Recombinant Bdellovibrio bacteriovorus Carboxylate-amine ligase Bd2263 (Bd2263)

Basic Research Questions

• What is Bdellovibrio bacteriovorus Carboxylate-amine ligase Bd2263 and what is its biochemical function?

  Bdellovibrio bacteriovorus Carboxylate-amine ligase Bd2263 (Bd2263) is an enzyme that belongs to the diverse superfamily of ATP-dependent carboxylate-amine ligases. These enzymes catalyze the ATP-dependent ligation of a carboxyl group from one substrate with an amino or imino group nitrogen from a second substrate. The catalytic mechanism involves the formation of acylphosphate intermediates, which is a characteristic feature of this enzyme class.

  Bd2263 is part of the ATP-grasp superfamily, which includes at least 15 groups of enzymes with similar catalytic functions but diverse substrate specificities. Other members of this superfamily include D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase. Within the B. bacteriovorus genome, Bd2263 is annotated in both the KEGG database (bba:Bd2263) and STRING database (264462.Bd2263).

• How does Bd2263 fit into the predatory lifecycle of Bdellovibrio bacteriovorus?

  B. bacteriovorus has a complex biphasic lifecycle consisting of:

  • Attack phase (AP): Free-living, flagellated cells seeking prey

  • Transition phase: Recognition and attachment to prey

  • Growth phase (GP): Intraperiplasmic growth within prey bacteria

  • Release phase: Progeny escape from the exhausted prey cell

  While the exact role of Bd2263 in this lifecycle has not been fully characterized in the available research, carboxylate-amine ligases typically function in peptide bond formation and modification of cellular components. Given the predatory nature of B. bacteriovorus, Bd2263 may be involved in:

  1. Cell wall modifications during invasion or exit from prey

  2. Peptidoglycan remodeling during intraperiplasmic growth

  3. Nutrient acquisition from prey components

  Unlike better-characterized enzymes such as DslA (Bd0314), which has been shown to specifically facilitate prey cell exit by acting on deacetylated peptidoglycan , the precise temporal expression and function of Bd2263 during the predatory lifecycle requires further investigation.

• What experimental model systems are available for studying Bd2263 function?

  Several experimental systems have been developed to study B. bacteriovorus enzymes like Bd2263:

  • Recombinant protein expression systems: Purified Bd2263 can be obtained through heterologous expression systems and used for biochemical characterization.

  • Genetic manipulation in B. bacteriovorus: Several genetic tools have been developed for B. bacteriovorus, including:

    • Conjugal plasmid transfer from E. coli to B. bacteriovorus

    • Suicide plasmids for targeted gene knockouts

    • Counterselection systems using sacB for markerless deletions

    • Complementation systems using autonomously replicating plasmids

  • Predation assays: Time-lapse microscopy can be used to monitor the predatory lifecycle and assess the impact of Bd2263 mutations on predation efficiency .

  • Host-independent mutants: B. bacteriovorus can be cultured axenically (without prey) as host-independent (HI) variants, which can simplify some experimental approaches .

Methodology and Experimental Design

• What are the recommended methods for expressing and purifying recombinant Bd2263?

  Based on established protocols for similar B. bacteriovorus enzymes, the following methodology is recommended:

  Expression system:

  • E. coli BL21(DE3) or equivalent strain with T7 RNA polymerase

  • pET-based expression vectors with 6xHis or other affinity tags

  • Expression at lower temperatures (16-25°C) to enhance solubility

  Purification protocol:

  1. Bacterial lysis using sonication or cell disruption in buffer containing:

     • 50 mM Tris-HCl, pH 8.0

     • 300 mM NaCl

     • 10% glycerol

     • 1 mM DTT

     • Protease inhibitor cocktail

  2. Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  3. Size exclusion chromatography to obtain homogeneous protein

  4. Storage at -80°C with 10-20% glycerol to maintain activity

  Recombinant Bd2263 is typically shipped with ice packs to maintain stability.

• What assays can be used to measure Bd2263 enzymatic activity?

  As an ATP-dependent carboxylate-amine ligase, Bd2263 activity can be assessed using several complementary approaches:

  ATP consumption assays:

  • Luciferase-based ATP detection assays

  • Malachite green phosphate detection (measures ADP production)

  • Coupled enzyme assays with pyruvate kinase and lactate dehydrogenase

  Product formation assays:

  • HPLC or LC-MS detection of reaction products

  • Radiometric assays using 14C or 3H-labeled substrates

  • Antibody-based detection methods if specific antibodies are available

  Substrate specificity analysis:

  • Varied carboxylate and amine substrates to determine preference

  • Kinetic analysis using Michaelis-Menten parameters

  • Inhibition studies with structural analogs or competitive inhibitors

  When designing these assays, researchers should consider the potential flexibility of Bd2263 with respect to both carboxyl and amino/thiol group-containing substrates, as this is a characteristic of this enzyme family.

• How can genetic manipulation be used to study Bd2263 function in B. bacteriovorus?

  Several genetic tools have been developed for B. bacteriovorus that can be applied to studying Bd2263:

  Knockout strategies:

  Complementation:

  For complementation studies, autonomously replicating plasmids like those based on IncQ origins (pMMB206) can be used to reintroduce wild-type or modified Bd2263 into knockout strains .

  Expression analysis:

  Real-time PCR can be used to monitor Bd2263 expression during different stages of the predatory lifecycle, similar to what has been done for other genes like Bd0314 (DslA), which shows upregulation during late stages of predation .

Advanced Research Questions

• What is known about the structural features of Bd2263 and how they relate to its catalytic mechanism?

  While the specific crystal structure of Bd2263 has not been reported in the provided search results, insights can be drawn from other members of the ATP-grasp superfamily. These enzymes typically share several structural features:

  • A characteristic ATP-grasp fold consisting of two α/β domains

  • A central β-sheet flanked by α-helices

  • A flexible "ATP-grasp" loop that closes over the active site upon ATP binding

  • Conserved residues for divalent cation coordination (typically Mg2+)

  • Binding pockets for both carboxylate and amine substrates

  The catalytic mechanism likely follows these steps:

  1. ATP binds in conjunction with a divalent metal ion

  2. The carboxylate substrate binds and is activated by ATP to form an acylphosphate intermediate

  3. The amine substrate attacks the activated carboxylate

  4. A peptide bond forms with release of phosphate and ADP

  Researchers investigating Bd2263 structure-function relationships should consider employing:

  • X-ray crystallography or cryo-EM for structural determination

  • Site-directed mutagenesis of predicted catalytic residues

  • Molecular dynamics simulations to understand substrate binding

  • Isothermal titration calorimetry (ITC) to quantify binding energetics

• How does Bd2263 compare to other enzymes in the B. bacteriovorus genome?

  B. bacteriovorus possesses an exceptionally large arsenal of hydrolytic enzymes and other proteins involved in its predatory lifestyle. The 3.8 Mb genome contains over 3,580 predicted open reading frames (ORFs), with a remarkably large secretome (42.4% of proteins contain predicted N-terminal signal sequences) .

  Within this context, Bd2263 represents one of approximately 293 potential lytic proteins encoded by B. bacteriovorus HD100, which include:

  • 150 annotated proteases and peptidases

  • 10 glycanases

  • 20 DNases

  • 9 RNases

  • 15 lipases

  While many B. bacteriovorus enzymes have been studied in detail (such as DslA, a deacetylation-specific lysozyme), Bd2263 represents an opportunity to further understand the diversity of enzymatic activities that enable the predatory lifestyle.

• What evidence exists for evolutionary selection on Bd2263 and related enzymes?

  Recent experimental evolution studies provide insights into how B. bacteriovorus adapts genetically during long-term coculture with prey bacteria. While Bd2263 was not specifically highlighted in these studies, related work has identified patterns of parallel evolution in B. bacteriovorus genes during experimental evolution.

  In one study, six replicate B. bacteriovorus populations were cocultured with Pseudomonas sp. NC02 for 40 passages (approximately 2,880 hours). Genome sequencing identified several genes that acquired high-frequency mutations across multiple independent populations, suggesting positive selection. These included genes encoding:

  • A sodium/phosphate cotransporter family protein (Bd2221)

  • A metallophosphoesterase (Bd0054)

  • A TonB family protein (Bd0396)

  • A hypothetical protein (Bd1601)

  While Bd2263 was not among these genes, the study demonstrates evolutionary pressures on B. bacteriovorus metabolism during predation. Similar approaches could be used to investigate selection on Bd2263 under different predatory conditions.

• What are the current challenges in working with Bd2263 and what methodological advances could help overcome them?

  Working with Bd2263 presents several challenges that require methodological solutions:

  Challenge 1: Limited knowledge of natural substrates

  • Solution: High-throughput substrate screening using metabolomic approaches

  • Method: Incubate Bd2263 with cellular extracts from prey bacteria and identify modified metabolites by LC-MS/MS

  Challenge 2: Difficulty in genetic manipulation

  • Solution: CRISPR-Cas9 based genome editing systems adapted for B. bacteriovorus

  • Method: Development of specialized delivery vectors and optimization of transformation conditions

  Challenge 3: Complex phenotype analysis

  • Solution: Single-cell tracking of predatory behavior

  • Method: Time-lapse microscopy with fluorescently labeled predator and prey cells

  Challenge 4: Structural characterization

  • Solution: Fragment-based drug design approaches to identify ligand binding sites

  • Method: Thermal shift assays, X-ray crystallography, and computational docking

  Challenge 5: Integration with systems biology data

  • Solution: Multi-omics approaches to contextualize Bd2263 function

  • Method: Integration of transcriptomics, proteomics, and metabolomics data with modeling of predatory behavior

• What statistical considerations are important when designing experiments involving Bd2263?

  When designing experiments to study Bd2263 function, several statistical considerations should be addressed:

  Sample size determination:

  • A minimum of n=5 independent samples per group should be used for statistical analysis

  • A priori power analysis should be conducted to determine appropriate sample sizes

  • The analysis should specify alpha (typically 0.05), power (typically 0.8), and expected effect size

  Appropriate controls:

  • Enzymatic assays should include:

    • No-enzyme controls

    • Heat-inactivated enzyme controls

    • Known substrate/product standards

    • Positive control enzymes from the same family

  Statistical testing:

  • Define significance thresholds before experiments (typically p<0.05)

  • Use appropriate post-hoc tests when making multiple comparisons

  • For complex designs with multiple factors, use multi-way ANOVA followed by appropriate post-hoc tests

  Data normalization considerations:

  • Individual experimental group values should not be normalized to matched control group values (which would eliminate variance in the control group)

  • Data transformation (e.g., log transformation) must be justified by showing it makes standard errors no longer proportional to means

  • Avoid normalizing to accommodate baseline differences between groups

  Replication versus pseudo-replication:

  • One sample tested in triplicate represents n=1, not n=3

  • Technical replicates should be used to test reliability of single values, not to inflate sample size

• How can Bd2263 be studied in the context of the complete B. bacteriovorus predatory lifecycle?

  To fully understand Bd2263's role in the predatory lifecycle, researchers should consider:

  Temporal expression analysis:

  • qRT-PCR to determine when Bd2263 is expressed during the predatory cycle

  • RNA-seq to place Bd2263 within broader transcriptional networks

  • Similar to studies of DslA (Bd0314), which demonstrated upregulation during late stages (180-240 min after infection)

  Spatial localization:

  • Immunofluorescence microscopy using antibodies against Bd2263

  • Fluorescent protein fusions to track localization during predation

  • Fractionation studies to determine if Bd2263 is secreted into prey

  Functional studies:

  • Creation of Bd2263 knockout strains to assess predation efficiency

  • Time-lapse microscopy to monitor predatory stages (similar to DslA studies that showed delayed exit time in mutants)

  • Complementation with wild-type or mutant variants to confirm phenotypes

  Interaction studies:

  • Pull-down assays to identify protein-protein interactions

  • Bacterial two-hybrid systems to screen for interacting partners

  • Cross-linking mass spectrometry to capture transient interactions

  By combining these approaches, researchers can develop a comprehensive understanding of Bd2263's role within the complex predatory lifecycle of B. bacteriovorus.

Potential Applications and Future Directions

• What potential biotechnological applications might utilize Bd2263?

  While specific applications of Bd2263 have not been directly reported, B. bacteriovorus enzymes have several potential applications:

  Biocatalysis:

  • ATP-dependent carboxylate-amine ligases like Bd2263 could be used for peptide bond formation in pharmaceutical synthesis

  • Engineering Bd2263 for altered substrate specificity could create novel biocatalysts for green chemistry applications

  Biofilm degradation:

  • B. bacteriovorus secretes enzymes that degrade biofilms of both Gram-negative and Gram-positive bacteria

  • If Bd2263 is involved in degradation of extracellular matrix components, it could be used in anti-biofilm technologies

  Antimicrobial applications:

  • B. bacteriovorus is being explored as a "living antibiotic"

  • Understanding Bd2263's role in predation could help optimize bacterial predators for therapeutic applications

  Biopolymer processing:

  • B. bacteriovorus has been applied to recovery of intracellular bioproducts like polyhydroxyalkanoates (PHAs)

  • If Bd2263 participates in polymer degradation, it could be utilized in bioplastic recycling or processing

• What are promising future research directions for understanding Bd2263 function?

  Several emerging approaches could advance our understanding of Bd2263:

  Structural biology approaches:

  • Cryo-EM to determine structure in different conformational states

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • Fragment-based screening to identify binding pockets

  Systems biology integration:

  • Multi-omics approaches combining:

    • Transcriptomics to identify co-regulated genes

    • Proteomics to map protein-protein interactions

    • Metabolomics to identify substrates and products

  • Computational modeling of B. bacteriovorus predation including Bd2263 function

  Synthetic biology applications:

  • Engineering B. bacteriovorus with modified Bd2263 variants

  • Developing minimal synthetic predators incorporating essential enzymes

  • Creating chimeric enzymes combining domains from different ligases

  High-throughput approaches:

  • Deep mutational scanning of Bd2263 to map functional residues

  • Microfluidic analysis of single-cell predation dynamics

  • Droplet-based enzyme evolution to develop enhanced variants

  By pursuing these directions, researchers can continue to unravel the fascinating biology of predatory bacteria and harness their unique enzymes for biotechnological applications.