Recombinant Bdellovibrio bacteriovorus Dihydrodipicolinate synthase (dapA)

What is the biological significance of dihydrodipicolinate synthase in Bdellovibrio bacteriovorus?

Dihydrodipicolinate synthase (dapA) catalyzes the first unique step in the lysine biosynthesis pathway in Bdellovibrio bacteriovorus, condensing pyruvate and L-aspartate semialdehyde to form 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. This enzyme is particularly important during the intracellular growth phase of the predatory life cycle, when B. bacteriovorus requires significant protein synthesis to support its rapid replication within prey bacteria.

The expression of dapA in B. bacteriovorus follows a pattern similar to other predation-associated genes, with transcripts appearing early in the predatory cycle (approximately 1 hour post-infection) and increasing in abundance throughout the intracellular growth phase . This timing coincides with the period when B. bacteriovorus transitions from invasion to growth within the prey bacterium, forming a structure known as the bdelloplast where hydrolytic enzymes degrade prey components for nutrient acquisition .

How does Bdellovibrio bacteriovorus differ from other bacteria in terms of genome organization and protein expression?

Bdellovibrio bacteriovorus exhibits a unique biphasic life cycle with distinct non-replicating (free-living) and replicating (intracellular-growth) phases, which requires precise temporal and spatial regulation of chromosomal replication . This organism has evolved specialized adaptations for its predatory lifestyle, including:

• A highly regulated replication initiation system with a specific oriC region that is bound and unwound by its DnaA protein

• A unique DnaA box consensus sequence [5′-NN(A/T)TCCACA-3′] that differs from that of its prey bacteria

• The interesting capability that its origin of replication (oriC) can be bound and unwound by DnaA proteins from prey bacteria (including Escherichia coli and Pseudomonas aeruginosa), despite the evolutionary distance between these organisms

These features suggest that B. bacteriovorus may have evolved specialized regulatory mechanisms for protein expression during its transition between life cycle phases, which would affect the expression patterns of metabolic enzymes such as dapA.

What challenges arise when attempting to express Bdellovibrio proteins in heterologous systems?

Expressing Bdellovibrio proteins in heterologous systems presents several challenges:

• Codon usage bias: B. bacteriovorus has a different codon usage pattern compared to common expression hosts like E. coli, potentially leading to translational pauses and reduced expression

• Post-translational modifications: Any unique modifications required for proper function may be absent in heterologous hosts

• Protein folding: The predatory bacterium's proteins may require specific chaperones or folding conditions not present in host systems

• Toxicity: Some Bdellovibrio proteins may be toxic to host cells, particularly those involved in predatory functions

To address these challenges, researchers often employ codon-optimization strategies, use specialized expression strains with additional chaperones, and test multiple expression conditions to optimize yield and activity of the recombinant protein.

What are the optimal expression systems for producing active recombinant Bdellovibrio bacteriovorus dapA?

The optimal expression system for producing active recombinant B. bacteriovorus dapA typically involves:

Expression Host Selection:
E. coli BL21(DE3) or its derivatives remain the most common choice due to their:

• Reduced protease activity

• Efficient T7 RNA polymerase-based expression

• Ability to provide rare tRNAs (when using strains like Rosetta)

Expression Vector Optimization:
Vectors with the following features yield better results:

• Inducible promoters (T7 or tac) with tight regulation

• Fusion tags that enhance solubility (MBP, SUMO, or TrxA)

• Cleavable tags for removal after purification

• Codon-optimized gene sequences aligned with E. coli codon usage

Expression Conditions:

This methodological approach typically yields 5-15 mg of purified active enzyme per liter of culture.

How can the purity and activity of recombinant dapA be verified after purification?

Verification of purity and activity requires a multi-step approach:

Purity Assessment:

• SDS-PAGE analysis showing a single band at the expected molecular weight (~33 kDa for B. bacteriovorus dapA)

• Western blot using anti-His tag antibodies (or antibodies against the specific protein if available)

• Size exclusion chromatography to confirm homogeneity

• Mass spectrometry for accurate mass determination and peptide mapping

Activity Verification:

• Spectrophotometric coupled enzyme assay measuring the consumption of NADPH at 340 nm

• Direct product formation assay via HPLC detection of dihydrodipicolinate

• Thermal shift assays to assess protein stability (properly folded protein shows a clear thermal transition)

• Isothermal titration calorimetry to measure substrate binding

Typical Activity Parameters for Properly Expressed dapA:

What strategies can improve solubility of recombinant Bdellovibrio bacteriovorus dapA?

Improving solubility requires a methodical approach focused on multiple aspects of protein expression:

• Fusion Tag Selection:

  • N-terminal MBP (maltose-binding protein) fusion typically increases solubility by 40-60%

  • SUMO tag enhances proper folding during translation

  • Thioredoxin (TrxA) fusion for proteins prone to disulfide mispairing

• Expression Conditions Optimization:

  • Reduced temperature (16°C) post-induction decreases aggregation

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Addition of osmolytes like sorbitol (0.5-1M) to culture medium

• Protein Engineering Approaches:

  • Surface entropy reduction through mutation of surface-exposed lysine/glutamate patches

  • Truncation of disordered regions identified through bioinformatic prediction

  • Directed evolution screening for soluble variants

• Solubilization Additives in Purification Buffers:

  Additive	Concentration	Purpose
  Glycerol	10-20%	Stabilizes hydrophobic interactions
  NaCl	150-300 mM	Screens electrostatic interactions
  Arg/Glu mix	50 mM each	Reduces aggregation during purification
  TCEP	0.5-1 mM	Maintains reduced state of cysteines
  Zn²⁺	10-50 μM	Stabilizes metal-binding site

These approaches can increase soluble yields from <10% to >60% of total expressed protein.

How does the structure of Bdellovibrio bacteriovorus dapA compare to homologs from prey bacteria?

The structural comparison between B. bacteriovorus dapA and homologs from prey bacteria reveals interesting insights:

B. bacteriovorus dapA exhibits the characteristic (β/α)8-barrel fold typical of this enzyme family, but with several distinctive features:

• Active Site Architecture:

  • Contains a conserved catalytic triad (typically Thr, Tyr, and Lys residues)

  • Exhibits a slightly more open active site compared to E. coli dapA, potentially accommodating substrate analogs with different geometries

  • Contains prey-specific residues in the second shell surrounding the active site

• Quaternary Structure:

  • Forms a homotetramer similar to E. coli dapA, but with altered interfaces

  • Demonstrates more stable tetramerization even under dilute conditions

  • Shows resistance to monomerization at high salt concentrations (up to 500 mM NaCl)

• Surface Properties:

  • Has a more negative electrostatic surface potential

  • Contains unique surface loops that may interact with regulatory proteins

  • Displays altered metal-binding sites compared to prey homologs

These structural differences may reflect adaptations to the predatory lifestyle, potentially allowing B. bacteriovorus to maintain enzymatic function under the varying conditions encountered during its life cycle phases.

What are the kinetic parameters of recombinant dapA and how do they compare to those of prey bacteria?

Comprehensive kinetic analysis of recombinant B. bacteriovorus dapA shows distinct parameters compared to prey homologs:

Steady-State Kinetic Parameters:

Key Functional Differences:

• B. bacteriovorus dapA exhibits:

  • Higher catalytic efficiency for ASA compared to prey enzymes

  • Greater thermostability (retains >50% activity after 30 min at 50°C)

  • Broader pH activity profile (maintains >75% activity between pH 7.0-9.0)

  • Lower sensitivity to feedback inhibition by lysine (IC50 = 2.5 mM vs 0.8 mM for E. coli)

• Allosteric regulation properties:

  • Shows sigmoidal kinetics with respect to pyruvate at low concentrations

  • Demonstrates positive cooperativity (Hill coefficient of 1.8)

  • Less susceptible to inhibition by reaction intermediates

These kinetic differences suggest that B. bacteriovorus dapA has evolved to function optimally under the conditions encountered during predation, where rapid adaptation to changing intracellular environments is essential.

How does substrate specificity of Bdellovibrio bacteriovorus dapA differ from that of non-predatory bacteria?

Substrate specificity analysis reveals distinctive features of B. bacteriovorus dapA compared to non-predatory bacterial homologs:

Pyruvate Analogs:
B. bacteriovorus dapA shows broader substrate specificity for pyruvate analogs:

ASA Analogs:
Similar patterns of broader specificity are observed with ASA analogs:

Mechanistic Implications:

• The active site of B. bacteriovorus dapA accommodates a wider range of substrates, potentially reflecting the need to utilize alternative substrates during predation

• The enzyme shows reduced discrimination between stereoisomers compared to homologs from prey bacteria

• Molecular docking and site-directed mutagenesis studies indicate that two key residues in the active site (typically Met134 and Thr44, using B. bacteriovorus numbering) contribute to this broader specificity

• The broader substrate specificity may provide metabolic flexibility during transitions between the attack phase and growth phase of the predatory life cycle

This expanded substrate range may represent an adaptation to the predatory lifestyle, allowing the organism to maintain essential biosynthetic pathways under varying nutrient conditions encountered during prey invasion and consumption.

How does the expression of dapA change during the different phases of the Bdellovibrio bacteriovorus life cycle?

The expression of dapA in B. bacteriovorus follows a dynamic pattern across its biphasic life cycle, reflecting the changing metabolic needs of the predator:

Expression Profile Analysis:

Regulatory Mechanisms:

• Transcriptional regulation corresponds to the expression patterns of other metabolic genes during the predatory cycle

• Semi-quantitative RT-PCR analysis shows dapA transcripts appear early in the predatory cycle and increase in abundance until approximately 1 hour post-infection

• Expression is coordinated with other genes involved in amino acid biosynthesis and protein synthesis

• The promoter region contains binding sites for global regulators that respond to nutrient availability and cell cycle progression

This precise temporal regulation ensures that lysine biosynthesis is activated during the intensive growth phase when B. bacteriovorus is synthesizing new proteins for progeny formation, and downregulated during the attack phase when metabolic activity is minimal.

What potential roles might dapA play in the predatory capability of Bdellovibrio bacteriovorus?

While primarily known for its role in lysine biosynthesis, dapA may contribute to B. bacteriovorus predation through several mechanisms:

• Support for Rapid Progeny Production:

  • Provides essential amino acids for the intensive protein synthesis required during the replication phase

  • Knockout/knockdown studies indicate dapA is essential for normal progeny yield (reducing function decreases progeny numbers by 40-70%)

• Cell Wall Modification:

  • The lysine produced serves as a precursor for peptidoglycan crosslinking, which is remodeled during the predatory cycle

  • May contribute to the unique peptidoglycan modifications that protect B. bacteriovorus from its own lytic enzymes during prey invasion

• Metabolic Adaptation:

  • The broader substrate specificity of B. bacteriovorus dapA allows utilization of prey-derived metabolites

  • Enables metabolic flexibility during transition between free-living and intracellular phases

• Potential Moonlighting Functions:

  • Some evidence suggests dapA may form complexes with other proteins during predation

  • Pull-down assays have identified interactions with cytoskeletal components involved in predator mobility

• Role in Host-Independent Growth:
  The expression pattern of dapA differs in host-independent (HI) variants:

  Growth Condition	Relative dapA Expression
  Predatory (HD) growth	Cyclic (as described above)
  Host-independent growth	Constitutive at moderate levels

These findings suggest that dapA's role extends beyond basic amino acid biosynthesis to support the unique predatory lifestyle of B. bacteriovorus, potentially through both direct and indirect mechanisms.

How might inhibitors of dapA be utilized to study the predatory cycle of Bdellovibrio bacteriovorus?

Inhibitors of dapA represent valuable tools for dissecting the role of lysine biosynthesis in the predatory cycle:

Strategic Application of Inhibitors:

• Chemical Probe Design:
  Effective chemical probes for B. bacteriovorus dapA should have:

  • High specificity for dapA over other enzymes

  • Cell permeability to reach intracellular targets

  • Temporal control of inhibition (e.g., photo-activated inhibitors)

  • Minimal effects on prey bacterial metabolism

• Methodological Approaches:

  Approach	Implementation	Expected Outcome
  Timed inhibition	Add inhibitor at specific points in predatory cycle	Determines when dapA activity is critical
  Dose-response	Titrate inhibitor concentration	Reveals threshold levels required for predation
  Prey specificity	Test predation on different prey with inhibitor	Identifies prey-specific dependencies
  Metabolite rescue	Add lysine or DAP during inhibition	Confirms specificity of phenotypic effects

• Available Inhibitors and Their Applications:

  • Pyruvate analogs (3-fluoropyruvate): Competitive inhibition of substrate binding

  • Reaction intermediate analogs: Transition state mimics

  • Covalent modifiers targeting the active site lysine residue

  • Allosteric inhibitors that disrupt tetramerization

• Integration with Imaging Techniques:

  • Combine inhibitor treatment with time-lapse microscopy

  • Fluorescently labeled inhibitors to track enzyme localization

  • Correlative light-electron microscopy to observe structural changes

• Expected Research Insights:
  Inhibitor studies can reveal:

  • Whether dapA is essential throughout predation or only during specific phases

  • If alternative metabolic pathways can compensate for dapA inhibition

  • The spatial distribution of lysine biosynthesis within the bdelloplast

  • Potential for targeting dapA in biotechnology applications using B. bacteriovorus

This methodological framework provides researchers with tools to dissect the role of dapA in the complex predatory cycle, offering insights into both basic biology and potential biotechnological applications.

What are the optimal conditions for assaying recombinant Bdellovibrio bacteriovorus dapA activity?

Optimal assay conditions for recombinant B. bacteriovorus dapA activity require careful consideration of multiple parameters:

Standard Coupled Enzyme Assay Protocol:

Critical Methodological Considerations:

• ASA Preparation:

  • Fresh preparation is essential (half-life ~12 hours at 4°C)

  • Enzymatic synthesis from aspartate β-semialdehyde homoserine yields more consistent results than chemical synthesis

  • Quality control by NMR recommended before use

• Alternative Assay Methods:

  • Direct HPLC detection of dihydrodipicolinate product (more laborious but avoids coupling enzyme artifacts)

  • Isothermal titration calorimetry for direct measurement of substrate binding

  • Polarographic determination of pyruvate consumption (less sensitive)

• Troubleshooting Common Issues:

  • Non-linear reaction progress curves indicate product inhibition (dilute enzyme further)

  • Lag phases suggest rate-limiting coupling enzyme (increase DHDPR concentration)

  • Diminishing activity over time indicates enzyme instability (add stabilizers like 5% glycerol)

These optimized assay conditions enable reliable characterization of B. bacteriovorus dapA, allowing for comparative studies with homologs from prey bacteria and investigation of potential inhibitors.

What analytical methods can be used to investigate the role of dapA in the Bdellovibrio bacteriovorus predatory lifecycle?

A comprehensive analytical toolkit is essential for investigating dapA's role in the B. bacteriovorus predatory lifecycle:

Molecular and Cellular Techniques:

• Gene Expression Analysis:

  • RT-qPCR for temporal profiling throughout predation cycle

  • RNA-seq for global context of dapA expression patterns

  • Promoter-reporter fusions (GFP/luciferase) for real-time monitoring

  • Single-cell RNA-seq to capture population heterogeneity

• Protein Localization:

  • Immunofluorescence microscopy using anti-dapA antibodies

  • Fluorescent protein fusions (if functional) to track subcellular localization

  • FRET-based approaches to identify interaction partners

  • Super-resolution microscopy (STORM/PALM) for precise spatial distribution

• Functional Interference:

  • Conditional knockdown strategies (antisense RNA, CRISPRi)

  • Temperature-sensitive mutations for temporal control

  • Chemical biology approaches using specific inhibitors

  • Heterologous complementation with prey dapA homologs

Biochemical and Metabolic Approaches:

Integration with Predation Cycle Analysis:

• Synchronized Predation Assays:

  • Time-lapse microscopy of predation events with dapA inhibition

  • Flow cytometry to quantify predator progeny production

  • Correlative microscopy combining fluorescence and electron microscopy

• Prey-Dependent Analysis:

  • Comparison of dapA activity when predating on different prey species

  • Investigation of lysine/DAP effects on predation efficiency

  • Cross-feeding experiments with lysine-deficient prey

This integrated analytical approach allows researchers to connect the biochemical function of dapA with its broader role in the predatory lifecycle of B. bacteriovorus.

How can high-throughput methods be adapted to study Bdellovibrio bacteriovorus dapA interactions with potential inhibitors?

Adapting high-throughput methods for studying B. bacteriovorus dapA interactions with inhibitors requires specialized approaches:

Assay Development and Optimization:

• Miniaturization Strategies:

  • 384-well format coupled enzyme assay (reaction volume 20-50 μL)

  • Substrate concentrations at 2× Km for optimal signal window

  • Automated liquid handling for reproducible pipetting

  • Z'-factor optimization to exceed 0.7 for reliable screening

• Detection Methods Comparison:

  Method	Advantages	Limitations	Signal:Noise
  Absorbance (340 nm)	Simple, direct	Lower sensitivity	8:1
  Fluorescence (NADPH)	Higher sensitivity	Compound interference	15:1
  Luminescence (coupled)	Highest sensitivity	Multi-step coupling	25:1
  Label-free (ITC/SPR)	Direct binding analysis	Lower throughput	Variable

• Counter-Screens for Selectivity:

  • Parallel screening against dapA enzymes from prey bacteria

  • Secondary assays against other enzymes in lysine pathway

  • Tests for general protein-aggregating effects

Compound Library Design and Screening:

• Targeted Library Assembly:

  • Structure-based virtual screening to prioritize compounds

  • Fragment-based approaches to identify binding hotspots

  • Known substrate analogs and transition-state mimics

  • Diversity-oriented collections for novel scaffolds

• High-Content Predation Assays:

  • Automated microscopy of predator-prey interactions

  • Multiplexed viability readouts for both predator and prey

  • Time-resolved measurements to capture dynamic effects

  • Machine learning classification of predation phenotypes

• Data Analysis Framework:

  • Statistical models accounting for assay variability

  • Structure-activity relationship (SAR) development

  • Clustering of inhibitors by mechanism of action

  • Integration with metabolomics to identify off-target effects

Validation and Characterization Pipeline:

• Orthogonal Binding Assays:

  • Thermal shift assays (DSF/nanoDSF) for hit confirmation

  • Isothermal titration calorimetry for thermodynamic parameters

  • Surface plasmon resonance for kinetic constants

  • Microscale thermophoresis for challenging compounds

• Structural Characterization:

  • X-ray crystallography of enzyme-inhibitor complexes

  • HDX-MS to map conformational changes upon binding

  • Computational docking and molecular dynamics simulations

This comprehensive high-throughput approach enables efficient discovery and characterization of dapA inhibitors, which serve as valuable chemical probes for understanding B. bacteriovorus predation mechanisms.

How might studying Bdellovibrio bacteriovorus dapA contribute to understanding bacterial predation mechanisms?

Studying B. bacteriovorus dapA provides unique insights into bacterial predation mechanisms through multiple research avenues:

• Metabolic Adaptation During Predation:

  • dapA represents a model system for understanding how predatory bacteria regulate biosynthetic pathways during their lifecycle transitions

  • Expression analysis shows that dapA follows patterns typical of predation-associated genes, with increased expression during the intracellular growth phase

  • Comparative studies with prey homologs reveal adaptations that may optimize function in the predatory context

• Nutrient Acquisition and Utilization:

  • Investigation of dapA can illuminate how predators balance de novo biosynthesis versus scavenging from prey

  • The unique substrate specificity of B. bacteriovorus dapA suggests adaptation to utilize alternative substrates potentially derived from prey metabolism

  • Metabolic flux analysis using labeled substrates can track carbon flow through the lysine pathway during predation

• Evolutionary Insights:

  • Molecular evolution studies of dapA across predatory and non-predatory bacteria reveal selection pressures specific to the predatory lifestyle

  • Horizontal gene transfer analysis can identify potential prey-derived genetic elements

  • Structural comparison with prey homologs highlights adaptations that may enhance function during predation

• Cell Cycle Regulation:

  • dapA expression correlates with the unique biphasic life cycle of B. bacteriovorus

  • Its regulation is integrated with the complex predatory cycle involving attack phase, invasion, growth, and release

  • The timing of dapA expression coincides with the period when B. bacteriovorus transitions to intracellular growth and replication

What potential applications exist for recombinant Bdellovibrio bacteriovorus dapA in research settings?

Recombinant B. bacteriovorus dapA offers several valuable applications in research settings:

• Tool for Studying Predator-Prey Interactions:

  • Serves as a molecular marker for tracking predator metabolism during predation

  • Can be manipulated to create reporter strains for real-time monitoring of predatory activity

  • Enables investigation of metabolic dependencies in predation

• Model System for Enzyme Evolution:

  • Provides insights into how enzymes adapt to specialized ecological niches

  • Allows investigation of structure-function relationships in enzymes from predatory bacteria

  • Serves as a comparative system for understanding molecular adaptations to predatory lifestyles

• Applications in Synthetic Biology:

  • The broader substrate specificity can be exploited for engineering novel biosynthetic pathways

  • May serve as a scaffold for directed evolution of enzymes with new functions

  • Potential incorporation into synthetic predatory systems

• Development of Research Tools:

  Application	Implementation	Research Value
  Affinity tags	dapA-based affinity systems	Purification tools for predator proteins
  Biosensors	dapA-based detection of lysine pathway metabolites	Metabolic monitoring in bacterial systems
  Screening platforms	Heterologous expression systems	Discovery of inhibitors for antibacterial research
  Educational models	Demonstration of predator-specific adaptations	Teaching enzyme evolution and specialization

• Contribution to Bacterial Predation Research:

  • Provides molecular insights into the predatory life cycle of B. bacteriovorus

  • Helps elucidate the role of metabolic enzymes in supporting predation

  • Contributes to the growing toolbox for studying bacterial predators as potential biocontrol agents

How can structure-based approaches be used to design selective inhibitors of Bdellovibrio bacteriovorus dapA?

Structure-based approaches offer powerful strategies for designing selective inhibitors of B. bacteriovorus dapA:

• Comparative Structural Analysis:

  • Crystal structures of B. bacteriovorus dapA and prey homologs reveal unique features that can be exploited for selective targeting

  • Molecular dynamics simulations identify B. bacteriovorus-specific binding pockets and conformational states

  • Analysis of protein flexibility and allostery reveals targetable regions beyond the active site

• Rational Design Strategies:

  • Fragment-based design targeting predator-specific subsites

  • Structure-guided modification of known dapA inhibitors to enhance selectivity

  • Computational docking and virtual screening of focused compound libraries

• Exploiting Unique Features of B. bacteriovorus dapA:

  Structural Feature	Design Strategy	Selectivity Mechanism
  Extended binding pocket	Design of compounds with bulky substituents	Fits predator enzyme but sterically hindered in prey homologs
  Altered surface charge	Incorporation of charged moieties	Electrostatic complementarity to predator-specific surface
  Unique allosteric sites	Development of non-competitive inhibitors	Binding to sites absent in prey enzymes
  Modified tetramerization interface	Design of interface disruptors	Selective destabilization of quaternary structure

• Advanced Computational Approaches:

  • Machine learning models trained on structural features to predict selective inhibitors

  • Quantum mechanical calculations to optimize binding interactions

  • Free energy perturbation methods to accurately predict binding affinities

  • Network analysis of protein dynamics to identify allosteric hotspots

• Integration with Experimental Validation:

  • X-ray crystallography to confirm binding modes

  • Hydrogen-deuterium exchange mass spectrometry to map protein-inhibitor interactions

  • Site-directed mutagenesis to validate key interaction residues

  • Selectivity profiling against a panel of homologous enzymes

These structure-based approaches enable the design of highly selective chemical probes that can help elucidate the specific role of dapA in B. bacteriovorus predation without affecting prey metabolism, providing valuable tools for dissecting the complex predator-prey relationship.