Recombinant Bdellovibrio bacteriovorus Phosphate import ATP-binding protein PstB (pstB)

What is the role of PstB protein in Bdellovibrio bacteriovorus phosphate transport?

PstB functions as the catalytic subunit of the phosphate-specific transport (Pst) system in B. bacteriovorus, coupling the energy of ATP hydrolysis to the import of phosphate across cellular membranes. The protein belongs to the ABC transporter superfamily and constitutes a critical component of the high-affinity Pi transporter in this predatory bacterium .

As part of the conserved core set of ABC systems in Bdellovibrio-and-like organisms (BALOs), PstB works in conjunction with other Pst system components to enable efficient phosphate uptake under phosphate-limited conditions. This ATP-related transport capability has been suggested to be an adaptation to the natural growth environment of BALOs within their prey (gram-negative bacteria) .

Research has demonstrated that the genes encoding these ABC systems occupy nearly 1.3% of the gene content in freshwater Bdellovibrio strains and about 0.7% in their saltwater counterparts .

How does the structure of Bdellovibrio bacteriovorus PstB compare to PstB proteins from other bacteria?

The PstB domain contains a highly conserved ATP binding cassette, which is also referred to as a nucleotide binding domain (NBD) . When examining the sequence architecture:

Analysis of PstB across different bacteria shows that while the catalytic core is highly conserved, adaptive modifications likely reflect the specific ecological niches and metabolic requirements of each organism .

What expression systems are recommended for producing recombinant Bdellovibrio bacteriovorus PstB?

For successful expression of recombinant B. bacteriovorus PstB, researchers should consider several expression systems optimized for membrane-associated proteins:

E. coli-based expression systems:

• BL21(DE3) strains with pET vector systems can be effective for PstB expression when optimized for membrane-associated proteins

• C41(DE3) or C43(DE3) strains are particularly useful as they were developed specifically for expressing toxic or membrane proteins

Expression optimization parameters:

• Induction temperature: 18-25°C (lower temperatures often improve proper folding)

• IPTG concentration: 0.1-0.5 mM (lower concentrations may yield better soluble protein)

• Growth media: Consider supplementation with extra phosphate sources when expressing phosphate-related proteins

• Induction time: Extended expression periods (16-20 hours) at lower temperatures

Researchers should note that the genetic toolbox for B. bacteriovorus is still limited despite six decades of research . While some success has been achieved with IncQ-type plasmids that autonomously replicate in predator cells, IncP-type plasmids maintained through integration via Campbell-like recombination may also be viable options for homologous expression .

What purification strategies are most effective for recombinant Bdellovibrio bacteriovorus PstB?

Purification of recombinant B. bacteriovorus PstB requires specific protocols tailored to this ATP-binding protein:

Recommended purification workflow:

• Cell lysis and membrane preparation:

  • Sonication or pressure-based disruption in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl₂, and protease inhibitors

  • Separation of membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

• Solubilization:

  • Membrane fractions containing PstB should be solubilized using mild detergents

  • Recommended detergents: n-dodecyl-β-D-maltoside (DDM) at 1% or n-octyl-β-D-glucopyranoside (OG) at 2%

  • Include 5 mM ATP or non-hydrolyzable ATP analogs to stabilize the protein

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) with Ni-NTA resin for His-tagged PstB

  • Wash buffers should contain 20-40 mM imidazole and 0.05% detergent

  • Elution with 250-300 mM imidazole

• Further purification:

  • Size exclusion chromatography to achieve higher purity and assess oligomeric state

  • Ion exchange chromatography as an optional polishing step

Maintaining the presence of specific divalent cations (15-25 mM Ca²⁺) throughout purification can enhance protein stability, as research suggests these concentrations are optimal for B. bacteriovorus physiological activity .

How can researchers assess the functional activity of purified recombinant Bdellovibrio bacteriovorus PstB?

Multiple complementary approaches can be employed to verify the functional activity of purified recombinant B. bacteriovorus PstB:

ATP binding and hydrolysis assays:

• ATP binding can be measured using fluorescent ATP analogs (TNP-ATP) or radioactively labeled ATP

• ATPase activity can be assessed through colorimetric phosphate release assays (malachite green) or coupled enzyme assays

• The PstB protein from related systems has been shown to bind and hydrolyze ATP, producing ADP in a specific manner

Reconstitution into proteoliposomes:

• Reconstitute purified PstB together with other Pst system components into liposomes

• Measure phosphate uptake using radioactively labeled phosphate (³²P) or fluorescent phosphate analogs

• Compare activity with known controls to establish specific activity levels

Thermal stability assays:

• Differential scanning fluorimetry (DSF) in the presence and absence of ATP, ADP, and phosphate

• Shifts in melting temperature can indicate functional ligand binding

Interaction studies:

• Bacterial two-hybrid analysis to assess interactions with PhoR and other system components, similar to methodologies used for studying related PhoU interactions

• Co-elution experiments to confirm protein-protein interactions within the Pst system

Research has demonstrated that in functional assays, the denatured PstB (e.g., urea-treated) does not bind ATP, providing a useful negative control .

What methods are most effective for studying Bdellovibrio bacteriovorus PstB interactions with other components of the phosphate transport system?

Several complementary techniques are effective for investigating B. bacteriovorus PstB interactions with other Pst system components:

In vitro methods:

• Pull-down assays: Using tagged versions of PstB to identify interaction partners from cell lysates or with purified candidate proteins

• Surface plasmon resonance (SPR): For quantitative measurement of binding affinities between PstB and other Pst components

• Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of binding interactions

• Crosslinking studies: Using chemical crosslinkers followed by mass spectrometry to identify interaction interfaces

In vivo methods:

• Bacterial two-hybrid (BTH) analysis: As demonstrated with the related PhoU protein, which interacts with PhoR through its PAS domain and with PstB

• Fluorescence resonance energy transfer (FRET): To visualize protein interactions in live cells using fluorescently tagged proteins

• Co-immunoprecipitation (Co-IP): To pull down protein complexes from native conditions

Research on related systems has shown that PstB interacts with other components of the phosphate signaling pathway, including PhoU, suggesting a similar interaction network may exist in B. bacteriovorus .

How does phosphate availability affect the expression and function of Bdellovibrio bacteriovorus PstB?

Phosphate availability significantly impacts the expression and function of B. bacteriovorus PstB through several regulatory mechanisms:

Expression regulation:

• Under phosphate-limited conditions, increased expression of the pst operon occurs

• The Pho regulon, which includes the pst genes, is controlled by the two-component system PhoR-PhoB

• When phosphate is scarce, PhoR phosphorylates PhoB, which then activates transcription of genes involved in phosphate acquisition, including pstB

Functional implications:

• In phosphate-rich environments, the Pst system functions primarily as a repressor of the Pho regulon

• During phosphate limitation, the system shifts to its transport function, where PstB drives ATP-dependent phosphate uptake

• This dual role as both transporter and regulator makes PstB particularly interesting for studying bacterial phosphate homeostasis

Analysis of BALOs genomes has revealed that the genes encoding these ABC systems occupy nearly 1.3% of the gene content in freshwater Bdellovibrio strains and about 0.7% in their saltwater counterparts, suggesting adaptation to different phosphate availability in these environments .

What are the key differences between PstB proteins from host-dependent and host-independent Bdellovibrio bacteriovorus strains?

Research comparing host-dependent (HD) and host-independent (HI) B. bacteriovorus strains has identified several differences in their PstB proteins:

Host-independent (HI) B. bacteriovorus strains, which can grow without prey in a saprophytic state, may have adapted their phosphate transport systems for this alternative lifestyle . Different sets of genes are essential for HD and HI growth , suggesting that PstB functionality may be adapted to these distinct metabolic states.

The expression of ATP-binding cassette transporters may differ between these growth modes as the HD form requires efficient nutrient acquisition from prey, while the HI form must obtain nutrients from the surrounding medium .

How can researchers generate mutations in Bdellovibrio bacteriovorus PstB for structure-function studies?

For structure-function studies of B. bacteriovorus PstB, researchers can employ several genetic manipulation strategies:

Site-directed mutagenesis approaches:

• For HD strains:

  • Expression of antisense RNA to downregulate pstB in wild-type obligate predator background

  • Counter-selectable marker strategy using suicide plasmids containing sacB

  • IncP plasmid pSET151-based tools for targeted gene modifications

• For HI strains:

  • Direct transformation with recombinant plasmids carrying the mutated pstB gene

  • Transposon mutagenesis using conjugation or electroporation with plasmids like pRL27

  • CRISPR-Cas9 approaches (although these are still being optimized for Bdellovibrio)

Key considerations:

• Different sets of genes are essential for HD and HI growth; therefore, HD strains can be used to inactivate genes essential for HI growth, while HI strains are preferable for manipulating predation-essential genes

• For conditional expression studies, synthetic riboswitches have been successfully employed in B. bacteriovorus and could be adapted for pstB manipulation

When designing mutations, focus on conserved motifs within PstB, such as the Walker A and Walker B motifs that are critical for ATP binding and hydrolysis, to understand the relationship between specific residues and protein function.

What is the significance of PstB in the ATP metabolism of Bdellovibrio bacteriovorus during its predatory lifecycle?

PstB plays a crucial role in the ATP metabolism of B. bacteriovorus during its complex predatory lifecycle:

ATP utilization during predation:

• PstB functions as an ATP-hydrolyzing enzyme that powers phosphate import, providing essential nutrients during the intraperiplasmic growth phase

• This ATP-dependent transport is critical for acquiring phosphate from prey contents

• Measurements of intracellular ATP levels have shown dynamic changes during the predation cycle, with ATP utilization systems like PstB being key components of energy management

Metabolic integration:

• Despite B. bacteriovorus showing low activities of most glycolytic enzymes, PstB and the Pst system show notably high activity

• This suggests a specialized role in predatory metabolism, where phosphate acquisition is prioritized

• Interestingly, studies have shown that B. bacteriovorus is unlikely to utilize polysaccharides as primary substrates for energy metabolism and instead respires amino acids for energy during intraperiplasmic growth

Lifecycle-specific functions:

• During the attack phase, ATP is primarily used for motility and prey detection

• After prey invasion, ATP utilization shifts to nutrient acquisition systems including the Pst system

• In the growth phase within the bdelloplast, PstB-driven phosphate import supports DNA replication and cell division

This metabolic adaptation allows B. bacteriovorus to efficiently utilize the limited resources available during its unique predatory lifecycle .

How do the kinetic properties of recombinant Bdellovibrio bacteriovorus PstB compare to PstB proteins from other prokaryotes?

Comparative analysis of kinetic properties between recombinant B. bacteriovorus PstB and PstB proteins from other prokaryotes reveals several distinct characteristics:

The PstB protein from B. bacteriovorus has been suggested to demonstrate specialized kinetic properties that reflect its predatory lifestyle, with potentially enhanced coupling between ATP hydrolysis and phosphate transport to maximize nutrient acquisition from prey .

Furthermore, the structure of B. bacteriovorus PstB suggests adaptations for function within the minimal genome context of this predatory bacterium, potentially representing "the minimal requirements for a functional PstB" . This may manifest in kinetic properties that prioritize efficiency over regulatory complexity when compared to PstB proteins from free-living bacteria.

What analytical techniques are recommended for characterizing the structural properties of recombinant Bdellovibrio bacteriovorus PstB?

Multiple analytical techniques can be employed to thoroughly characterize the structural properties of recombinant B. bacteriovorus PstB:

High-resolution structural techniques:

• X-ray crystallography:

  • Recommended for atomic-level resolution of PstB structure

  • Has been successfully applied to related bacterial ABC transporters

  • May require optimization of crystallization conditions with and without ATP/ADP

• Cryo-electron microscopy (cryo-EM):

  • Appropriate for visualizing PstB in complex with other components of the Pst system

  • Particularly valuable for capturing different conformational states during the ATP hydrolysis cycle

Solution-based structural techniques:

Spectroscopic methods:

• Nuclear magnetic resonance (NMR) spectroscopy:

  • For analyzing dynamics and ligand binding

  • May be challenging for the full-length protein but applicable to domains

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • For mapping conformational changes and solvent accessibility

  • Particularly useful for identifying ATP-binding-induced conformational changes

Biophysical characterization:

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • For determining oligomeric state and monodispersity

  • Critical for confirming proper folding and assembly

• Differential scanning calorimetry (DSC) and fluorimetry (DSF):

  • For assessing thermal stability and ligand binding

Researchers have successfully employed X-ray crystallography to solve structures of related proteins from B. bacteriovorus, such as phosphoglucose isomerase at high resolution (1.67-1.74Å) , suggesting similar approaches would be applicable to PstB.

What role does PstB play in the phosphate signaling pathway of Bdellovibrio bacteriovorus?

PstB functions as a key component in the phosphate signaling pathway of B. bacteriovorus, integrating transport function with regulatory roles:

Signal transduction involvement:

• PstB interacts with the PhoU protein, which serves as an intermediary between the Pst transport system and the PhoR-PhoB two-component regulatory system

• This interaction is crucial for sensing environmental phosphate levels and transmitting this signal to regulate gene expression

• Research on related systems has demonstrated that PstB interacts with PhoU, which in turn interacts with PhoR through its PAS domain

Regulatory mechanisms:

• Under phosphate-replete conditions, the Pst system, including PstB, forms a repression complex with PhoU and PhoR that prevents PhoR from phosphorylating PhoB

• When phosphate becomes limiting, conformational changes in the Pst system disrupt this repression complex

• PhoR then phosphorylates PhoB, which activates transcription of the Pho regulon genes

Physiological implications:

• This signaling pathway allows B. bacteriovorus to adapt to varying phosphate availability during its lifecycle

• The system likely plays a crucial role during transitions between the attack phase and growth phase within prey

• Freshwater Bdellovibrio strains have more ABC systems than saltwater strains, suggesting environmental adaptation of these signaling pathways

The involvement of PstB in both transport and signaling makes it a critical node in the regulatory network that controls phosphate homeostasis in B. bacteriovorus .

How does temperature affect the stability and activity of recombinant Bdellovibrio bacteriovorus PstB?

Temperature has significant effects on both the stability and activity of recombinant B. bacteriovorus PstB, with important implications for experimental design:

Thermal stability profile:

• Recombinant B. bacteriovorus PstB likely exhibits optimal stability within the temperature range that supports the bacterium's predatory activity (30-35°C)

• Above this range, protein denaturation and aggregation may occur progressively

• Below this range, decreased flexibility may reduce catalytic efficiency

Temperature-dependent activity:

• ATP hydrolysis rates by PstB show temperature dependence, with activity measurements ideally performed at physiologically relevant temperatures

• Activation energy for ATP hydrolysis can be determined using Arrhenius plots across different temperatures

• Coupling between ATP hydrolysis and phosphate transport may be affected differently by temperature changes

Experimental considerations:

• For purification, working at 4°C is generally recommended to minimize proteolysis and denaturation

• For activity assays, temperatures between 25-37°C are appropriate, with 30-35°C being optimal based on the physiological temperature preference of B. bacteriovorus

• For stability studies, differential scanning fluorimetry can provide precise melting temperatures (Tm) under various buffer conditions

Research on B. bacteriovorus physiology has established that the optimal temperature range for growth and reproduction is 30-35°C , suggesting that PstB would exhibit maximal activity within this temperature range in its native context.

What genome engineering approaches can be used to modify the PstB gene in Bdellovibrio bacteriovorus for functional studies?

Several genome engineering strategies can be applied to modify the pstB gene in B. bacteriovorus for functional studies:

Established methods:

• Homologous recombination:

  • Using suicide vectors like pSSK10 containing counterselectable markers (sacB)

  • Generation of markerless deletion mutants using pK18 mobsacB

  • These approaches allow for precise modifications without leaving selection markers

• Transposon mutagenesis:

  • Using transposon-containing plasmids like pRL27 delivered by conjugation or electroporation

  • Particularly effective for HI strains of B. bacteriovorus

  • Useful for generating random mutation libraries to identify functional domains

• Antisense RNA expression:

  • For downregulation of pstB in wild-type obligate predator backgrounds

  • Allows study of essential genes where knockout would be lethal

Emerging technologies:

• Synthetic riboswitches:

  • Have been successfully used for regulated expression of flagellar genes in B. bacteriovorus

  • Could be adapted for conditional expression or repression of pstB

• CRISPR-Cas9 systems:

  • Being developed for B. bacteriovorus though not yet widely implemented

  • Would allow precise genome editing without selection markers

Delivery methods:

• Conjugation is the most common method for delivering recombinant DNA into B. bacteriovorus

• Electroporation has been shown to be equally efficient for introducing plasmids into HI B. bacteriovorus strains

Researchers should note that different sets of genes are essential for HD and HI growth modes, which determines the appropriate strain background for genetic manipulations .

How can researchers quantitatively assess phosphate transport activity mediated by recombinant Bdellovibrio bacteriovorus PstB?

Researchers can employ several quantitative approaches to measure phosphate transport activity mediated by recombinant B. bacteriovorus PstB:

In vitro reconstitution assays:

• Proteoliposome-based transport assays:

  • Reconstitute purified PstB along with other Pst system components (PstS, PstA, PstC) into liposomes

  • Measure uptake of radioactively labeled phosphate (³²P) or fluorescent phosphate analogs

  • Monitor time-dependent accumulation inside vesicles using filtration or centrifugation techniques

  • Calculate initial rates and kinetic parameters (Km, Vmax)

• ATPase activity coupling:

  • Measure ATP hydrolysis rates using colorimetric assays (malachite green) or coupled enzyme systems

  • Correlate ATP hydrolysis with phosphate transport to determine coupling efficiency

  • Compare rates with and without phosphate gradient to establish transport-specific activity

Whole-cell assays:

• Radioactive phosphate uptake:

  • Express recombinant PstB in phosphate transport-deficient cells

  • Measure uptake of ³²P over time

  • Calculate transport rates normalized to protein expression levels

• Growth complementation:

  • Transform phosphate transport-deficient strains with recombinant PstB

  • Assess growth rescue under phosphate-limited conditions

  • Quantify growth rates as an indirect measure of transport activity

Data analysis:

• Transport kinetics should be analyzed using appropriate models (Michaelis-Menten, Hill equation)

• Consider the effect of membrane potential and pH on transport rates

• Account for background transport through other phosphate transporters

These approaches can be tailored to investigate specific aspects of PstB function, such as substrate specificity, ATP dependence, and the effects of mutations on transport activity.

What are the challenges in expressing and purifying functional Bdellovibrio bacteriovorus PstB, and how can researchers overcome them?

Researchers face several challenges when expressing and purifying functional B. bacteriovorus PstB, with specific strategies available to address each issue:

Expression challenges:

• Membrane association:

  • Challenge: PstB has membrane-associated domains that can cause aggregation

  • Solution: Use specialized E. coli strains (C41/C43), lower expression temperatures (16-18°C), and mild induction conditions

• Protein toxicity:

  • Challenge: Overexpression may disrupt host cell physiology

  • Solution: Use tightly regulated expression systems, consider cell-free expression systems, or employ secretion-based expression strategies

• Codon usage bias:

  • Challenge: Different codon preferences between B. bacteriovorus and expression hosts

  • Solution: Optimize codons for the expression host or use Rosetta strains that supply rare tRNAs

Purification challenges:

• Maintaining native conformation:

  • Challenge: Loss of activity during extraction from membranes

  • Solution: Use gentle detergents (DDM, LMNG), include stabilizing additives (glycerol, specific lipids)

• Preserving ATP-binding activity:

  • Challenge: ATP-binding site sensitivity to purification conditions

  • Solution: Include ATP or non-hydrolyzable analogs during purification; avoid urea treatment which has been shown to eliminate ATP binding

• Co-purification with interacting proteins:

  • Challenge: Native interactions with other Pst components

  • Solution: Use high-salt washes, design constructs that minimize interaction surfaces, or co-express with interacting partners

Stability and storage:

• Buffer optimization:

  • Challenge: Poor stability in standard buffers

  • Solution: Screen buffer conditions using differential scanning fluorimetry; include Mg²⁺ and Ca²⁺ (15-25 mM optimal)

• Preventing aggregation:

  • Challenge: Tendency to aggregate during concentration and storage

  • Solution: Add glycerol (10-15%), use appropriate detergents, store at protein-specific optimal temperature

These approaches can be combined and optimized based on specific experimental goals and the intended applications of the purified protein.

How does the PstB protein from Bdellovibrio bacteriovorus contribute to its predatory lifestyle?

The PstB protein from B. bacteriovorus plays several critical roles that support its unique predatory lifecycle:

Nutrient acquisition during predation:

• PstB powers the high-affinity phosphate transport system that enables efficient phosphate uptake from prey cytoplasm

• This is particularly important as B. bacteriovorus grows and reproduces within the periplasm of host bacteria, where it must efficiently extract nutrients from the prey cytoplasm

• The ATP-dependent phosphate transport capability has been suggested to be an adaptation to the natural growth environment of BALOs within their gram-negative bacterial prey

Adaptation to phosphate-limited environments:

• The freshwater Bdellovibrio strains have more ABC systems (typically around 51) compared to saltwater Bacteriovorax strains (28 or less)

• This difference likely reflects adaptation to different phosphate availability in these environments

• These high-affinity Pi transporters enable BALOs to efficiently uptake phosphate under Pi-limited conditions

Metabolic integration:

• Despite B. bacteriovorus being unlikely to utilize polysaccharides as primary substrates for energy metabolism (instead respiring amino acids during intraperiplasmic growth), high activities of PstB and phosphate transport systems have been observed

• This suggests a specialized metabolic strategy where phosphate acquisition is prioritized for nucleic acid synthesis and energy metabolism

Signal transduction:

• PstB likely participates in phosphate sensing and signaling, allowing the predator to adapt its metabolism based on phosphate availability

• This sensing mechanism would be particularly important during transitions between the free-swimming attack phase and the intraperiplasmic growth phase

The integration of PstB into both metabolic and regulatory networks highlights its importance in the complex predatory lifecycle of B. bacteriovorus .

What bioinformatic approaches are useful for analyzing the evolutionary relationships between Bdellovibrio bacteriovorus PstB and related proteins?

Several bioinformatic approaches can be employed to analyze evolutionary relationships between B. bacteriovorus PstB and related proteins:

Sequence-based analyses:

• Multiple sequence alignment (MSA):

  • Tools: Clustal Omega, MUSCLE, T-Coffee

  • Identify conserved domains, motifs, and functionally important residues

  • Particularly focus on ATP-binding and catalytic sites

• Phylogenetic tree construction:

  • Methods: Maximum Likelihood, Bayesian inference, Minimum Evolution

  • Software: MEGA, PhyML, MrBayes

  • Apply bootstrap analysis (>1000 replicates) to assess tree reliability, as used in BALOs ABC transporter studies

• Ortholog/paralog identification:

  • Tools: OrthoMCL, OrthoFinder, BLAST-based approaches

  • Identify PstB homologs across bacterial species and distinguish between orthologs and paralogs

Structure-based approaches:

• Homology modeling:

  • Use related crystal structures as templates

  • Software: SWISS-MODEL, Phyre2, I-TASSER

  • Validate models with tools like PROCHECK and MolProbity

• Structural alignment:

  • Compare predicted or experimental structures of PstB proteins

  • Tools: PyMOL, UCSF Chimera

  • Identify structurally conserved regions despite sequence divergence

Functional prediction:

• Domain architecture analysis:

  • Tools: PFAM, InterPro, SMART

  • Compare domain organization across species

• Coevolution analysis:

  • Identify co-evolving residues that may be functionally linked

  • Tools: PSICOV, DCA, EVcouplings

Research on BALOs has shown that phylogenetic analysis of ABC proteins can assign them into separate families with high bootstrap supports (>50%), providing a framework for evolutionary studies of PstB .

How can researchers evaluate the potential role of Bdellovibrio bacteriovorus PstB in developing antimicrobial applications?

Researchers can employ several approaches to evaluate the potential role of B. bacteriovorus PstB in antimicrobial applications:

Functional inhibition studies:

• Target validation:

  • Generate conditional pstB mutants in B. bacteriovorus

  • Assess impact on predatory efficiency against various Gram-negative pathogens

  • Quantify effects on predation rates, bdelloplast formation, and prey killing

• Small molecule screening:

  • Develop high-throughput assays for PstB ATP hydrolysis activity

  • Screen compound libraries for specific PstB inhibitors

  • Evaluate effects of hits on predatory capacity against antibiotic-resistant pathogens

Genetic enhancement approaches:

• Overexpression studies:

  • Engineer B. bacteriovorus strains with enhanced PstB expression

  • Compare predatory efficiency and host range with wild-type strains

  • Assess impact on phosphate acquisition and energy metabolism

• Protein engineering:

  • Design PstB variants with enhanced activity or stability

  • Test engineered strains against biofilms and antibiotic-resistant bacteria

  • Evaluate in relevant infection models

Application-specific evaluations:

• Biofilm penetration:

  • Assess the role of phosphate metabolism in biofilm predation

  • Compare wild-type and PstB-modified strains in their ability to disrupt biofilms

  • Note that B. bacteriovorus has demonstrated effectiveness against biofilms formed by colistin-resistant Gram-negative bacteria

• In vivo efficacy:

  • Test modified strains in animal infection models

  • Evaluate safety, efficacy, and immune responses

  • Compare with conventional antibiotics and wild-type B. bacteriovorus

Regulatory considerations:

• Safety assessment:

  • Evaluate potential off-target effects of PstB modification

  • Assess horizontal gene transfer risks

  • Monitor for emergence of resistance mechanisms