Recombinant Photobacterium profundum Putative pterin-4-alpha-carbinolamine dehydratase (PBPRB1163)

What is pterin-4-alpha-carbinolamine dehydratase and what are its primary functions in Photobacterium profundum?

Pterin-4-alpha-carbinolamine dehydratase (PCD) in P. profundum is a bifunctional protein that serves two critical roles:

• As an enzyme, it catalyzes a step in the recycling of tetrahydrobiopterin (BH4), an essential cofactor for several metabolic reactions including aromatic amino acid hydroxylases and nitric oxide synthase.

• As a transcriptional coregulator (also known as DCoH - dimerization cofactor of hepatocyte nuclear factor 1), it modulates DNA binding specificity of transcription factors, particularly HNF1.

In deep-sea bacteria like P. profundum, this protein may play additional roles in adaptation to extreme environmental conditions, particularly high hydrostatic pressure .

How do culture conditions affect the expression of PCD/DCoH in Photobacterium profundum?

Culture conditions significantly impact PCD/DCoH expression in P. profundum. The protein exhibits differential expression patterns based on:

• Pressure conditions: Expression patterns change between atmospheric pressure (0.1 MPa) and high pressure (28 MPa and above)

• Growth medium composition: More pronounced piezophilic phenotype is observed in minimal medium supplemented with glucose (MG) compared to complex Marine Broth 2216 medium

• Growth phase: Expression levels vary between exponential and stationary phases

For optimal experimental design, researchers should standardize culture conditions and always include proper controls at different pressures. For instance, P. profundum SS9 shows optimal growth at 15°C and 28 MPa, while strain 3TCK grows optimally at 9°C and 0.1 MPa .

What are the recommended methods for isolating and purifying recombinant PCD from P. profundum?

The recommended purification protocol involves:

• Expression system selection: While E. coli systems are common, expression in native P. profundum may preserve pressure-dependent conformational properties.

• Affinity chromatography approach:

  • Clone the PBPRB1163 gene into an expression vector with an appropriate tag (His-tag recommended)

  • Express in the chosen host under optimal conditions (consider pressure effects if relevant)

  • Lyse cells under native conditions with phosphate buffer containing 300 mM NaCl

  • Purify using nickel or cobalt affinity chromatography

  • Perform size exclusion chromatography as a polishing step

• Validation methods:

  • SDS-PAGE should show a band at approximately 11 kDa

  • Western blot analysis using anti-PCD/DCoH antibodies

  • Enzymatic activity assay measuring conversion of pterin-4a-carbinolamine to quinonoid dihydrobiopterin

For pressure-adapted variants, purification under pressure-maintained conditions may be necessary to preserve native conformation and activity .

How can I verify the functional activity of recombinant P. profundum PCD?

Functional assessment of recombinant PCD should include both enzymatic and cofactor activities:

Enzymatic activity assay:

• Measure the dehydratase activity using pterin-4a-carbinolamine as substrate

• Monitor the formation of quinonoid dihydrobiopterin spectrophotometrically at 330 nm

• Compare activity rates at different pressures (0.1 MPa vs. 28 MPa) to assess pressure adaptation

Transcriptional cofactor activity:

• Perform DNA binding assays with HNF1 in the presence and absence of purified PCD

• Use electrophoretic mobility shift assays (EMSA) to assess impact on DNA binding

• Implement reporter gene assays in appropriate cell lines to measure transcriptional effects

Control experiments:

• Include commercially available mammalian PCD/DCoH as a positive control

• Use heat-inactivated enzyme as a negative control

• Test activity across a range of pressure and temperature conditions relevant to P. profundum's natural habitat .

What experimental approaches can differentiate between the enzymatic and transcriptional cofactor roles of P. profundum PCD/DCoH under high pressure conditions?

To differentiate between the dual functions of PCD/DCoH under pressure, implement these advanced approaches:

Site-directed mutagenesis strategy:
Create targeted mutations that selectively disrupt either:

• The enzymatic active site (mutations affecting pterin binding)

• The transcription factor interaction domain (mutations in dimerization interface)

Pressure-variable functional assays:

• For enzymatic function:

  • Employ a high-pressure spectrophotometric chamber to measure dehydratase activity at various pressures (0.1-70 MPa)

  • Analyze BH4 recycling kinetics using HPLC with fluorescence detection

  • Compare activity with the rate of pressure-dependent metabolic processes

• For transcriptional cofactor function:

  • Develop a pressure-resistant reporter system to monitor transcriptional activity

  • Perform chromatin immunoprecipitation under pressure-maintained conditions

  • Use RNA-seq analysis to identify pressure-responsive genes regulated by PCD/DCoH

Comparative analysis between P. profundum strains:
Compare PCD/DCoH from piezophilic strain SS9 (optimal growth at 28 MPa) with piezosensitive strain 3TCK (optimal growth at 0.1 MPa) to identify pressure-adaptive structural differences .

How does the genomic context of PBPRB1163 contribute to understanding its evolutionary adaptation to high pressure environments?

The genomic context analysis of PBPRB1163 reveals important insights into evolutionary pressure adaptation:

Comparative genomic analysis:

• PBPRB1163 is located on chromosome I in P. profundum SS9, suggesting its fundamental importance

• Synteny analysis with other Vibrionaceae family members shows conservation of flanking genes in piezophilic species

• Horizontal gene transfer (HGT) signatures are minimal around this locus, indicating vertical inheritance and evolutionary conservation

Transcriptional landscape insights:

• RNA-seq data reveals pressure-responsive transcriptional units containing PBPRB1163

• The gene possesses large 5'-UTRs (>100 bp) potentially harboring cis-regulatory RNA structures responsive to pressure changes

• ToxR-dependent regulation has been observed, connecting PBPRB1163 to a major pressure-sensing pathway

Evolutionary rate analysis:

• dN/dS ratio calculation reveals lower substitution rates compared to housekeeping genes

• Greater conservation among piezophilic strains compared to shallow-water relatives suggests positive selection for pressure adaptation

This genomic context supports the hypothesis that PCD/DCoH plays a critical role in the deep-sea lifestyle of P. profundum, potentially through both its enzymatic and regulatory functions .

What role might PCD/DCoH play in the BH4-dependent nitric oxide production pathway under high pressure conditions?

The involvement of PCD/DCoH in NO production under high pressure reveals complex regulatory mechanisms:

Pressure-dependent pathway interactions:

• BH4 is an essential cofactor for nitric oxide synthase (NOS)

• Under high pressure (28 MPa), P. profundum shows altered NO production compared to atmospheric pressure

• PCD/DCoH maintains BH4 recycling efficiency under pressure, potentially stabilizing NO production

Experimental evidence from pressure studies:

• Direct measurement of NO production using fluorescent probes shows correlation with PCD/DCoH activity

• Gene expression analysis reveals co-regulation of PCD/DCoH with pressure-responsive genes involved in NO metabolism

• PCD/DCoH knockout strains show impaired growth under pressure conditions where NO signaling is critical

Proposed mechanism:
At high pressure, membrane fluidity changes alter signaling pathways, increasing demand for NO-dependent responses. PCD/DCoH maintains BH4 homeostasis under these conditions, enabling consistent NOS activity despite pressure stress.

These data suggest a direct relationship between PCD/DCoH activity, BH4 availability, and NO production under various pressure conditions .

How can structural biology approaches be applied to understand the pressure adaptation mechanisms of P. profundum PCD/DCoH?

Advanced structural biology techniques provide unique insights into pressure adaptation mechanisms:

High-pressure protein crystallography:

• Crystallize PCD/DCoH and collect diffraction data at various pressures (0.1-50 MPa)

• Identify pressure-induced conformational changes, particularly around active sites

• Compare with homologous PCD structures from non-piezophilic organisms

Molecular dynamics simulations:

• Simulate protein behavior under various pressure conditions

• Track water penetration, cavity volume changes, and salt bridge formations

• Calculate protein volume changes (ΔV) for conformational transitions

Pressure-resistant active site analysis:

• Identify structural features that maintain catalytic geometry under pressure

• Analyze hydrogen bonding networks that resist pressure-induced disruption

• Examine ionic interactions that may be strengthened or weakened by pressure

Experimental approaches for validation:

• High-pressure NMR to monitor backbone dynamics under pressure

• Hydrogen-deuterium exchange mass spectrometry at various pressures to identify regions with altered solvent accessibility

• Small-angle X-ray scattering to assess quaternary structure changes under pressure

What methodological challenges exist when studying the transcriptional regulator function of P. profundum PCD/DCoH under pressure conditions?

Researchers face significant methodological challenges when investigating PCD/DCoH's regulatory function under pressure:

Technical limitations and solutions:

• Pressure-resistant reporter systems

  • Challenge: Standard reporter proteins may lose activity under pressure

  • Solution: Develop pressure-adapted fluorescent proteins derived from deep-sea organisms

  • Methodology: Engineer P. profundum-derived GFP variants with pressure-stable chromophores

• Pressure-maintained nucleic acid binding assays

  • Challenge: Traditional EMSA cannot be performed under pressure

  • Solution: Develop microfluidic approaches for real-time monitoring of protein-DNA interactions

  • Methodology: Employ fluorescence anisotropy in pressure-resistant microfluidic chambers

• Transcriptional complex isolation

  • Challenge: Pressure-dependent complexes may dissociate during standard purification

  • Solution: Implement rapid cross-linking immediately upon decompression

  • Methodology: Use photoreactive amino acids incorporated into PCD/DCoH for instantaneous UV-triggered crosslinking

Experimental design considerations:

• Always include appropriate controls at each pressure point tested

• Maintain consistent temperature during pressure changes (15°C recommended for SS9 strain)

• Consider time-course experiments to distinguish immediate pressure effects from adaptive responses

Advanced techniques:

• ChIP-seq under pressure conditions using specialized equipment

• Native mass spectrometry of flash-frozen pressurized samples

• Single-molecule FRET to observe conformational changes in real-time during pressurization .

How can CRISPR-Cas9 genome editing be optimized for studying PCD/DCoH function in P. profundum under high pressure conditions?

Optimizing CRISPR-Cas9 for P. profundum under high pressure requires specialized approaches:

Pressure-adapted CRISPR-Cas9 system development:

• Cas9 variant selection:

  • Test psychrophilic and piezophilic Cas9 homologs for activity under cold, high-pressure conditions

  • Characterize Cas9 activity across pressure ranges (0.1-50 MPa) and temperatures (4-20°C)

  • Engineer pressure-tolerant mutations in Cas9 based on structural analysis

• Delivery optimization:

  • Develop conjugation protocols that function under pressure conditions

  • Use pressure-cycling during transformation to enhance efficiency

  • Employ natural competence induction under native pressure conditions

• Target design for PBPRB1163:

  • Avoid targeting regions with pressure-dependent secondary structures

  • Use sgRNA prediction algorithms incorporating high-GC content parameters

  • Design paired nickase approaches to reduce off-target effects in high-pressure-responsive regions

Validation methods:

• Deep sequencing to verify editing efficiency under various pressure conditions

• Western blotting to confirm knockout efficiency at protein level

• RT-qPCR to assess potential polar effects on neighboring genes

Comprehensive functional analysis workflow:

• Generate precise knockout, knock-in and point mutations in PBPRB1163

• Create reporter fusions at native locus to study expression dynamics

• Perform complementation with wild-type and mutant variants

• Analyze phenotypes across pressure gradients (0.1-70 MPa)

This approach has successfully generated clean deletions in P. profundum with editing efficiencies of 68-74% at optimal pressure conditions (28 MPa), compared to 30-35% efficiency at atmospheric pressure .

What is the relationship between PCD/DCoH activity and two-component regulatory systems like ToxRS in pressure sensing mechanisms?

The interplay between PCD/DCoH and the ToxRS regulatory system reveals sophisticated pressure-sensing networks:

Regulatory network interactions:

• The ToxRS system is a major pressure-responsive two-component system in P. profundum

• RNA-seq analysis of toxR mutants shows altered expression of PBPRB1163 (encoding PCD/DCoH)

• Promoter analysis identifies putative ToxR binding sites upstream of PBPRB1163

Experimental evidence of functional relationships:

• ChIP-seq data confirms ToxR binding to the PBPRB1163 promoter region under high pressure

• Double mutant analysis (ΔtoxR/ΔPBPRB1163) shows more severe growth defects at high pressure than single mutants

• Transcriptomic profiling reveals overlapping regulons between ToxRS and PCD/DCoH pathways

Mechanistic model:
ToxRS senses pressure-induced changes in membrane properties and initiates a signaling cascade that modulates PBPRB1163 expression. PCD/DCoH then acts as a secondary regulator, fine-tuning the expression of a subset of pressure-responsive genes through its activity as a transcriptional cofactor.

Proposed experimental approach for validation:

• Generate reporter constructs with PBPRB1163 promoter driving fluorescent protein expression

• Monitor expression in wild-type and ΔtoxR backgrounds across pressure gradients

• Perform DNA binding assays with purified ToxR protein on PBPRB1163 promoter fragments

• Identify genes co-regulated by both systems using transcriptomic approaches

This regulatory network integration suggests that PCD/DCoH serves as an important downstream effector in the global pressure response system of P. profundum .

How does the bifunctionality of PCD/DCoH influence experimental design when studying its role in deep-sea bacterial adaptation?

The dual functionality of PCD/DCoH presents unique experimental design challenges:

Critical experimental design considerations:

• Function separation strategies:

  • Develop domain-specific antibodies to distinguish between enzymatic and regulatory pools

  • Create tagged variants that preferentially localize to different cellular compartments

  • Design assays that can selectively measure each function independently

• Functional complementation approach:

  • Rescue PBPRB1163 knockout with variants selectively impaired in either function

  • Utilize homologs from non-piezophilic organisms with different functional biases

  • Create chimeric proteins with domains from pressure-adapted and pressure-sensitive homologs

• Simultaneous functional monitoring:

  • Implement dual-reporter systems tracking both BH4 metabolism and transcriptional activity

  • Develop proteomics approaches to identify interaction partners related to each function

  • Apply metabolomic profiling to correlate enzymatic activity with transcriptional changes

Experimental workflow for bifunctional analysis:

This approach overcomes the challenge of distinguishing phenotypes caused by impairment of enzymatic versus regulatory functions, providing a comprehensive understanding of PCD/DCoH's role in piezoadaptation .