Recombinant Bdellovibrio bacteriovorus Putative phosphotransferase Bd1093 (Bd1093)

What is Bdellovibrio bacteriovorus and why is its Bd1093 protein significant for research?

Bdellovibrio bacteriovorus is a small Deltaproteobacterium that has gained scientific attention for its unique ability to prey on other Gram-negative bacteria. This predatory bacterium enters the periplasmic space of prey bacteria and consumes them from within, making it a promising candidate as a "living antibiotic" against antibiotic-resistant infections . The Bd1093 protein is annotated as a putative phosphotransferase, specifically a pyruvate phosphate dikinase regulatory protein (PPDK regulatory protein) with EC numbers 2.7.11.32 and 2.7.4.27 . Understanding the function of Bd1093 could provide valuable insights into the predatory mechanism of B. bacteriovorus and potentially contribute to novel antibiotic strategies.

What expression systems are most effective for recombinant Bd1093 production?

Several expression systems have been employed for the production of recombinant Bd1093, each with distinct advantages:

For most research applications, E. coli-based expression with BirA technology for in vivo biotinylation has proven particularly useful for functional studies, allowing site-specific biotinylation through an AviTag . When designing expression constructs, include a purification tag (typically His-tag) and consider codon optimization for the expression host to enhance protein yield.

What structural and biochemical properties of Bd1093 are important to consider in experimental design?

When designing experiments involving Bd1093, several key properties should be considered:

• Molecular characteristics:

  • Theoretical molecular weight: 34 kDa

  • Observed molecular weight: 65-80 kDa under reducing conditions

  • Full amino acid sequence comprises residues 1-281

• Structural features:

  • Contains putative ATP-binding sites

  • Likely includes catalytic domains for both kinase (EC 2.7.11.32) and phosphotransferase (EC 2.7.4.27) activities

  • Proper folding is critical for enzymatic activity

• Stability considerations:

  • Store lyophilized protein at -20°C to -70°C for long-term stability

  • After reconstitution, store at 2-8°C for up to 1 month

  • Avoid repeated freeze-thaw cycles as they may compromise protein activity

When planning experiments, account for these properties to ensure optimal protein handling and experimental outcomes.

How can I verify the activity of purified recombinant Bd1093?

Verifying the enzymatic activity of recombinant Bd1093 requires appropriate assays for its putative phosphotransferase function:

• Standard phosphotransferase activity assay:

  • Reaction buffer: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT

  • ATP concentration: 0.5-1 mM

  • Substrate concentration: 0.1-0.5 mM (potentially pyruvate-containing compounds)

  • Detection methods: malachite green assay for phosphate release or coupled enzyme assay

• Binding assays:

  • Surface plasmon resonance (SPR) to measure interaction with potential substrates

  • Isothermal titration calorimetry (ITC) to determine binding affinity and thermodynamics

  • Microscale thermophoresis for detecting interactions in solution

• Critical controls:

  • No-enzyme control to account for spontaneous ATP hydrolysis

  • Heat-inactivated enzyme control

  • Known phosphotransferase with similar function as positive control

It's important to note that substrate specificity remains largely uncharacterized, so screening with various potential substrates may be necessary.

What purification strategies yield the highest purity of recombinant Bd1093?

For optimal purification of recombinant Bd1093, a multi-step approach is recommended:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged Bd1093

  • Buffer conditions: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 10-250 mM imidazole gradient

• Intermediate purification:

  • Ion exchange chromatography (typically anion exchange)

  • Buffer conditions: 20 mM Tris-HCl pH 8.0, 50-500 mM NaCl gradient

• Polishing step:

  • Size exclusion chromatography (SEC)

  • Buffer conditions: PBS with trehalose as a stabilizing agent

Throughout purification, maintain temperature at 4°C and include protease inhibitors during initial lysis. The final product should achieve >85% purity as assessed by SDS-PAGE .

How can I design experiments to determine the physiological substrates of Bd1093?

Identifying the physiological substrates of Bd1093 requires a comprehensive experimental approach:

• Phosphoproteomic analysis:

  • Generate a Bd1093 knockout strain of B. bacteriovorus

  • Compare phosphoproteomes of wild-type and knockout strains using SILAC labeling

  • Identify differentially phosphorylated proteins via LC-MS/MS

  • Confirm direct interaction using recombinant proteins

• Proximity-based labeling:

  • Create a Bd1093-BioID fusion expressed in B. bacteriovorus

  • Allow biotinylation of proximal proteins during predation cycle

  • Purify biotinylated proteins and identify via mass spectrometry

  • Validate interactions through co-immunoprecipitation

• In vitro validation:

  • Express and purify candidate substrates

  • Perform in vitro phosphorylation assays using γ-³²P-ATP

  • Map phosphorylation sites by mass spectrometry

  • Generate phospho-specific antibodies for in vivo validation

This multi-faceted approach compensates for the limitations of individual methods and increases confidence in identified substrates.

What role might Bd1093 play in the predatory lifecycle of Bdellovibrio bacteriovorus?

Based on its annotation as a putative phosphotransferase with potential regulatory functions, Bd1093 may play critical roles in the predatory lifecycle:

• Metabolic regulation during predation:

  • As B. bacteriovorus transitions between free-living and intraperiplasmic stages, significant metabolic rewiring occurs

  • Phosphorylation-based regulation by Bd1093 may help coordinate these metabolic shifts

  • PPDK (pyruvate phosphate dikinase) regulation could be particularly important for energy metabolism during predation

• Experimental approaches to determine lifecycle roles:

  • Time-course analysis of Bd1093 expression during predation stages

  • Construction of conditional knockouts to assess predation efficiency

  • Fluorescently tagged Bd1093 to track subcellular localization during predation

  • Metabolomic analysis of wild-type vs. Bd1093 mutants during predation

• Potential significance for predator-prey dynamics:

  • If Bd1093 regulates energy metabolism, it may influence predation efficiency

  • Could be involved in sensing prey availability or quality

  • May contribute to the transition between attack phase and growth phase

These hypotheses can be tested through carefully designed genetic and biochemical experiments focusing on predatory fitness.

How do mutations in Bd1093 affect the predatory abilities of Bdellovibrio bacteriovorus?

To investigate the effects of Bd1093 mutations on predatory function:

• Systematic mutagenesis approach:

  • Generate catalytic site mutants (e.g., D137A) to disrupt phosphotransferase activity

  • Create domain deletion variants to identify functional regions

  • Use CRISPR-Cas9 to introduce point mutations at conserved residues

• Predation phenotype analysis:

  • Quantitative predation assays comparing wild-type and mutant strains

  • Time-lapse microscopy to assess predation kinetics

  • Competition assays between mutant and wild-type predators

  • Biofilm predation assays to test predatory efficiency in structured environments

• Molecular and cellular analyses:

  • Phosphoproteomic comparison between wild-type and mutant strains

  • Transcriptomic analysis to identify compensatory mechanisms

  • Metabolic flux analysis to detect alterations in energy utilization

These approaches can reveal the specific contributions of Bd1093 to the predatory lifestyle of B. bacteriovorus.

What technical challenges exist in crystallizing Bd1093 for structural studies?

Structural studies of Bd1093 face several technical challenges:

• Protein stability and solubility issues:

  • The discrepancy between theoretical (34 kDa) and observed (65-80 kDa) molecular weights suggests unusual structural properties

  • Potential aggregation during concentration

  • Difficulty maintaining enzymatic activity during purification

• Crystallization strategies:

  • Surface entropy reduction (SER) to replace surface lysine and glutamate residues with alanine

  • Co-crystallization with substrates or ATP analogs to stabilize specific conformations

  • Screening different constructs (full-length vs. domains) to identify crystallizable fragments

  • Use of crystallization chaperones such as antibody fragments

• Alternative structural approaches:

  • Cryo-electron microscopy for full-length protein structure

  • NMR spectroscopy for individual domains

  • Small-angle X-ray scattering (SAXS) for solution conformation

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

Systematic screening of crystallization conditions with various additives and protein constructs offers the best chance of success for obtaining diffraction-quality crystals.

How can I design experiments to analyze the kinetic parameters of Bd1093?

Rigorous kinetic analysis of Bd1093 requires careful experimental design:

• Steady-state kinetics:

  • Measure initial reaction velocities at varying substrate concentrations

  • Determine Km and kcat using Michaelis-Menten equation

  • Analyze potential cooperativity using Hill equation

  • Establish optimal reaction conditions (pH, temperature, buffer components)

• Pre-steady-state kinetics:

  • Use stopped-flow techniques to measure rapid kinetics

  • Determine rate constants for individual steps in the reaction mechanism

  • Identify rate-limiting steps in the catalytic cycle

• Inhibition studies:

  • Test product inhibition patterns

  • Use transition state analogs to probe mechanism

  • Analyze competitive vs. non-competitive inhibition

• Data analysis and modeling:

  • Fit experimental data to appropriate kinetic models

  • Use global fitting approaches for complex kinetic schemes

  • Validate models through prediction and experimental testing

These approaches will provide insights into the catalytic mechanism and efficiency of Bd1093, which could inform its physiological role in B. bacteriovorus.

What are the recommended controls when studying interactions between Bd1093 and potential binding partners?

When investigating protein-protein interactions involving Bd1093, proper controls are essential:

• Negative controls:

  • Non-specific proteins of similar size and charge

  • Heat-denatured Bd1093

  • Competitive binding with excess unlabeled protein

  • Irrelevant prey bacterial proteins for predation-specific studies

• Specificity controls:

  • Mutated binding sites in both Bd1093 and putative partners

  • Truncated protein constructs to map interaction domains

  • Cross-linking distance controls

  • Competition with predicted binding peptides

• Methodology-specific controls:

  • For pull-down assays: beads-only, tag-only, and reverse pull-down controls

  • For fluorescence resonance energy transfer (FRET): donor-only and acceptor-only controls

  • For surface plasmon resonance: reference channel and buffer injection controls

  • For yeast two-hybrid: autoactivation and non-specific activation controls

Implementing these controls helps distinguish genuine interactions from experimental artifacts and increases confidence in identified binding partners.

How can differential phosphoproteomic analysis be used to study Bd1093 function?

Differential phosphoproteomics offers a powerful approach to understand Bd1093 function:

• Experimental design:

  • Compare wild-type B. bacteriovorus with Bd1093 knockout or catalytically inactive mutants

  • Use SILAC labeling for quantitative comparison

  • Isolate samples at different stages of the predatory lifecycle

  • Include appropriate biological and technical replicates

• Sample preparation workflow:

  • Extract total protein from bacterial cultures

  • Digest proteins using trypsin or Lys-C

  • Enrich for phosphopeptides using TiO₂ or IMAC

  • Fractionate samples to increase phosphoproteome coverage

• Mass spectrometry analysis:

  • LC-MS/MS analysis using high-resolution mass spectrometers

  • Data-dependent acquisition for discovery

  • Parallel reaction monitoring for targeted validation

  • Quantify changes in phosphorylation stoichiometry

• Bioinformatic analysis:

  • Identify differentially phosphorylated proteins

  • Map phosphorylation sites to functional domains

  • Perform motif analysis to identify Bd1093 recognition sequences

  • Conduct pathway enrichment analysis to identify regulated processes

This approach can reveal the broader impact of Bd1093 on cellular phosphorylation networks during predation.

What potential biotechnological applications might emerge from studying Bd1093?

Understanding Bd1093 could lead to several innovative biotechnological applications:

• Engineered predatory bacteria:

  • Modified Bd1093 activity could enhance predation efficiency against specific pathogens

  • Development of predatory bacteria with expanded host range for biocontrol applications

  • Creating predators with tunable predation rates for environmental applications

• Novel enzymatic tools:

  • Engineered Bd1093 variants with altered substrate specificity

  • Development of biosensors based on Bd1093 phosphotransferase activity

  • Creation of synthetic signaling pathways incorporating Bd1093 domains

• Therapeutic applications:

  • Understanding Bd1093's role in predation could inform development of "living antibiotics"

  • Potential target for enhancing B. bacteriovorus predation against antibiotic-resistant pathogens

  • Design of small molecule modulators of predatory activity for combination therapies

• Methodological advances:

  • Development of new protein-protein interaction detection methods

  • Novel approaches for studying bacterial predator-prey dynamics

  • Improved methods for expression and characterization of challenging bacterial proteins

These potential applications highlight the broader significance of fundamental research on Bd1093 and B. bacteriovorus predatory mechanisms.