Recombinant Bdellovibrio bacteriovorus Pantothenate synthetase (panC)

What is Bdellovibrio bacteriovorus and what makes its pantothenate synthetase significant?

Bdellovibrio bacteriovorus is a predatory delta-proteobacterium that invades the periplasmic space of other Gram-negative bacteria, where it replicates and eventually lyses the host cell. It represents a unique bacterial predation system with applications as a potential biocontrol agent against pathogenic bacteria, including multidrug-resistant strains.

The pantothenate synthetase (panC) of B. bacteriovorus catalyzes the final step in pantothenate (vitamin B5) biosynthesis, which is a critical precursor for coenzyme A (CoA) and acyl carrier protein. This pathway is particularly significant because it is absent in mammals (who obtain pantothenate through diet) but essential for bacterial metabolism, making it a potential antimicrobial target .

B. bacteriovorus panC likely plays a crucial role in supporting the unique predatory lifecycle, as CoA-dependent processes are essential for energy production and cellular remodeling during the transition between free-living and intraperiplasmic growth phases. Understanding its structure and function could provide insights into predator-specific metabolic adaptations.

How does the pantothenate biosynthesis pathway function in bacterial metabolism?

Pantothenate synthetase (panC) catalyzes the ATP-dependent condensation of D-pantoate and β-alanine to generate pantothenate through the formation of an intermediate, pantoyl adenylate . This reaction represents the final step in the four-step pantothenate biosynthesis pathway (involving enzymes encoded by panB, panC, panD, and panE genes).

The importance of this pathway in bacterial metabolism stems from pantothenate's role as a precursor for:

• Coenzyme A (CoA): Essential for various intracellular processes including fatty acid metabolism, the citric acid cycle, and cellular respiration

• Acyl carrier protein: Required for fatty acid synthesis and polyketide biosynthesis

• Secondary metabolites: Including non-ribosomal peptides and signaling molecules

This pathway is particularly critical for microorganisms since, unlike mammals who acquire pantothenate through diet, bacteria must synthesize it de novo. In predatory bacteria like B. bacteriovorus, this pathway likely supports the metabolic demands of the predatory lifecycle, including rapid transitions between attack phase and growth phase.

What genetic tools are available for studying B. bacteriovorus proteins?

Several genetic tools have been developed for studying B. bacteriovorus, which can be applied to investigate recombinant panC:

• Mobilizable vector plasmids:

  • pSUP404.2: Contains two plasmid replicons (p15A ori and RSF1010 replication region), shows high transfer frequency (10^-1 to 10^-4), and maintains stable inheritance without selective pressure. It contains an average of seven copies per genome as determined by quantitative PCR .

  • pSUP202: Shows lower transfer frequency (10^-5 to 10^-6) and is less stable in B. bacteriovorus due to its pMB1 origin .

• Antibiotic selection markers:

  • pSUP404.2 carries both chloramphenicol and kanamycin resistance genes, which can be used for selection or insertional inactivation in cloning experiments .

• Expression systems:

  • E. coli hypersecretor Tol-pal mutants have been successfully used for extracellular production of B. bacteriovorus proteins .

  • Pseudomonas putida hypersecretor Tol-pal mutants also provide an alternative expression system .

These tools enable several experimental approaches including gene knockout studies, heterologous expression for biochemical characterization, tagged protein production for localization studies, and site-directed mutagenesis for structure-function analysis.

What expression systems yield optimal results for recombinant B. bacteriovorus panC?

While specific optimization data for panC expression is not directly provided in available literature, successful expression approaches for other B. bacteriovorus proteins suggest the following strategy:

• Host selection:

  • Primary recommendation: E. coli hypersecretor Tol-pal mutants have demonstrated success with extracellular production of B. bacteriovorus proteins .

  • Alternative system: Pseudomonas putida hypersecretor Tol-pal mutants offer an additional expression platform with potentially better folding of predatory bacterial proteins .

  • Standard systems: BL21(DE3) derivatives including Arctic Express (for low-temperature expression) or Origami (for disulfide bond formation) may be considered.

• Vector design considerations:

  • Codon optimization for the expression host

  • Fusion partners to enhance solubility (MBP, GST, SUMO)

  • Signal sequences for extracellular secretion in Tol-pal mutant systems

  • Inducible promoter systems with titratable expression levels

• Expression conditions optimization:

  • Temperature range: 16-25°C often improves solubility

  • Inducer concentration: Lower IPTG levels (0.1-0.5 mM) for slower expression

  • Media composition: Rich media for high cell density

  • Duration: Extended expression time (16-24 hours) at lower temperatures

• Purification strategy:

  • Immobilized metal affinity chromatography for His-tagged constructs

  • Size exclusion chromatography for final purification

  • Buffer optimization including glycerol, reducing agents, and salt concentration

When testing expression systems, small-scale parallel trials with multiple constructs and conditions, followed by solubility assessment and activity assays, provide the most efficient path to optimization.

How can isothermal titration calorimetry be optimized for studying substrate binding in recombinant panC?

Isothermal titration calorimetry (ITC) provides valuable thermodynamic data on substrate binding to enzymes like pantothenate synthetase. Optimizing ITC for B. bacteriovorus panC requires careful consideration of:

• Sample preparation requirements:

  • Protein purity: >95% homogeneity by SDS-PAGE and size exclusion

  • Protein concentration: 10-20 μM in cell (optimized based on binding affinity)

  • Buffer composition: Identical for protein and ligands, low heat of ionization

  • Protein stability: Verified by dynamic light scattering before experiments

• Experimental design for multi-substrate enzyme:

  • Sequential binding studies: Individual experiments for ATP, D-pantoate, and β-alanine

  • Order-of-addition experiments: Determine sequential binding mechanisms

  • Temperature selection: 25°C standard, with range studies for enthalpy-entropy relationships

  • Control titrations: Buffer-into-buffer and buffer-into-protein baselines

• Data analysis considerations:

  • Binding model selection: Single-site binding for individual substrates; sequential or cooperative models for multiple substrates

  • Parameter extraction: Binding affinity (Kd), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n)

  • Integrated analysis with enzyme kinetics data

Example experimental protocol:

• Prepare 15 μM B. bacteriovorus panC in 50 mM phosphate buffer, pH 7.5, 150 mM NaCl, 5% glycerol

• Prepare 300 μM ATP in identical buffer

• Load protein into sample cell and ATP into syringe

• Program injection schedule: 0.5 μL initial injection followed by 19 injections of 2 μL

• Analyze data using appropriate binding model

• Repeat with D-pantoate and β-alanine

This approach provides comprehensive thermodynamic characterization of substrate binding, revealing insights into the catalytic mechanism and potential unique adaptations of B. bacteriovorus panC.

What strategies overcome protein solubility issues when expressing recombinant panC?

Predatory bacterial proteins like B. bacteriovorus pantothenate synthetase often present solubility challenges during recombinant expression. The following systematic approach addresses these challenges:

• Expression system modifications:

  • Fusion partners: MBP, GST, SUMO, or thioredoxin tags significantly enhance solubility

  • Expression temperature: Reducing to 16-20°C slows folding and prevents aggregation

  • Induction strategy: Lower inducer concentrations and extended expression time

  • Host strain selection: C41/C43(DE3) for toxic proteins or SHuffle for disulfide formation

• Protein engineering approaches:

  • Remove hydrophobic patches through site-directed mutagenesis

  • Create truncated constructs based on domain predictions

  • Increase surface charged residues to enhance solubility

  • Utilize bioinformatic tools (AGGRESCAN, SOLpro) to predict aggregation-prone regions

• Buffer optimization:

  • Solubilizing additives: 5-10% glycerol, 50-300 mM NaCl, 0.1-1% non-ionic detergents

  • Stabilizing cofactors: ATP or non-hydrolyzable analogs, magnesium ions

  • pH screening: Test range from pH 6.5-8.0, typically away from the isoelectric point

  • Reducing agents: DTT or β-mercaptoethanol if cysteine residues are present

• Refolding strategies (if inclusion bodies form):

  • Gentle solubilization: 8M urea or 6M guanidinium hydrochloride

  • Refolding methods: Dilution, dialysis, on-column refolding

  • Additives during refolding: L-arginine, glycerol, low concentrations of detergents

  • Chaperone co-expression: GroEL/ES or DnaK/J/GrpE systems

Systematic testing through a decision tree approach (starting with fusion tags and temperature optimization, then buffer screening, and finally refolding if necessary) provides the most effective path to obtaining soluble, active enzyme.

How might mutations in the catalytic domain affect B. bacteriovorus predatory lifecycle?

The relationship between panC mutations and B. bacteriovorus predation requires systematic investigation through targeted mutagenesis and phenotypic analysis. Based on pantothenate synthetase's role in CoA biosynthesis, the following effects might be observed:

• Potential predatory phase impacts:

  • Attack phase: Reduced motility due to energy limitations from impaired CoA production

  • Prey recognition: Potentially altered sensing capabilities due to membrane composition changes

  • Invasion: Delayed or inefficient penetration into prey periplasm

  • Bdelloplast formation: Compromised ability to modify prey cell structure during early stages

• Growth phase consequences:

  • Replication rate: Slower proliferation within prey due to limited fatty acid metabolism

  • Resource utilization: Inefficient use of prey components requiring CoA-dependent pathways

  • Progeny development: Reduced number or viability of progeny cells

  • Lysis timing: Altered synchronization of prey cell lysis

• Experimental design for investigation:

  • Site-directed mutagenesis targeting key residues (ATP binding, catalytic residues, substrate specificity)

  • Complementation with wild-type panC or controlled expression systems

  • Predation efficiency assays measuring prey killing rates and progeny yields

  • Time-lapse microscopy tracking predatory cycle progression

• Expected phenotypes based on mutation severity:

  • Mild mutations: Extended predatory cycle duration with normal completion

  • Moderate mutations: Reduced predation efficiency with partial cycle completion

  • Severe mutations: Complete inhibition of predatory lifecycle

This research would provide fundamental insights into the metabolic requirements for bacterial predation and potentially identify key regulatory points in the predator-prey interaction.

How does the predatory efficiency of B. bacteriovorus correlate with its ability to kill multidrug-resistant pathogens?

B. bacteriovorus has demonstrated remarkable predatory capabilities against multidrug-resistant (MDR) clinical pathogens, including both planktonic cultures and biofilms. The correlation between predatory efficiency and antimicrobial resistance includes:

• Predation efficacy against MDR planktonic bacteria:

  • Most efficient predation observed against drug-resistant E. coli, with a 3.11 log10 reduction in viability

  • Significant reductions also observed for K. pneumoniae, P. aeruginosa, and A. baumannii

  • Predation efficacy varies by species but appears independent of their antibiotic resistance profile

• Biofilm prevention capabilities:

  • Co-culture with B. bacteriovorus reduced biofilm formation of E. coli by 65.2%

  • Reduction in K. pneumoniae, P. aeruginosa, and A. baumannii biofilm formation by 37.1%, 44.7%, and 36.8%, respectively

  • Prevention mechanism likely involves early predation of planktonic cells before attachment

• Established biofilm disruption:

  • B. bacteriovorus significantly reduced established biofilms of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii by 83.4%, 81.8%, 83.1%, and 79.9%, respectively

  • Scanning electron microscopy confirmed physical disruption of biofilm architecture

  • Effective penetration of predator into mature biofilm structures

• Unique predatory mechanisms against Gram-positive bacteria:

  • First evidence of B. bacteriovorus survival with a Gram-positive prey (S. aureus)

  • Novel "epibiotic" foraging strategy observed through field emission scanning electron microscopy

  • Mean attachment time of 185 seconds to S. aureus cells

  • Reduction of S. aureus biofilms by 74% (static) and 46% (flow) at 24h and 20h, respectively

These findings demonstrate that B. bacteriovorus predation effectiveness is largely independent of the antibiotic resistance status of prey bacteria, suggesting its potential as an alternative approach to conventional antimicrobials.

What crystallization strategies are most effective for structural studies of B. bacteriovorus panC?

Obtaining diffraction-quality crystals of B. bacteriovorus panC presents specific challenges that require systematic approaches:

• Protein production optimization:

  • Expression construct design: Remove flexible regions (N/C termini), consider domain boundaries

  • Purification strategy: Multi-step chromatography (IMAC, ion exchange, size exclusion)

  • Sample homogeneity: Verify by dynamic light scattering and analytical size exclusion

  • Stability screening: Thermal shift assays to identify optimal buffer conditions

• Crystallization approach:

  • Initial screening: Commercial sparse matrix screens (400-1000 conditions)

  • Optimization strategy: Grid screens varying precipitant, pH, additives, and protein concentration

  • Advanced techniques: Microseeding, streak seeding, and counter-diffusion methods

  • Alternative approaches: In situ proteolysis, surface entropy reduction mutations

• Co-crystallization considerations:

  • Substrate/product complexes: ATP, D-pantoate, β-alanine, pantothenate

  • Transition state analogs: Non-hydrolyzable ATP analogs, pantoyl adenylate mimics

  • Inhibitor complexes: For enzyme mechanism studies

  • Metal ion inclusion: Magnesium or manganese for active site coordination

• Crystal handling and data collection:

  • Cryoprotection optimization: Glycerol, ethylene glycol, or PEG based

  • Crystal mounting: MicroMesh for microcrystals, loop size matching for larger crystals

  • Data collection strategy: Multiple orientations, appropriate exposure times

  • Remote synchrotron access: Higher intensity beams for small or weakly diffracting crystals

If standard approaches fail, alternative structural methods should be considered:

• Cryo-electron microscopy for proteins recalcitrant to crystallization

• Small-angle X-ray scattering (SAXS) for solution structure and conformational states

• Nuclear magnetic resonance (NMR) for smaller domains or fragments

Integrating these approaches provides the best opportunity for successful structural characterization of this predatory bacterial enzyme.

How can site-directed mutagenesis enhance the catalytic efficiency of B. bacteriovorus panC?

Rational enzyme engineering through site-directed mutagenesis offers opportunities to enhance pantothenate synthetase activity for research or biotechnological applications. A strategic approach includes:

• Target site identification methods:

  • Structural analysis: Active site residues, substrate channel, and conformational change mediators

  • Sequence conservation analysis: Comparison with high-efficiency pantothenate synthetases

  • Computational prediction: Molecular dynamics to identify rate-limiting steps

  • Evolutionary analysis: Ancestral sequence reconstruction to identify beneficial mutations

• Strategic mutation targets:

  • Substrate binding optimization: Enhance affinity for ATP, D-pantoate, or β-alanine

  • Catalytic residue refinement: Optimize proton transfer and transition state stabilization

  • Product release facilitation: Reduce energy barriers for conformational changes

  • Enzyme stability enhancement: Introduce stabilizing interactions or remove destabilizing ones

• Mutagenesis approaches:

  • Rational design: Single mutations based on structural/biochemical knowledge

  • Semi-rational design: Site-saturation mutagenesis at key positions

  • Combinatorial approaches: Multiple mutations tested in parallel

  • Directed evolution: Creating focused libraries for screening

• Activity screening methods:

  • High-throughput colorimetric assays for pantothenate formation

  • Coupled enzyme assays measuring ATP consumption

  • Thermal stability assays to identify stabilizing mutations

  • In vivo complementation in pantothenate auxotrophs

Expected outcomes table for potential mutations:

This systematic approach to panC engineering could yield variants with significantly enhanced catalytic performance for potential biotechnological applications in pantothenate or CoA precursor production.

How do genomic approaches identify unique features of B. bacteriovorus panC for selective targeting?

Comparative genomic approaches provide powerful tools for identifying distinctive features of B. bacteriovorus panC that could enable selective targeting. A comprehensive strategy includes:

• Sequence-based comparative analysis:

  • Multiple sequence alignment across diverse bacterial phyla

  • Phylogenetic placement within pantothenate synthetase evolution

  • Identification of predatory bacteria-specific sequence motifs

  • Conservation scoring to highlight B. bacteriovorus-unique residues

• Structural bioinformatics approaches:

  • Homology modeling based on crystallized pantothenate synthetases

  • Binding site architecture comparison with non-predatory bacteria

  • Surface electrostatics and hydrophobicity mapping

  • Molecular dynamics simulations to identify conformational differences

• Functional genomics integration:

  • Transcriptomic analysis during different predatory cycle stages

  • Proteomic identification of post-translational modifications

  • Metabolomic profiling of pantothenate pathway intermediates

  • Protein-protein interaction network differences

• Targetability assessment:

  • Comparison with human pantothenate kinase and transporters

  • Off-target prediction against human microbiome species

  • Druggability analysis of unique binding sites

  • Fragment-based screening against identified pockets

Key areas of potential uniqueness:

• Substrate specificity determinants that may differ in predatory bacteria

• Regulatory elements specific to the predatory lifestyle

• Post-translational modifications unique to B. bacteriovorus

• Protein dynamics reflecting the rapid metabolic transitions during predation

These approaches would identify the most promising structural and functional differences for developing selective inhibitors or biotechnological applications specific to predatory bacterial pantothenate synthetases.

What is the potential for B. bacteriovorus as a live therapeutic against pancreatic cancer?

While direct evidence linking B. bacteriovorus to pancreatic cancer therapy is not provided in the search results, we can synthesize information about its predatory capabilities with pancreatic cancer research to explore this innovative therapeutic direction:

• Rationale for investigating B. bacteriovorus in pancreatic cancer:

  • Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis and limited treatment options

  • Bacterial-based therapies represent an emerging approach in cancer treatment

  • B. bacteriovorus's ability to invade other cells and produce hydrolytic enzymes could potentially be harnessed against cancer cells

• Potential mechanisms of action:

  • Direct predation on tumor-associated bacteria (bacterial microbiome within tumors)

  • Production of tumor-inhibiting enzymes and metabolites

  • Immune system modulation in the tumor microenvironment

  • Potential modification to target specific pancreatic cancer markers

• Experimental models for investigation:

  • Panc-1 pancreatic carcinoma cell cultures represent an established in vitro model

  • Organoid systems combining cancer and bacterial components

  • Patient-derived xenograft models

  • Genetically engineered mouse models of pancreatic cancer

• Safety and delivery considerations:

  • Genetic modification to ensure specificity for target cells

  • Engineered kill-switches or sensitivity to standard antibiotics

  • Localized delivery methods to pancreatic tumors

  • Prevention of systemic inflammatory responses

• Combination therapy potential:

  • Synergy with conventional chemotherapy (platinum-based therapeutics)

  • Enhanced efficacy in homologous recombination deficient tumors

  • Potential for combination with immunotherapy

  • Targeted delivery of therapeutic compounds via B. bacteriovorus

This innovative approach would require extensive preclinical validation, addressing safety concerns, delivery challenges, and efficacy parameters before clinical translation. Collaborations between predatory bacteria researchers and pancreatic cancer specialists would be essential for developing this potential therapeutic strategy.