Recombinant Photobacterium profundum HTH-type transcriptional repressor PurR (purR)

What is the function of PurR in Photobacterium profundum?

PurR in P. profundum likely functions as a transcriptional repressor involved in regulating purine metabolism genes, similar to its homolog in Bacillus subtilis. The repressor typically controls genes involved in purine synthesis, transport, and metabolism by binding to specific DNA sequences (PurBoxes) in the upstream control regions of affected genes. In B. subtilis, the PurR-PurBox system regulates transcription of genes encoding enzymes for synthesis of IMP from PRPP (α-d-5-phosphoribosyl-1-pyrophosphate) and synthesis of AMP from IMP . Given the conservation of this regulatory system across many bacteria, P. profundum PurR likely serves a similar function but may exhibit adaptations related to deep-sea high-pressure environments.

What is the typical structural organization of bacterial PurR proteins?

Based on crystallographic studies of B. subtilis PurR, these proteins are typically organized as dimers with two primary domains. The C-terminal domain belongs to the phosphoribosyltransferase (PRT) structural family and contains a binding site for the inducer PRPP. The N-terminal domain belongs to the winged-helix family of DNA binding proteins and is responsible for specific interaction with regulatory DNA sequences . The winged-helix domain presents a positively charged surface that likely binds specific DNA sequences in the recognition site, while another positively charged surface surrounds the PRPP binding site at the opposite end of the dimer . This structural arrangement enables PurR to function as a repressor that responds to PRPP levels.

How does pressure adaptation in P. profundum potentially affect PurR structure and function?

P. profundum is a piezophilic (pressure-loving) bacterium that has evolved numerous adaptations for life in the deep sea. Although not specifically studied for PurR, pressure adaptation in P. profundum proteins often involves modifications that maintain protein flexibility and function under high hydrostatic pressure . These adaptations may include changes in amino acid composition to stabilize protein structure while maintaining necessary flexibility for conformational changes during DNA and effector binding. Given that P. profundum can maintain functionality at pressures up to 150 MPa , its PurR protein likely contains specific sequence and structural adaptations that preserve DNA-binding and regulatory functions under these extreme conditions.

What are the optimal expression systems for producing recombinant P. profundum PurR?

For effective recombinant expression of P. profundum PurR, researchers should consider:

• Expression host selection: E. coli BL21(DE3) strains are typically suitable for initial attempts, but given the high GC content often found in marine bacterial genes, codon optimization may be necessary.

• Vector design: A pET-based vector containing a 6×His tag for purification is recommended, with optimization of the promoter strength to prevent formation of inclusion bodies.

• Expression conditions: Low-temperature induction (16-18°C) with reduced IPTG concentration (0.1-0.5 mM) often enhances solubility of recombinant proteins from piezophilic bacteria.

• Co-expression strategy: Consider co-expression with chaperones (GroEL/GroES) that can assist proper folding of piezophilic proteins when expressed at atmospheric pressure.

To validate expression, perform small-scale test expressions analyzing samples by SDS-PAGE and Western blotting using anti-His antibodies before scaling up production.

What methodologies are recommended for studying PurR-DNA interactions under high pressure?

Investigating PurR-DNA interactions under high pressure requires specialized approaches:

Table 1: Methodologies for Studying PurR-DNA Interactions Under High Pressure

When designing experiments, it's crucial to include appropriate controls using mesophilic PurR proteins (e.g., from E. coli) that would show pressure sensitivity, as comparative references. The experimental design should systematically evaluate binding at different pressure points (0.1, 30, 60, 90, 120, and 150 MPa) to establish a comprehensive pressure-response profile.

How should researchers approach crystallization of P. profundum PurR for structural studies?

Crystallizing P. profundum PurR presents unique challenges due to its adaptation to high pressure environments. A systematic approach should include:

• Protein preparation: Ensure extremely high purity (>95%) through multiple chromatography steps (affinity, ion exchange, and size exclusion).

• Stability screening: Before crystallization attempts, conduct thermal shift assays to identify buffer conditions that maximize protein stability.

• Crystallization strategies:

  • Conventional screening at atmospheric pressure with commercial sparse matrix screens

  • Specialized high-pressure crystallization chambers for trials at 30-50 MPa

  • Co-crystallization with DNA fragments containing putative PurBox sequences

  • Co-crystallization with PRPP, which likely serves as the inducer

• Crystal handling: Crystals formed under high pressure should be carefully transitioned to atmospheric pressure to prevent damage.

For structure determination, consider multi-wavelength anomalous dispersion (MAD) phasing with selenomethionine-labeled protein, similar to the approach used for B. subtilis PurR . Analysis should focus on identifying structural features that contribute to pressure adaptation by comparison with mesophilic homologs.

What strategies are recommended for identifying PurR binding sites in the P. profundum genome?

To identify PurR binding sites (PurBoxes) in the P. profundum genome, employ a multi-faceted approach:

• Bioinformatic prediction: Perform genome-wide searches using position weight matrices derived from known bacterial PurR binding motifs. In B. subtilis, PurBoxes are found upstream of genes involved in purine metabolism , providing a starting template.

• Chromatin immunoprecipitation sequencing (ChIP-seq): Using antibodies against recombinant P. profundum PurR, perform ChIP-seq under varying pressure conditions (atmospheric vs. high pressure) to identify pressure-dependent binding patterns.

• DNase I footprinting: Conduct protection assays on candidate promoter regions to precisely define the boundaries of PurR binding sites.

• Electrophoretic mobility shift assays (EMSA): Confirm direct binding of recombinant PurR to predicted binding sites and determine binding affinities under different pressure conditions.

• Reporter gene assays: Construct transcriptional fusions with predicted PurR-regulated promoters to verify functional repression in vivo.

Comparing results from these complementary approaches will provide a robust map of the PurR regulon in P. profundum and reveal how it may differ from mesophilic bacteria.

How can researchers effectively design mutagenesis studies to identify key functional residues in P. profundum PurR?

A systematic mutagenesis approach should target residues in both the DNA-binding and PRPP-binding domains:

• Homology-based targeting: Align P. profundum PurR with B. subtilis PurR and identify conserved residues in the winged-helix domain and around the PRPP binding site . These represent primary targets for mutagenesis.

• Pressure-specific adaptation targeting: Identify amino acids unique to P. profundum PurR when compared to mesophilic homologs, particularly focusing on residues that might confer pressure resistance.

• Mutant design strategy:

  • Alanine scanning of conserved regions

  • Conservative substitutions (e.g., Lys→Arg) to assess charge requirements

  • Non-conservative substitutions to test functional hypotheses

  • Introduction of residues from mesophilic PurR proteins to test pressure adaptation

• Functional assays for mutants:

  • DNA binding assays at various pressures

  • PRPP binding assays to determine affinity changes

  • Structural stability assessments under pressure

This approach will identify residues critical for DNA binding, PRPP interaction, and pressure adaptation, providing insights into the molecular mechanisms of PurR function in deep-sea environments.

How does P. profundum PurR compare to PurR proteins from other pressure-adapted bacteria?

Comparative analysis of PurR proteins from various pressure-adapted bacteria reveals evolutionary patterns:

Table 2: Comparison of PurR Proteins from Piezophilic and Mesophilic Bacteria

When conducting comparative studies, researchers should:

• Perform phylogenetic analysis of PurR sequences from bacteria inhabiting different depth zones

• Analyze selection pressures on different domains using dN/dS ratios

• Conduct ancestral sequence reconstruction to identify key evolutionary transitions

• Compare pressure stability profiles across homologs experimentally

Such analyses will reveal convergent and divergent adaptations to high-pressure environments among PurR proteins.

What biochemical assays are most informative for comparing the pressure-dependence of PurR activity?

To effectively compare pressure effects on PurR activity across species:

• DNA binding assays under pressure:

  • Fluorescence anisotropy measurements using labeled DNA containing PurBox sequences

  • High-pressure electrophoretic mobility shift assays

  • Surface plasmon resonance with pressure cells

• Structural stability assays:

  • Intrinsic fluorescence monitoring under variable pressure

  • FTIR spectroscopy at high pressure to track secondary structure changes

  • Limited proteolysis to assess conformational changes under pressure

• Functional assays:

  • In vitro transcription assays under variable pressure conditions

  • Reporter gene assays in pressure-adaptable expression systems

• Binding kinetics:

  • Stopped-flow measurements with pressure jumps to determine association/dissociation rates

  • Isothermal titration calorimetry under pressure

These assays should be performed across a pressure range (0.1-150 MPa) similar to that used in motility studies with P. profundum , with temperature controlled to isolate pressure effects from temperature effects.

What are the challenges in developing a high-pressure in vitro transcription system to study PurR regulation?

Developing a high-pressure in vitro transcription system presents several technical challenges:

• Equipment limitations:

  • Need for specialized high-pressure vessels with optical access

  • Requirement for pressure-resistant connections for reagent addition

  • Challenge of maintaining consistent temperature during pressure changes

• Biochemical considerations:

  • Pressure effects on RNA polymerase activity independent of PurR

  • Altered buffer properties (pH shifts) under pressure

  • Changed reaction kinetics affecting optimal template and enzyme concentrations

• Experimental design:

  • Need for pressure-stable fluorescent reporters for real-time monitoring

  • Requirement for appropriate controls to distinguish direct pressure effects from PurR-mediated effects

  • Challenge of sample recovery for analysis without decompression artifacts

To address these challenges, researchers should:

• Adapt high-pressure microscopic chambers similar to those used for motility studies in P. profundum

• Include control transcription templates not regulated by PurR

• Develop a staged approach starting with atmospheric pressure validation before introducing pressure variables

How can researchers accurately measure PRPP binding to P. profundum PurR under high-pressure conditions?

Accurately measuring PRPP binding to PurR under high pressure requires specialized techniques:

• High-pressure fluorescence approaches:

  • Intrinsic tryptophan fluorescence if residues are proximal to the binding site

  • Site-directed labeling with pressure-stable fluorophores near the PRPP binding site

  • Fluorescent PRPP analogs with similar binding properties

• Isothermal titration calorimetry (ITC) under pressure:

  • Specialized high-pressure ITC cells

  • Systematic correction for pressure-dependent heat effects

  • Comparative analysis with mesophilic PurR proteins

• Surface plasmon resonance with pressure cell:

  • Immobilize PurR and measure PRPP binding kinetics

  • Determine pressure effects on association and dissociation rates

  • Compare with structural homologs from mesophilic bacteria

• Equilibrium dialysis with pressure treatment:

  • Pre-equilibrate PRPP-PurR binding under pressure

  • Rapid sample processing after decompression

  • Control experiments to account for decompression effects

These methods should be calibrated using known PRPP-binding proteins with established pressure responses to ensure accuracy of measurements under extreme conditions.

How should researchers interpret discrepancies between in vitro and in vivo studies of P. profundum PurR under pressure?

When confronting discrepancies between in vitro and in vivo results:

• Systematic analysis of differences:

  • Compare precise pressure conditions between experiments

  • Evaluate buffer composition differences that might affect pressure responses

  • Consider cellular factors absent in vitro (molecular crowding, interacting proteins)

• Modified experimental approaches:

  • Develop cell extracts retaining cellular complexity for in vitro studies

  • Use cellular reporter systems designed to isolate PurR effects

  • Conduct parallel experiments with mesophilic PurR to establish baseline pressure effects

• Integrated data analysis framework:

  • Develop mathematical models incorporating both datasets

  • Weight observations based on experimental robustness

  • Identify specific conditions where discrepancies arise

• Validation strategies:

  • Design hybrid experiments bridging in vitro and in vivo conditions

  • Perform genetic manipulations to test specific hypotheses arising from discrepancies

  • Use structural biology approaches to directly observe conformational states

Careful consideration of these factors will help resolve apparent contradictions and develop a more complete understanding of pressure effects on PurR function.

What statistical approaches are most appropriate for analyzing pressure-dependent changes in PurR binding affinity?

For robust statistical analysis of pressure-dependent binding data:

• Model selection:

  • Use non-linear regression models accounting for cooperative binding

  • Apply pressure-modified binding equations incorporating volume changes

  • Compare model fits using Akaike Information Criterion (AIC) to identify optimal models

• Experimental design considerations:

  • Ensure sufficient data points across the pressure range (minimum 6-8 pressure points)

  • Perform technical replicates (n≥3) and biological replicates (n≥3)

  • Include appropriate controls for pressure effects on assay components

• Advanced statistical approaches:

  • Bayesian hierarchical modeling to account for experiment-to-experiment variability

  • Bootstrap resampling to establish confidence intervals for binding parameters

  • Principal component analysis to identify patterns in multi-parameter datasets

• Visualization strategies:

  • 3D surface plots showing binding affinity, pressure, and temperature relationships

  • Residual plots to identify systematic deviations from models

  • Comparative visualization with mesophilic control proteins

By applying these statistical approaches, researchers can confidently identify true pressure-dependent effects on PurR binding affinity and distinguish them from experimental artifacts.