Recombinant Bdellovibrio bacteriovorus Adenylosuccinate synthetase (purA)

What is Adenylosuccinate synthetase (purA) and what is its function in Bdellovibrio bacteriovorus?

Adenylosuccinate synthetase (purA, EC 6.3.4.4) in Bdellovibrio bacteriovorus is a critical enzyme in purine nucleotide biosynthesis that catalyzes the first committed step in the conversion of IMP to AMP. Also known as AMPSase or AdSS, this enzyme facilitates the reaction between IMP, aspartate, and GTP to form adenylosuccinate, which is subsequently converted to AMP . In the predatory bacterium B. bacteriovorus, purA likely plays an essential role in nucleotide metabolism during its unique life cycle, particularly during the intraperiplasmic growth phase when the predator undergoes rapid DNA replication within its prey . The enzyme has been identified in the B. bacteriovorus HD100 strain (ATCC 15356 / DSM 50701 / NCIB 9529) with the UniProt accession number Q6MN06 .

How does purA from B. bacteriovorus differ structurally from its homologs in other bacteria?

The purA protein from B. bacteriovorus consists of 432 amino acids with a complete sequence identified through recombinant expression . Comparative analysis reveals that while the enzyme maintains the core catalytic domains found in other bacterial adenylosuccinate synthetases, B. bacteriovorus purA has evolved specific structural adaptations that potentially reflect its predatory lifestyle. Unlike non-predatory bacteria, B. bacteriovorus must rapidly mobilize and utilize nucleotide resources from prey bacteria, which may explain certain unique features in its purA enzyme. The full sequence of the recombinant protein reveals conserved residues involved in substrate binding and catalysis, though detailed structural studies comparing it to homologs from non-predatory bacteria are still emerging in the scientific literature .

What expression systems have been successfully used for recombinant production of B. bacteriovorus purA?

Recombinant B. bacteriovorus purA has been successfully expressed in mammalian cell expression systems, yielding protein with greater than 85% purity as determined by SDS-PAGE . Researchers have developed protocols for expressing the full-length protein (amino acids 1-432) with various tag configurations determined during the manufacturing process. While mammalian expression systems appear to be the preferred method for commercial production, bacterial expression systems have also been employed in research settings, particularly when studying enzyme kinetics or structure-function relationships. When expressing recombinant purA, researchers should consider codon optimization for the selected expression system and ensure proper folding through controlled induction and temperature conditions .

How can I design experiments to assess purA activity in the context of B. bacteriovorus predation efficiency?

To investigate the relationship between purA activity and B. bacteriovorus predation efficiency, researchers should consider a multi-faceted experimental approach:

• Genetic manipulation strategy: Create a conditional knockdown or controlled expression system for purA using markerless deletion techniques developed specifically for B. bacteriovorus . This can be accomplished using suicide plasmids like pSSK10 (derived from pDS132) carrying the sacB gene for counterselection.

• Quantitative predation assays: Compare predation rates between wild-type and purA-modulated strains using time-lapse microscopy and viable count methods. Measure predatory capacity against multiple Gram-negative prey species to assess spectrum changes.

• Metabolomic profiling: Analyze nucleotide pools during different stages of the predatory cycle using LC-MS/MS to determine how purA expression levels affect AMP biosynthesis during intraperiplasmic growth.

• In vitro enzyme kinetics: Purify recombinant purA and assess activity parameters under varying conditions (pH, temperature, ion concentrations) that mimic the intraperiplasmic environment to understand how predatory lifestyle influences enzyme function.

• Co-culture competition experiments: Perform mixed population studies where wild-type and purA-modulated B. bacteriovorus compete for the same prey population to determine fitness effects.

This experimental framework allows for comprehensive assessment of purA's role in the predatory efficiency of B. bacteriovorus while controlling for confounding variables .

What are the challenges in studying B. bacteriovorus purA compared to homologous enzymes from non-predatory bacteria?

Studying B. bacteriovorus purA presents several unique challenges compared to homologous enzymes from non-predatory bacteria:

These challenges necessitate specialized approaches that consider the unique biology of predatory bacteria while leveraging techniques from both molecular enzymology and bacterial physiology .

How can purA activity be accurately measured in complex experimental systems involving B. bacteriovorus and prey bacteria?

Accurately measuring purA activity in predator-prey systems requires methods that can distinguish between predator and prey contributions while maintaining physiological relevance:

• Spectrophotometric coupled assays: Adapt traditional adenylosuccinate synthetase assays to measure activity in filtered predator-prey co-cultures. This approach links AMP production to NADH oxidation through coupling enzymes, allowing continuous monitoring at 340 nm.

• Radiolabeled substrate incorporation: Use 14C-labeled aspartate to track substrate incorporation into adenylosuccinate, enabling sensitive detection even in complex mixtures.

• Predator-specific antibody precipitation: Develop antibodies against B. bacteriovorus purA to immunoprecipitate the enzyme from mixed cultures before activity measurements.

• Quantitative RT-PCR: Measure purA expression levels using B. bacteriovorus-specific primers (similar to the approach used with rpsL as a load control in related research) . This requires careful RNA extraction from predator-prey co-cultures using specialized protocols.

• Metabolic labeling: Employ heavy isotope labeling of either predator or prey to distinguish metabolites originating from each organism in mass spectrometry analysis.

For optimal results, researchers should implement controls including heat-inactivated samples, purA-deficient mutants, and prey-only cultures to establish baseline measurements and account for matrix effects .

What are the most effective methods for creating purA mutants in B. bacteriovorus for functional studies?

Creating purA mutants in B. bacteriovorus requires specialized genetic tools adapted for this predatory bacterium:

• Suicide vector strategy: Utilize pK18mobsacB-based suicide vectors that carry the sacB gene for counterselection on sucrose-containing media. This two-step approach allows for scarless, markerless gene deletions or site-directed mutations .

• Conjugation protocol: Transfer the suicide vector via conjugation using E. coli S17-1 λpir or SM10 λpir donor strains. The optimized protocol involves:

  • Growing donor cells to log phase in LB media with appropriate antibiotics

  • Growing B. bacteriovorus in co-culture with prey

  • Filtering and pelleting the B. bacteriovorus cells

  • Mixing donor and predator cells on nitrocellulose filters placed on peptone yeast agar

  • Selecting transconjugants on DNB (Diluted Nutrient Broth) supplemented with appropriate antibiotics

• Verification methods: Confirm successful mutations using:

  • PCR with primers flanking the deletion site

  • RT-PCR to verify changes in expression

  • Functional assays to assess phenotypic changes

  • Whole-genome sequencing to rule out off-target effects

• Conditional expression systems: For essential genes like purA, consider inducible expression systems that allow titration of gene expression rather than complete knockouts.

These methods have been successfully applied to create deletion mutants in other genes in B. bacteriovorus and can be adapted for purA studies .

What considerations are important when expressing recombinant B. bacteriovorus purA for structural studies?

For successful structural studies of recombinant B. bacteriovorus purA, researchers should consider:

• Expression system selection: Mammalian cell expression systems have been successfully used to produce high-purity (>85%) B. bacteriovorus purA . Consider codon optimization for the chosen expression system to maximize yield.

• Protein stability and storage: The recombinant protein has demonstrated stability with a shelf life of approximately 6 months at -20°C/-80°C in liquid form and 12 months when lyophilized . Minimize freeze-thaw cycles by preparing smaller working aliquots.

• Buffer composition: For structural studies, buffer optimization is critical. Initial reconstitution in deionized sterile water to 0.1-1.0 mg/mL with 5-50% glycerol has been recommended for long-term storage . For crystallography, systematically screen buffer conditions to identify those promoting crystal formation.

• Purification strategy: Tag selection should be carefully considered—while tags facilitate purification, they may interfere with structural studies. Include proteolytic tag removal steps and additional purification by size exclusion chromatography to achieve >95% homogeneity.

• Protein quality assessment: Before initiating structural studies, confirm enzyme activity through in vitro assays and assess protein homogeneity using dynamic light scattering and analytical ultracentrifugation.

• Co-crystallization considerations: For mechanistic insights, attempt co-crystallization with substrates (IMP, aspartate) or substrate analogs to capture different catalytic states.

These considerations help ensure that the recombinant protein maintains its native structure and enzymatic properties, which is essential for valid structural interpretations .

How does purA from B. bacteriovorus compare functionally to analogous enzymes in non-predatory bacteria?

The adenylosuccinate synthetase (purA) from B. bacteriovorus likely exhibits functional adaptations reflecting its predatory lifestyle compared to homologs in non-predatory bacteria:

• Substrate affinity and specificity: While the core catalytic mechanism remains conserved, B. bacteriovorus purA may exhibit altered substrate affinity profiles optimized for function during intraperiplasmic growth within prey bacteria. This adaptation could allow efficient nucleotide synthesis using prey-derived precursors.

• Regulatory mechanisms: Unlike non-predatory bacteria that typically regulate purA through feedback inhibition by AMP, B. bacteriovorus may employ modified regulatory mechanisms to coordinate purine biosynthesis with its biphasic life cycle (attack phase versus growth phase).

• Environmental tolerance: The enzyme likely functions efficiently within the unique biochemical environment of the prey periplasm, potentially showing different pH or ion dependencies compared to homologs from free-living bacteria.

• Protein-protein interactions: B. bacteriovorus purA may participate in predator-specific protein-protein interactions that coordinate nucleotide synthesis with other aspects of intraperiplasmic growth.

• Evolutionary convergence/divergence: Comparative genomic analyses suggest that while B. bacteriovorus maintains core metabolic pathways like nucleotide synthesis, these pathways often show unique adaptations. For instance, B. bacteriovorus shows distinctive tRNA synthetase patterns, suggesting similar specialization might occur in purA .

Further experimental evidence is needed to fully characterize these functional differences, which could provide insights into the evolutionary adaptations enabling the predatory lifestyle .

What can B. bacteriovorus purA tell us about the evolution of predatory behavior in bacteria?

The study of B. bacteriovorus purA offers valuable insights into bacterial predatory evolution:

• Metabolic adaptation signatures: Analysis of purA sequence and activity patterns may reveal molecular signatures of adaptation to predatory metabolism. The enzyme must function efficiently during rapid intraperiplasmic growth, potentially showing optimizations for utilizing prey-derived metabolites .

• Horizontal gene transfer assessment: Comparative genomic analysis of purA sequences across predatory and non-predatory bacteria can reveal whether this essential metabolic gene shows evidence of horizontal gene transfer during the evolution of predatory behavior.

• Metabolic repurposing: The unique life cycle of B. bacteriovorus requires dramatic metabolic shifts between attack phase and growth phase. purA regulation may show evidence of repurposing common metabolic pathways for predatory function.

• Conservation across predatory species: Comparing purA across different predatory bacteria (Bdellovibrio, Bacteriovorax, Micavibrio) can identify conserved adaptations associated specifically with predatory lifestyles versus convergent evolution.

• Auxiliary pathway integration: The relationships between purA and predator-specific pathways—such as those involved in prey recognition, invasion, and intraperiplasmic growth—may provide insights into the stepwise evolution of predatory behavior.

Understanding these aspects of purA evolution contributes to the broader picture of how metabolic enzymes are adapted and integrated into complex behavioral strategies like bacterial predation .

How can recombinant purA be utilized in studying B. bacteriovorus as a potential antimicrobial agent?

Recombinant purA can serve as a valuable tool in developing B. bacteriovorus as an antimicrobial agent through several research applications:

• Metabolic engineering marker: Recombinant purA, tagged with fluorescent proteins, can serve as a metabolic activity marker to monitor predator vitality in antimicrobial applications. This allows researchers to track active predation in complex environments like biofilms or in vivo systems.

• Strain optimization: Understanding purA's role in nucleotide metabolism during predation can guide genetic engineering efforts to create enhanced predatory strains. Modulated expression of purA or enzyme variants with altered kinetic properties could potentially improve predation efficiency against antibiotic-resistant pathogens .

• Host range determination: Comparative enzymatic studies of purA across multiple B. bacteriovorus strains may reveal correlations between enzyme properties and predatory host range, helping identify optimal strains for targeting specific pathogens.

• Biofilm penetration enhancement: Insights from purA metabolism could inform strategies to improve predator activity within biofilms, where metabolic adaptability is crucial for effective predation of embedded bacteria .

• Synergy with antibiotics: Understanding nucleotide metabolism in B. bacteriovorus can help identify potential synergistic combinations with traditional antibiotics that target nucleotide synthesis pathways.

• Safety assessment: Recombinant purA can be used to develop antibodies for tracking B. bacteriovorus in safety studies, monitoring clearance rates from host tissues in animal models, and assessing potential immune responses .

These applications leverage fundamental enzymology to advance the applied potential of B. bacteriovorus as a novel approach to combat antibiotic-resistant infections .

What role might purA play in the cell-lytic applications of engineered B. bacteriovorus?

The adenylosuccinate synthetase (purA) likely plays several important roles in the cell-lytic applications of engineered B. bacteriovorus:

• Metabolic bottleneck management: When engineering B. bacteriovorus as a lytic agent for recovering intracellular bioproducts from prey bacteria, purA activity may represent a potential metabolic bottleneck during rapid predator replication. Optimizing purA expression could enhance lytic efficiency, particularly at high prey densities .

• Energy conservation for lytic processes: purA functions at a critical junction of nucleotide and energy metabolism. Engineering this pathway could redirect ATP utilization toward enhanced production of lytic enzymes rather than nucleotide biosynthesis, potentially increasing product recovery yields .

• Synchronized lysis control: Developing inducible systems linked to purA regulation could allow temporal control over the predatory cycle, enabling synchronized lysis of prey populations at optimal times for product recovery.

• Reduced prey product consumption: Similar to how B. bacteriovorus has been engineered to prevent degradation of polyhydroxyalkanoates (PHAs) by knockout of specific depolymerases, understanding purA's role in prey nucleotide utilization could guide strategies to reduce consumption of valuable nucleic acid-based products from prey .

• Predictive metabolic modeling: Incorporating purA kinetics into metabolic models of predator-prey interactions could enable prediction of optimal harvest times and conditions for maximum product recovery.

This understanding has direct applications in developing B. bacteriovorus as an external cell-lytic agent for recovering valuable intracellular bioproducts from various Gram-negative bacterial cultures .

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

Several promising research directions involving B. bacteriovorus purA merit further investigation:

• Structural biology: Determining the crystal structure of B. bacteriovorus purA would reveal predator-specific adaptations and facilitate structure-based design of engineered variants with enhanced properties for biotechnological applications.

• Metabolic regulation during predation: Investigating how purA activity is regulated during the transition between attack phase and growth phase could provide fundamental insights into the metabolic reprogramming that enables predatory behavior.

• Systems biology integration: Incorporating purA into comprehensive metabolic models of B. bacteriovorus predation would allow in silico prediction of genetic modifications to enhance predatory efficiency or expand prey range.

• Synthetic biology applications: Exploring purA as part of minimal gene sets required for predation could guide the development of simplified, synthetic predatory systems with customized properties.

• Comparative enzymology: Systematic comparison of kinetic and regulatory properties of purA across predatory and non-predatory bacteria could reveal convergent evolutionary solutions to the challenges of predatory metabolism.

• Inhibitor development: Identifying specific inhibitors of B. bacteriovorus purA could provide valuable research tools for dissecting predation mechanisms and potentially lead to methods for controlling predatory bacteria in specific applications.

• Immunological interactions: Investigating how purA and other B. bacteriovorus enzymes interact with mammalian immune systems could advance the development of predatory bacteria as safe therapeutic agents .

These research directions would advance both fundamental understanding of bacterial predation and applied use of predatory bacteria in biotechnology and medicine .

What are common challenges when working with recombinant B. bacteriovorus purA and how can they be addressed?

Researchers working with recombinant B. bacteriovorus purA often encounter several challenges that can be systematically addressed:

For optimal results, researchers should reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add glycerol to a final concentration of 5-50% for long-term storage at -20°C/-80°C, while avoiding repeated freeze-thaw cycles .

What controls should be included when studying purA function in B. bacteriovorus predation experiments?

Rigorous experimental design for studying purA function in B. bacteriovorus predation requires comprehensive controls:

• Genetic controls:

  • Wild-type B. bacteriovorus (positive control for normal predation)

  • Complemented purA mutants (to verify phenotype restoration)

  • Non-predatory mutant controls (e.g., motility-deficient strains as negative controls)

  • Heat-killed predators (to distinguish predation from passive effects)

• Prey controls:

  • Multiple prey species (to assess host range impacts)

  • Prey-only cultures (baseline for growth without predation)

  • E. coli-specific controls for PCR and RNA extraction procedures to ensure B. bacteriovorus-specific amplification

• Enzymatic controls:

  • Purified recombinant purA with validated activity (positive control)

  • Heat-inactivated enzyme preparations (negative control)

  • Known purA inhibitors (chemical validation of specific activity)

  • Alternative pathway controls (to assess metabolic compensation)

• Experimental validation controls:

  • RNA extraction controls with RNase-free DNase treatment and cleanup to ensure absence of contaminating DNA

  • Load controls for gene expression studies (e.g., rpsL gene expression as used in related B. bacteriovorus research)

  • Time course sampling (to capture dynamic changes during predation cycle)

  • Technical replicates (minimum triplicate) and biological replicates (minimum triplicate from independent cultures)

These controls ensure that observed phenotypes can be confidently attributed to purA function rather than experimental artifacts or secondary effects .

How does purA function integrate with other metabolic pathways during the B. bacteriovorus predatory cycle?

The adenylosuccinate synthetase (purA) functions within an intricate network of metabolic pathways during the B. bacteriovorus predatory cycle:

• Nucleotide metabolism integration: purA catalyzes a critical step in purine biosynthesis, connecting IMP metabolism to AMP production. During intraperiplasmic growth, this pathway likely experiences increased flux to support rapid DNA replication as the predator multiplies within its prey .

• Energy metabolism coupling: The purA reaction consumes GTP, linking purine biosynthesis with energy metabolism. This connection becomes particularly significant during the transition from attack phase (where energy is primarily directed toward motility) to growth phase (where energy supports macromolecular synthesis) .

• Amino acid metabolism interface: purA utilizes aspartate as a substrate, creating a direct link between nucleotide biosynthesis and amino acid metabolism. This connection is potentially significant given B. bacteriovorus's complex amino acid requirements and its unique tRNA synthetase systems, including non-discriminating aspartyl-tRNA synthetase with GatCAB for asparagine synthesis .

• Prey resource utilization: B. bacteriovorus must efficiently convert prey-derived metabolites into predator biomass. purA likely plays a role in channeling prey-derived purine precursors into predator nucleotide pools during the intraperiplasmic growth phase .

• Cell cycle coordination: Nucleotide synthesis must be coordinated with other aspects of B. bacteriovorus growth and division within the prey cell. purA activity may be regulated in concert with cell cycle checkpoints unique to this predatory bacterium .

Understanding these integrative aspects of purA function provides a more complete picture of the metabolic adaptations enabling the predatory lifestyle of B. bacteriovorus .

What interdisciplinary approaches can advance our understanding of B. bacteriovorus purA in predatory mechanisms?

Advancing knowledge of B. bacteriovorus purA requires diverse interdisciplinary approaches:

Collaborative research integrating these approaches would yield comprehensive understanding of how purA contributes to predatory mechanisms and how this knowledge can be translated into biotechnological and medical applications .

What are the most significant unresolved questions about purA in B. bacteriovorus that merit further investigation?

Despite progress in understanding B. bacteriovorus biology, several crucial questions about purA remain unanswered:

Addressing these questions will advance both fundamental understanding of bacterial predation and practical applications of predatory bacteria.

How might future technological advances enhance our ability to study and utilize B. bacteriovorus purA?

Emerging technologies will substantially enhance research on B. bacteriovorus purA:

• Advanced imaging technologies: Super-resolution microscopy and correlative light-electron microscopy will enable visualization of purA localization during the predatory cycle with nanometer precision, potentially revealing functional microdomains within the predator cell.

• Single-cell metabolomics: Technologies for analyzing metabolite profiles in individual bacterial cells will allow researchers to track dynamic changes in nucleotide metabolism during predation at unprecedented resolution.

• Genome editing advances: CRISPR-Cas systems optimized for predatory bacteria will facilitate precise genetic manipulation of purA and interacting genes, enabling creation of subtle mutations for structure-function studies.

• Protein engineering platforms: Directed evolution approaches combined with high-throughput screening will enable development of purA variants with enhanced properties for biotechnological applications.

• Microfluidic predation chambers: Custom microfluidic devices will allow controlled observation and manipulation of individual predator-prey interactions while monitoring metabolic activities.

• Artificial intelligence applications: Machine learning algorithms applied to predation datasets will help identify patterns and predictors of successful predation, potentially highlighting previously unrecognized roles for purA.

• In situ structural biology: Techniques like cryo-electron tomography will reveal the native structure and interactions of purA within intact B. bacteriovorus cells during different predatory stages.

• Synthetic biology frameworks: Development of genetic circuit design tools optimized for predatory bacteria will enable precise control over purA expression and activity for customized predatory functions .

These technological advances will both deepen fundamental understanding of B. bacteriovorus biology and expand practical applications in biotechnology, agriculture, and medicine.