Superdex 200 column

  • Elute with buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Verify purity by SDS-PAGE (target >95% purity)

Quality control assessment:

• Verify enzymatic activity using aminoacylation assay

• Confirm identity by mass spectrometry

• Assess oligomeric state by native PAGE or analytical gel filtration

• Check for contaminating nucleases or proteases

Typical yield from E. coli expression systems is 2-5 mg of purified protein per liter of culture, with final purity exceeding 85% by SDS-PAGE analysis .

How can I design experiments to investigate the role of thrS in B. bacteriovorus predation efficiency?

To investigate the role of thrS in B. bacteriovorus predation efficiency, consider the following experimental approaches:

Genetic manipulation approaches:

• Conditional expression system:

  • Create an inducible thrS construct using the PJ^ExD/EliR promoter/regulator system

  • Titrate expression levels to identify threshold requirements for predation

  • Monitor predation efficiency at different expression levels

• Domain swapping:

  • Generate chimeric thrS proteins by swapping domains with thrS from non-predatory bacteria

  • Evaluate how domain replacements affect predation efficiency

  • Identify domains critical for predatory lifestyle

• Site-directed mutagenesis:

  • Target conserved catalytic residues or predator-specific residues

  • Evaluate effects on aminoacylation activity and predation

  • Create structure-function maps of residues critical for predation

Predation assay designs:

• Quantitative predation efficiency measurement:

  • Co-culture B. bacteriovorus strains with prey bacteria (E. coli, Pseudomonas, etc.)

  • Monitor prey killing via viable counts, optical density, or live/dead staining

  • Calculate predation efficiency as reduction in prey population over time

• Microscopy-based approaches:

  • Use fluorescently labeled prey to visualize predation events

  • Implement time-lapse microscopy to track predator-prey interactions

  • Quantify predation parameters (attachment time, invasion time, bdelloplast formation)

• Metabolic labeling:

  • Use amino acid analogs (e.g., BONCAT) to track protein synthesis during predation

  • Correlate thrS activity with protein synthesis rates at different predatory stages

  • Identify prey-derived vs. predator-synthesized proteins

Data analysis framework:

What approaches can be used to study the potential interactions between B. bacteriovorus thrS and prey-derived molecules?

Several complementary approaches can investigate interactions between B. bacteriovorus thrS and prey-derived molecules:

Biochemical interaction studies:

• Pull-down assays:

  • Immobilize tagged recombinant thrS on appropriate resin

  • Incubate with prey cell lysates or fractions

  • Elute and identify interacting molecules by mass spectrometry

• Surface plasmon resonance (SPR):

  • Immobilize thrS on sensor chip

  • Flow prey-derived fractions or candidate molecules

  • Measure binding kinetics and affinity constants

• Isothermal titration calorimetry (ITC):

  • Directly measure thermodynamic parameters of thrS-ligand interactions

  • Determine binding stoichiometry and specificity

  • Characterize interactions with prey-derived nucleotides or metabolites

Structural approaches:

• X-ray crystallography:

  • Crystallize thrS alone and in complex with prey-derived molecules

  • Solve structures to identify binding sites and conformational changes

  • Compare with thrS structures from non-predatory bacteria

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

  • Monitor conformational changes in thrS upon interaction with prey molecules

  • Identify regions with altered solvent accessibility

  • Map potential allosteric regulation sites

Functional assays:

• Enzymatic activity modulation:

  • Test thrS activity in presence of prey cell extracts or fractions

  • Identify inhibitory or stimulatory effects on aminoacylation

  • Characterize specificity of effects for B. bacteriovorus thrS vs. other synthetases

• Prey-specific adaptation studies:

  • Compare thrS activity when B. bacteriovorus is grown on different prey species

  • Identify prey-specific adaptations in enzyme kinetics or substrate preference

  • Correlate with transcriptomic or proteomic changes during predation

Bioinformatic analyses:

• Conduct comparative genomic analyses across predatory and non-predatory bacteria

• Identify predator-specific sequence motifs or domains in thrS

• Model potential interaction sites based on sequence conservation patterns

This multi-faceted approach can reveal whether B. bacteriovorus thrS has evolved specific adaptations for interaction with prey-derived molecules, potentially contributing to the bacterium's predatory efficiency and host range.

What are the latest developments in understanding the role of thrS in B. bacteriovorus biology?

Recent advances in understanding B. bacteriovorus thrS function have emerged from several complementary research directions:

Regulatory mechanisms during predation cycle:

• Transcriptomic studies have revealed stage-specific expression patterns of thrS during the predatory lifecycle

• thrS expression appears coordinated with other translation machinery components during the transition from attack phase to growth phase

• Evidence suggests specific regulatory mechanisms may control thrS activity to support the rapid protein synthesis required during prey consumption

Genomic and evolutionary insights:

• Comparative genomic analyses have identified predator-specific features in thrS sequences

• These adaptations may reflect optimization for the unique metabolic demands of predatory bacteria

• Recent reclassification of Bdellovibrio from Deltaproteobacteria to class Oligoflexia (with proposals for a distinct phylum Bdellovibrionota) has prompted reexamination of evolutionary relationships among predatory bacteria translation machinery

Technological developments:

• New genetic tools specifically adapted for B. bacteriovorus manipulation have enabled more sophisticated studies

• The Golden Standard (GS) hierarchical assembly cloning technique has been adapted for B. bacteriovorus HD100

• Characterization of constitutive (P^BG37) and inducible (PJ^ExD/EliR) promoters now allows controlled gene expression

• Tn7 transposon-based chromosomal integration systems provide stable genetic modification options

These advances provide a foundation for future studies exploring the specific roles of thrS in supporting B. bacteriovorus' predatory lifestyle and potential therapeutic applications.

How might thrS function in B. bacteriovorus differ between host-dependent and host-independent growth modes?

The function of thrS in B. bacteriovorus likely shows significant differences between host-dependent (HD) and host-independent (HI) growth modes:

Differential expression patterns:

• In HD mode, thrS expression must coordinate with predatory lifecycle stages

• In HI mode, expression likely follows patterns more similar to conventional bacteria

• Transcriptomic studies suggest different regulatory networks govern translation machinery in these distinct growth modes

Functional adaptations:

• HD growth requires rapid protein synthesis during the bdelloplast stage to support filamentous growth and progeny formation

• HI growth may involve more conventional translation regulation responsive to nutrient availability

• Different sets of genes are essential for HD versus HI growth, suggesting potential functional divergence in core processes including translation

Metabolic context differences:

Experimental evidence and research directions:

• HI strains have been instrumental in studying B. bacteriovorus genetics by enabling cultivation without prey

• Transposon mutagenesis in HI isolates has helped identify genes essential for different growth modes

• Comparative studies of thrS activity between HD and HI strains could reveal adaptation mechanisms

• Engineering thrS variants optimized for either growth mode might enhance research applications

Understanding these differences has practical implications for both fundamental research and potential applications of B. bacteriovorus as a biocontrol agent or living antibiotic.

What are the most promising future research directions involving B. bacteriovorus thrS?

Several promising research directions involving B. bacteriovorus thrS warrant further investigation:

Structural biology and drug development:

• High-resolution structural characterization of B. bacteriovorus thrS

• Comparative structural analyses with pathogen thrS enzymes

• Structure-guided design of selective inhibitors targeting pathogen synthetases while sparing predator enzymes

• Exploration of potential moonlighting functions or bioactive peptides derived from thrS

Synthetic biology applications:

• Engineering B. bacteriovorus strains with enhanced thrS activity for improved predation efficiency

• Development of biosensors using thrS-based reporters to monitor predator activity in complex environments

• Creation of predator strains with expanded prey range through synthetase modifications

• Design of genetic circuits controlling thrS expression to modulate predator behavior

Therapeutic development:

• Investigation of thrS role in predator survival within mammalian hosts

• Optimization of thrS function for enhanced predator persistence in therapeutic contexts

• Study of potential immunomodulatory effects of B. bacteriovorus proteins including thrS

• Development of combination therapies pairing predatory bacteria with conventional antibiotics

Fundamental biological questions:

• Elucidation of translation regulation mechanisms during predatory lifecycle transitions

• Investigation of potential horizontal gene transfer between predator and prey affecting translation machinery

• Understanding how thrS activity is coordinated with other cellular processes during predation

• Exploration of the evolutionary history of thrS in predatory bacteria

Emerging methodologies enabling these directions:

• Cryo-electron microscopy for high-resolution structural studies

• Single-cell RNA-seq to capture transcriptional dynamics during predation

• Advanced genetic tools specifically optimized for B. bacteriovorus

• In vivo imaging techniques to track predator activity in complex environments

These research directions could significantly advance both fundamental understanding of predatory bacteria and their practical applications in biotechnology and medicine.