Recombinant Photobacterium profundum Vitamin B12 import ATP-binding protein BtuD (btuD)

What is Photobacterium profundum BtuD and what is its functional role in bacterial physiology?

Photobacterium profundum BtuD functions as the ATP-binding protein component of the BtuCD complex, an ATP-binding cassette (ABC) transporter specialized for vitamin B12 import. This protein belongs to the broader family of ABC transporters that utilize ATP hydrolysis to drive substrate translocation across cell membranes. In the specific case of the BtuCD system, BtuD provides the energetic driving force through ATP binding and hydrolysis that enables vitamin B12 uptake, a critical micronutrient for bacterial metabolism .

P. profundum is a deep-sea bacterium that has evolved to thrive under high hydrostatic pressure and low temperature conditions . As such, its BtuD protein likely possesses adaptations that enable optimal functionality under these extreme environments, though specific pressure-related modifications have not been fully characterized in the available literature.

What are the conserved motifs and key functional domains in P. profundum BtuD?

Like other members of the ABC transporter family, P. profundum BtuD likely contains several highly conserved motifs that are essential for ATP binding and hydrolysis:

• Walker A motif (P-loop): Critical for ATP binding

• Walker B motif: Essential for ATP hydrolysis

• Q-loop: Involved in communication between the nucleotide-binding domain and the transmembrane domain

• H-loop: Contains a conserved histidine important for the hydrolysis mechanism

• D-loop: Facilitates interaction between the two nucleotide-binding domains

Specific research on point mutations supports the essential nature of these domains. Notably, the E159A mutation in the Walker B motif has been shown to significantly impair ATP hydrolysis activity while preserving ATP binding capability in related systems .

What are the optimal conditions for heterologous expression of recombinant P. profundum BtuD?

Based on methodologies applied to related proteins from P. profundum, optimal expression of recombinant BtuD would likely require:

• Expression Host Selection:

  • E. coli strains designed for membrane protein expression (e.g., C41/C43(DE3) or Lemo21(DE3))

  • Consideration of cold-adapted expression hosts for proteins evolved in low-temperature environments

• Expression Conditions:

  • Lower induction temperatures (15-20°C) to mimic the natural cold environment of P. profundum

  • Extended expression periods (24-48 hours) at reduced temperatures

  • Induction at lower OD values to prevent inclusion body formation

• Growth Media Considerations:

  • Marine broth supplementation may improve expression of proteins from marine bacteria

  • Addition of osmolytes that mimic high-pressure environments

Research with other P. profundum proteins has demonstrated successful complementation in E. coli expression systems, suggesting functional conservation despite the pressure-adapted nature of the original host .

What purification strategy is most effective for obtaining active recombinant P. profundum BtuD?

A methodological approach for purification would include:

• Initial Extraction:

  • Gentle detergent solubilization if co-expressed with membrane components

  • Carefully optimized lysis conditions considering the pressure-adapted nature of the protein

• Chromatography Sequence:

  • Immobilized metal affinity chromatography (IMAC) using an engineered His-tag

  • Ion exchange chromatography for removal of nucleotide contaminants

  • Size exclusion chromatography for final polishing and buffer exchange

• Critical Considerations:

  • Maintaining appropriate nucleotide concentrations throughout purification

  • Temperature control during all purification steps

  • Inclusion of stabilizing agents (glycerol, specific lipids)

Research protocols for similar ATP-binding proteins suggest maintaining ATP analogs or non-hydrolyzable ATP derivatives during purification to enhance stability .

How can researchers accurately measure the ATPase activity of recombinant P. profundum BtuD?

Several methodological approaches can be employed to measure ATPase activity:

• Coupled Enzyme Assays:

  • NADH-coupled assay where ATP hydrolysis is linked to NADH oxidation

  • Continuous spectrophotometric monitoring at 340 nm

• Phosphate Release Assays:

  • Malachite green assay for detecting released inorganic phosphate

  • Radioactive [γ-32P]ATP hydrolysis assays for enhanced sensitivity

• Experimental Design Considerations:

  • Comparison of activity in detergent solution vs. reconstituted proteoliposomes

  • Analysis across a range of ATP concentrations to determine kinetic parameters

  • Assessment of cooperativity through Hill coefficient determination

Published data for BtuCD systems shows significantly different ATPase activities depending on whether the protein is in detergent solution or reconstituted into liposomes, with maximal rates of approximately 0.17 ± 0.04 μmol·mg−1·min−1 observed in proteoliposomes for related systems . This highlights the importance of the membrane environment for proper functional characterization.

What methods can be used to study vitamin B12 transport mediated by recombinant P. profundum BtuD?

Transport activity can be assessed through several complementary approaches:

• Radiolabeled Substrate Transport:

  • Incorporation of the BtuCD complex into proteoliposomes

  • Monitoring uptake of [57Co]cyanocobalamin or other radiolabeled vitamin B12 derivatives

  • Rapid filtration to separate transported substrate from the bulk solution

• Spheroplast-Based Transport Assays:

  • Generation of spheroplasts from cells expressing recombinant BtuCD

  • Addition of BtuF and radiolabeled vitamin B12

  • Measurement of time-dependent accumulation through filtration and washing steps

• Whole-Cell Complementation:

  • Rescue of vitamin B12-dependent growth in auxotrophic strains

  • Comparative growth curve analysis under varying vitamin B12 concentrations

Transport efficiency can be quantified by calculating the ATP/vitamin B12 ratio, which has been determined to be approximately 37 ATP molecules hydrolyzed per vitamin B12 molecule transported in related systems .

How does high hydrostatic pressure affect the structure and function of P. profundum BtuD?

As a deep-sea bacterium, P. profundum has evolved to function optimally under high hydrostatic pressure conditions. The specific adaptations of BtuD to pressure are not fully characterized, but several methodological approaches can be employed:

• Comparative Pressure Studies:

  • Analysis of ATP hydrolysis rates under varying pressure conditions

  • Structural studies using high-pressure NMR or X-ray crystallography

  • Molecular dynamics simulations to predict pressure effects on protein dynamics

• Experimental Approaches:

  • Utilization of pressure vessels for bacterial growth at various pressures (0.1-30 MPa)

  • Comparison of HP/LP (high-pressure/low-pressure) ratios between wild-type and mutant strains

  • Assessment of protein stability under pressure using fluorescence-based thermal shift assays

P. profundum SS9 serves as an established model for high-pressure adaptation, with specific growth conditions requiring pressure vessels and specialized heat-sealed bulbs for maintenance at elevated pressures (typically 15°C at pressures of 0.1 MPa vs. 30 MPa) .

What amino acid substitutions in P. profundum BtuD might contribute to its high-pressure adaptation?

While specific data for BtuD is limited, general principles of high-pressure adaptation in proteins from piezophiles (pressure-loving organisms) suggest several potential adaptations:

Research with P. profundum has established methodologies for high-pressure experiments that could be applied specifically to BtuD characterization .

How can researchers use transcriptomic approaches to understand the regulation of btuD expression in P. profundum?

RNA-seq methodologies provide powerful tools for analyzing btuD expression under varying conditions:

• Experimental Design Considerations:

  • Comparison of expression under varying pressures (0.1 vs. 30 MPa)

  • Analysis across growth phases and nutrient conditions

  • Examination of regulatory mutants (e.g., toxR mutants) for impact on btuD expression

• Technical Methodology:

  • Massively parallel cDNA sequencing (RNA-seq) as demonstrated for P. profundum

  • Careful RNA extraction protocols optimized for pressure-treated cells

  • Appropriate bioinformatic analysis pipelines for pressure-responsive gene identification

• Regulatory Analysis:

  • Investigation of promoter elements controlling btuD expression

  • Identification of pressure-responsive transcription factors

  • Analysis of small RNA regulators that may influence btuD expression

RNA-seq has been successfully applied to characterize the transcriptional landscape of P. profundum, revealing complex expression patterns and identifying previously unknown genes .

What experimental approaches can determine the interaction between BtuD and other components of the vitamin B12 import system?

Multiple complementary techniques can characterize protein-protein interactions:

• Biochemical Interaction Studies:

  • Pull-down assays using immobilized BtuD to capture interaction partners

  • Surface plasmon resonance for measuring binding kinetics

  • Blue native PAGE for analyzing intact complexes

• Structural Biology Approaches:

  • Cryo-electron microscopy of the assembled complex

  • X-ray crystallography of component pairs (e.g., BtuC-BtuD)

  • Cross-linking mass spectrometry to identify interaction interfaces

• Functional Interaction Studies:

  • BtuF association assays in the presence/absence of ATP

  • Reconstitution experiments with purified components

  • FRET-based approaches to monitor conformational changes during transport

Research has demonstrated that ATP binding influences the association between BtuCD and the substrate-binding protein BtuF, suggesting a mechanism for coupling ATP hydrolysis to transport .

What is the significance of the ATP/vitamin B12 ratio in evaluating transport efficiency for mutant BtuD proteins?

The ATP/vitamin B12 ratio serves as a critical parameter for evaluating transporter efficiency:

• Methodological Determination:

  • Simultaneous measurement of ATP hydrolysis and vitamin B12 transport rates

  • Calculation of the stoichiometric relationship between ATP consumed and substrate transported

  • Comparison between wild-type and mutant transporters

• Interpretation Framework:

  Transporter Variant	ATP/Vitamin B12 Ratio	Transport Efficiency
  Wild-type BtuCD	~25-30	Reference Standard
  BtuC2-tandem BtuD	~37	Moderately Reduced
  Single-site E159A Mutant	~37	Severely Reduced
  Double-site E159A Mutant	Not Measurable	No Transport

• Mechanistic Insights:

  • Analysis of uncoupled ATP hydrolysis (ATP hydrolysis without transport)

  • Correlation between structural perturbations and changes in coupling efficiency

  • Identification of residues critical for maintaining the correct ATP/transport ratio

Research with single-site and double-site Walker B motif mutants (E159A/Q) has revealed important insights about the asymmetric nature of ATP hydrolysis in the BtuCD transporter, demonstrating that even a single intact ATP site can support transport, albeit at reduced efficiency .

How can structural biology and computational modeling be integrated to understand the pressure adaptations of P. profundum BtuD?

An integrative structural biology approach would include:

• Experimental Structure Determination:

  • X-ray crystallography under varying pressure conditions

  • High-pressure NMR studies to detect pressure-induced conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to identify pressure-sensitive regions

• Computational Approaches:

  • Molecular dynamics simulations under different pressure conditions

  • Comparative modeling with mesophilic homologs

  • Prediction of pressure-sensitive residues and domains

• Validation Strategy:

  • Site-directed mutagenesis of predicted pressure-sensitive residues

  • Functional testing of mutants across pressure ranges

  • Iterative refinement of computational models based on experimental data

This integrated approach would provide mechanistic insights into how high-pressure adaptation is achieved at the molecular level in P. profundum proteins.

What genetic tools are available for manipulating btuD in P. profundum to study its function under different pressure conditions?

Several genetic tools have been developed for P. profundum manipulation:

• Transformation Methods:

  • Conjugation protocols using E. coli donor and helper strains

  • Specific adaptation of conjugation for P. profundum (e.g., incubation at 20°C for 40 h followed by selective plating)

  • Transformation efficiency optimization for high-pressure adapted bacteria

• Genetic Manipulation Tools:

  • Transposon mutagenesis libraries for identifying pressure-related phenotypes

  • Plasmid vectors optimized for P. profundum (e.g., pFL190, a broad-host-range plasmid)

  • Inducible expression systems (e.g., arabinose-inducible promoters)

• Phenotypic Analysis:

  • Growth assessment under varying pressure conditions using specialized pressure vessels

  • HP/LP ratio determination for mutant strains

  • Complementation studies to confirm gene function

Research has demonstrated successful genetic manipulation of P. profundum, including the creation of transposon mutant libraries that revealed genes involved in pressure adaptation .