Recombinant Desulfovibrio vulgaris Phosphate import ATP-binding protein PstB (pstB)

What is Desulfovibrio vulgaris and what role does PstB play in its metabolism?

Desulfovibrio vulgaris is an anaerobic, sulfate-reducing bacterium found ubiquitously in nature. It serves as a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and has significant economic impacts including biocorrosion of metal infrastructure and bioremediation of toxic metal ions . The genome of D. vulgaris Hildenborough strain is 3,570,858 base pairs and contains genes for a network of novel c-type cytochromes that connect multiple periplasmic hydrogenases and formate dehydrogenases .

How does the PstB protein contribute to phosphate transport in Desulfovibrio vulgaris?

PstB operates as part of the complete Pst system, which typically consists of four components arranged as shown in the table below:

The transport mechanism follows these steps:

• PstS binds phosphate in the periplasm

• PstS docks with the PstA/PstC membrane complex

• PstB binds ATP, inducing conformational changes in PstA/PstC

• The conformational change transfers phosphate across the membrane

• ATP hydrolysis resets the system for another transport cycle

This high-affinity transport system allows D. vulgaris to efficiently acquire phosphate even in environments where this essential nutrient is limited.

What are the challenges in expressing recombinant Desulfovibrio vulgaris PstB in heterologous systems?

Expressing recombinant proteins from anaerobic bacteria like D. vulgaris in heterologous systems presents several challenges:

• Codon usage bias: D. vulgaris has different codon preferences compared to common expression hosts like E. coli. Based on search result , successful expression of D. vulgaris proteins has required codon optimization in some cases.

• Protein folding issues: Proteins from anaerobic organisms may not fold properly in aerobic expression hosts due to differences in the intracellular environment.

• Expression toxicity: Overexpression of membrane-associated proteins like PstB can be toxic to host cells.

• Maintaining solubility: ABC transporter components often have hydrophobic regions that can lead to aggregation.

These challenges can be addressed through strategies such as:

• Using specialized expression strains

• Employing lower expression temperatures (16-25°C)

• Utilizing solubility-enhancing fusion tags

• Codon optimization for the expression host

Search result describes successful expression of a D. vulgaris cytochrome c3 in Shewanella oneidensis MR-1 under both aerobic and anaerobic conditions, suggesting that this host might also be suitable for expressing PstB with yields of 0.3-0.5 mg of protein per gram of cells .

How can researchers design effective experimental approaches to study the in vivo function of PstB?

Based on the experimental design principles outlined in search results , , and , a systematic approach for studying PstB function should include:

• Define your variables:

  • Independent variable: PstB activity/expression level

  • Dependent variable: Phosphate transport efficiency, growth rate

  • Control variables: Growth conditions, media composition

• Generate testable hypotheses:

  • Example: "Mutations in the Walker A motif of PstB will reduce phosphate uptake under phosphate-limited conditions"

• Design manipulation strategies:

  • Gene deletion/complementation studies

  • Site-directed mutagenesis of key catalytic residues

  • Controlled expression systems

• Select appropriate measurement methods:

  • Radiolabeled phosphate uptake assays

  • Growth rate determination in phosphate-limited media

  • ATP hydrolysis assays with purified protein

• Implement genetic tools for D. vulgaris:
  Search result describes generalized schemes for high-throughput manipulation of D. vulgaris genome, including:

  • Custom suicide vectors with reusable DNA parts

  • Gateway recombination system for construct assembly

  • Creation of gene fusions with affinity purification tags

What methods are most effective for analyzing ATP hydrolysis activity of recombinant PstB?

To effectively analyze the ATP hydrolysis activity of recombinant PstB, researchers should employ a multi-method approach:

• Colorimetric phosphate release assays:

• Coupled enzyme assays:

  • Pyruvate kinase/lactate dehydrogenase system

  • ATP hydrolysis coupled to NADH oxidation

  • Real-time monitoring via absorbance at 340 nm

• Experimental protocol optimization:

  • Buffer conditions: 20-50 mM Tris-HCl (pH 7.5), 50-150 mM NaCl, 5 mM MgCl₂

  • Temperature range: Test at both physiological (37°C) and ambient (25°C) temperatures

  • Substrate concentration: 0.1-5 mM ATP with Michaelis-Menten kinetic analysis

• Data analysis and reporting:

  • Calculate Km, Vmax, and kcat values

  • Determine effects of potential inhibitors or activators

  • Compare wild-type to site-directed mutant variants

When performing these assays, include appropriate controls such as no-enzyme blanks, heat-inactivated enzyme, and Walker A motif mutants (e.g., K43A) as negative controls. This systematic approach will provide comprehensive characterization of PstB's enzymatic properties.

What are the optimal conditions for expressing and purifying recombinant Desulfovibrio vulgaris PstB?

Based on information from search results and , an optimized protocol for expression and purification of recombinant D. vulgaris PstB should include:

Expression system:

• Host: E. coli BL21(DE3) cells

• Vector: pET-based with T7 promoter system

• Fusion tags: 6×His tag for purification , consider adding solubility enhancers (SUMO, MBP) if needed

Expression protocol:

• Culture in LB or 2×YT media with appropriate antibiotics

• Grow at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.1-0.5 mM IPTG

• Continue expression at reduced temperature (16-20°C) for 16-20 hours

• Harvest cells by centrifugation (4,000-6,000 × g, 4°C, 15-20 minutes)

Purification strategy:

• Initial IMAC purification (Ni-NTA):

  • Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM MgCl₂, protease inhibitors

  • Wash buffer: Same as lysis buffer with 20-40 mM imidazole

  • Elution buffer: Same as lysis buffer with 250 mM imidazole

• Size exclusion chromatography:

  • Buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT, 5 mM MgCl₂

Search result indicates that properly purified recombinant PstB should have a purity of >85% as assessed by SDS-PAGE.

How can researchers establish an experimental system to study PstB-mediated phosphate transport?

To establish a robust experimental system for studying PstB-mediated phosphate transport, researchers should consider:

• In vivo transport assays:

  • Develop a D. vulgaris strain with tagged or modified PstB using approaches from search result

  • Measure ³²P-labeled phosphate uptake under various conditions

  • Compare wild-type, pstB knockout, and complemented strains

• Reconstituted liposome system:

  • Purify all components of the Pst system (PstS, PstA, PstC, PstB)

  • Reconstitute into liposomes with controlled lipid composition

  • Measure ATP-dependent phosphate transport into liposomes

• Experimental design considerations based on search results and :

  • Control variables: pH, temperature, ionic strength

  • Independent variables: ATP concentration, phosphate concentration, PstB variants

  • Dependent variables: Transport rate, ATP hydrolysis rate

• Data collection and analysis:

  • Time-course measurements for kinetic analysis

  • Calculation of transport efficiency (phosphate transported per ATP hydrolyzed)

  • Statistical comparison between experimental conditions

What protein-protein interaction methods are most suitable for studying PstB interactions within the Pst system?

Based on search result , several effective approaches have been developed for studying protein-protein interactions in D. vulgaris:

• Affinity purification-based methods:

• Protocol for SPA purification of PstB complexes (based on search result ):

  • Culture D. vulgaris strains with SPA-tagged PstB in 1 liter of 2× LS4D medium under anaerobic conditions

  • Harvest cells and prepare lysates under native conditions

  • Apply lysate to anti-FLAG beads

  • Elute bound proteins using TEV protease

  • Apply TEV eluate to IgG beads or Streptactin Superflow beads

  • Analyze by mass spectrometry to identify interaction partners

• Complementary approaches:

  • Bacterial two-hybrid systems adapted for D. vulgaris

  • Crosslinking mass spectrometry

  • FRET-based interaction studies with fluorescently labeled components

Search result demonstrates that creating chromosomally tagged proteins in D. vulgaris is feasible using custom suicide vectors with reusable DNA parts, providing a solid foundation for studying PstB interactions in its native context.

How can researchers systematically investigate the relationship between PstB function and D. vulgaris metabolism?

To systematically investigate the relationship between PstB function and D. vulgaris metabolism, researchers should implement a multi-level experimental approach:

• Genetic manipulation strategies (based on search result ):

  • Generate pstB knockout strains using suicide vectors and homologous recombination

  • Create strains with PstB variants containing specific mutations in functional domains

  • Develop controllable expression systems for PstB

• Phenotypic characterization:

  • Growth curves under varying phosphate concentrations

  • Competitive fitness assays with wild-type strain

  • Phosphate uptake measurements using radiolabeled phosphate

  • Metabolomic profiling to assess global metabolic changes

• Systems biology integration:

  • Transcriptomic analysis to identify genes affected by PstB disruption

  • Proteomic analysis to detect changes in protein expression patterns

  • Flux balance analysis to model the effects on metabolic pathways

• Experimental design considerations:

  • Follow design principles from search results , , and

  • Include appropriate controls and replicates

  • Ensure statistical power through proper sample sizing

  • Control for batch effects and environmental variables

This comprehensive approach will provide insights into how PstB function influences not only phosphate acquisition but also broader aspects of D. vulgaris metabolism and physiology.