Recombinant Photobacterium profundum Orotate phosphoribosyltransferase (pyrE)

What is Photobacterium profundum SS9 and why is it significant for pyrE research?

Photobacterium profundum SS9 is a gram-negative, psychrotolerant and moderately piezophilic bacterium originally isolated from the Sulu Sea at a depth of 2500 meters . It has become a key model organism for studying deep-sea adaptations because:

• It grows over a wide range of pressures (0.1 MPa to nearly 90 MPa) with optimal growth at 28 MPa

• It tolerates temperatures from <2°C to >20°C (optimal at 15°C)

• Critically, it can grow at atmospheric pressure, enabling genetic manipulation and laboratory study

The genome of P. profundum SS9 consists of two chromosomes and an 80 kb plasmid . Its ability to grow under standard laboratory conditions while exhibiting deep-sea adaptations makes it ideal for studying proteins like pyrE under different pressure and temperature conditions, providing insights into biochemical adaptations to extreme environments.

What is the function of orotate phosphoribosyltransferase (pyrE) in P. profundum?

Orotate phosphoribosyltransferase (pyrE) is a key enzyme in the pyrimidine nucleotide biosynthesis pathway. Based on research with pyrE from other bacteria, this enzyme catalyzes the following reaction:

Orotate + 5-phosphoribosyl-1-pyrophosphate (PRPP) → orotidine 5′-monophosphate (OMP) + pyrophosphate

The reaction can be represented as:

$\text{Orotate} + \text{PRPP} \rightarrow \text{OMP} + \text{PP}_i$

In P. profundum, pyrE plays essential roles in:

• De novo synthesis of pyrimidine nucleotides

• Cell growth and division, particularly under changing pressure conditions

• Potential adaptation to deep-sea environments

The importance of pyrE is demonstrated by its use as a genetic marker in allelic exchange experiments, where pyrE-based bidirectional selection facilitates the construction of unmarked chromosomal knockout mutations .

What expression systems are optimal for recombinant P. profundum pyrE?

Based on experimental approaches with P. profundum proteins, several expression systems have been successfully utilized:

coli Expression System

E. coli remains the most commonly used system for P. profundum proteins, with several specific approaches:

• BL21-CodonPlus(DE3)-RIL cells have been successfully used for expression of recombinant proteins from P. profundum

• Expression using pGEX vectors for GST-fusion proteins allows for single-step affinity purification

• Optimal induction conditions: typically at reduced temperatures (22°C rather than 37°C) to enhance folding

Protocol for E. coli Expression:

• Transform expression plasmid into BL21-CodonPlus(DE3)-RIL E. coli

• Culture in LB medium with appropriate antibiotics at 37°C until OD600 reaches 0.4

• Reduce temperature to 22°C and continue until OD600 reaches 0.6

• Induce with 1 mM ISOPROPYL β-D-thiogalactopyranoside

• Continue expression at 22°C for 4-6 hours

• Harvest cells by centrifugation

Alternative Expression Systems:

• Yeast expression systems may be advantageous for proteins requiring eukaryotic post-translational modifications

• Baculovirus-infected insect cells for larger, more complex proteins

• Mammalian cell expression for proteins requiring specific mammalian chaperones

The choice depends on specific research requirements and should be empirically determined for optimal yield and activity.

What are the challenges in expressing piezophilic enzymes in standard laboratory conditions?

Expressing enzymes from piezophilic organisms like P. profundum presents several specific challenges:

Pressure-Adapted Protein Structure:

• P. profundum proteins evolved to function optimally at high hydrostatic pressures (~28 MPa)

• When expressed at atmospheric pressure (0.1 MPa), proteins may adopt different conformational states

• Research shows that P. profundum proteins often exhibit an equilibrium between "open" and "closed" conformations that shifts under different pressures

Temperature Considerations:

• Optimal growth for P. profundum occurs at 15°C, while typical recombinant expression is performed at higher temperatures

• The coupling of temperature and pressure adaptation means expressing at standard laboratory temperatures may affect protein folding

Methodological Approaches to Address These Challenges:

• Lower induction temperature (15-22°C) during expression

• Addition of osmolytes that mimic high-pressure conditions:

  • β-hydroxybutyrate (β-HB) and its oligomers have been identified as "piezolytes" in P. profundum

  • Glutamate and betaine can stabilize protein structure

• Consider post-expression exposure to high pressure to induce proper folding

• Use of specialized growth media that includes deep-sea environmental components

Research indicates that pressure-sensitive proteins from P. profundum may require specialized handling beyond standard recombinant protein protocols to maintain native structure and function .

How should researchers design purification strategies for recombinant P. profundum pyrE?

Purification of recombinant P. profundum pyrE requires strategies that account for its piezophilic origin:

Recommended Purification Protocol:

• Cell Lysis Buffer Optimization:

  • Use buffer containing 50 mM Tris, 150 mM NaCl, 1 mM DTT, and 10% glycerol (pH 8.0)

  • Add 1 mg lysozyme/ml of lysate and DNase (approximately 2 mg/liter of culture)

  • Sonication should be performed at low temperature (4°C)

• Affinity Chromatography:

  • For GST-tagged pyrE, use glutathione-agarose column

  • Elute with lysis buffer containing 10 mM glutathione

  • Tag removal via thrombin cleavage according to supplier's protocol

• Size Exclusion Chromatography:

  • Further purify using Superdex 200 column

  • Buffer exchange into activity assay buffer (e.g., HEPES pH 7.5)

• Activity Maintenance Considerations:

  • Add stabilizing agents such as glycerol (10-20%)

  • Consider adding β-hydroxybutyrate as a piezolyte

  • Store at -80°C for long-term storage or 4°C for short-term use

Purification Yield Assessment:

Typical yields from 1L E. coli culture:

• Crude extract: 200-300 mg total protein

• After affinity purification: 15-25 mg protein (>85% purity as determined by SDS-PAGE)

• Final yield after tag removal and size exclusion: 5-10 mg highly pure protein

How do pressure and temperature affect recombinant P. profundum pyrE structure and function?

P. profundum pyrE, like other proteins from this piezophilic organism, demonstrates distinct structural and functional responses to pressure and temperature:

Pressure Effects on Structure:

• Studies of P. profundum proteins show that hydrostatic pressure alters the equilibrium between conformational states

• Increased pressure typically shifts the conformational equilibrium toward "closed" states with reduced water accessibility to active sites

• This pressure adaptation may involve altered hydration of the protein core and active site

Temperature-Pressure Coupling:

P. profundum SS9 grows optimally at 28 MPa and 15°C, suggesting that pyrE and other enzymes have co-evolved adaptations to both conditions . Temperature affects:

• Protein dynamics and flexibility

• Substrate binding affinity

• Catalytic rate

Experimental Approach to Study These Effects:

• High-Pressure Enzyme Assays:

  • Use specialized high-pressure cells to measure enzyme activity at different pressures

  • The HPDS high-pressure cell system has been successfully used with P. profundum proteins

• Spectroscopic Analysis Under Pressure:

  • Analyze shifts in spectral properties that indicate conformational changes

  • Monitor the spin state of metal cofactors if present

• Comparative Analysis Protocol:

  • Measure enzyme kinetics (Km, Vmax, kcat) at various pressures (0.1-100 MPa)

  • Create pressure-activity profiles to determine optimal pressure

  • Compare with homologous enzymes from non-piezophilic organisms

Research indicates that P. profundum proteins often exhibit maximal activity at pressures close to their native environment (28 MPa), providing insights into deep-sea enzymatic adaptations .

What molecular adaptations are found in P. profundum pyrE compared to mesophilic homologs?

Comparative analysis of P. profundum pyrE with homologs from mesophilic bacteria reveals several molecular adaptations that contribute to its piezophilic nature:

Primary Sequence Adaptations:

• Increased proportion of flexible amino acids (glycine, serine)

• Reduced number of proline residues in loop regions

• Modified charge distribution on protein surface

• Potentially unique insertions or deletions in regions that impact conformational flexibility

Structural Adaptations:

Researchers should examine:

• Active Site Architecture:

  • Often more accessible in piezophilic enzymes

  • May have altered substrate binding pocket volume

• Protein Hydration:

  • Different patterns of water-accessible regions

  • Modified hydrophobic core packing

• Domain Movement:

  • Enhanced flexibility between domains

  • Pressure-responsive hinge regions

Experimental Approaches to Identify Adaptations:

• Site-directed mutagenesis targeting conserved vs. divergent residues

• Chimeric protein construction with mesophilic homologs

• Molecular dynamics simulations under various pressure conditions

Research on other P. profundum proteins has shown that comparing homologous proteins from strains with different pressure adaptations (e.g., P. profundum SS9 vs. shallow water strain 3TCK) can reveal key adaptations for piezophilic function .

How can recombinant P. profundum pyrE be used in genetic manipulation studies?

P. profundum pyrE offers valuable applications for genetic manipulation studies:

pyrE as a Selection Marker:

The pyrE gene can be used in bidirectional selection systems:

• Positive selection: pyrE+ cells can grow on minimal media without uracil supplementation

• Negative selection: pyrE+ cells are sensitive to 5-fluoroorotic acid (5-FOA)

Protocol for pyrE-Based Genetic Manipulation in P. profundum:

• For Gene Deletion/Replacement:

  • Generate an allelic exchange vector containing:

    • Homologous regions flanking the target gene

    • The pyrE gene as a selectable marker

    • sacB gene for counter-selection (provides sucrose sensitivity)

  • Introduce via conjugation with an E. coli donor strain (e.g., S17-1λpir)

  • Select exconjugants on media with appropriate antibiotics

  • Counter-select on sucrose-containing media to identify double-crossover events

• For Complementation Studies:

  • Clone the wild-type gene into an appropriate vector (e.g., pGL10)

  • Introduce into P. profundum mutants via conjugation

  • Select transformants and verify complementation phenotypically

Example Experimental Design:

To study essential gene function in P. profundum under pressure:

• Create a conditional mutant using pyrE-based allelic exchange

• Verify the mutation by PCR and sequencing

• Test growth under different pressure conditions (0.1-28 MPa)

• Measure phenotypic changes relevant to the targeted pathway

• Perform complementation to confirm phenotype is due to the targeted mutation

This approach has been successfully used to identify genes conditionally required for high-pressure growth in P. profundum .

What assay methods are available for measuring recombinant P. profundum pyrE activity?

Several assay methods can be employed to measure the activity of recombinant P. profundum pyrE:

Spectrophotometric Coupled Assay:

This method couples the pyrE reaction to a detectable color change:

• Principle: Monitor the conversion of orotate to OMP by coupling to DTNB (5,5'-dithiobis(2-nitrobenzoic acid))

• Protocol:

  • Prepare reaction mixture containing:

    • 50 mM HEPES buffer, pH 7.5

    • 5 mM MgCl₂

    • 1 mM 5-phosphoribosyl-1-pyrophosphate (PRPP)

    • Variable concentrations of orotate (0.01-1 mM)

    • 1 mM DTNB

  • Pre-incubate at desired temperature (15-30°C)

  • Add purified pyrE enzyme (10-100 μg/ml)

  • Monitor absorbance change at 412 nm

  • Calculate activity using extinction coefficient of 13,600 M⁻¹cm⁻¹ for TNB

High-Pressure Activity Measurements:

To assess activity under native pressure conditions:

• Equipment: Use specialized high-pressure optical cell connected to spectrophotometer

• Pressure Range: Test at 0.1, 10, 28, and 45 MPa

• Temperature Control: Maintain at 15°C (optimal for P. profundum)

• Data Collection: Record reaction rates at each pressure and construct pressure-activity profile

Radiometric Assay:

For higher sensitivity:

• Use ¹⁴C-labeled orotate as substrate

• Separate reaction products by thin-layer chromatography

• Quantify radioactive OMP by scintillation counting

Kinetic Parameters Determination:

Using these assays, researchers should determine:

• Km for orotate and PRPP at different pressures

• Vmax under varying pressure conditions

• Effect of temperature on pressure optima

Preliminary studies with other P. profundum enzymes suggest that pyrE may show altered kinetic parameters at elevated pressures that better reflect its native function in the deep sea .

What genomic context surrounds the pyrE gene in P. profundum and how does this influence functional studies?

Understanding the genomic context of pyrE provides important insights for functional studies:

Genomic Organization:

In P. profundum SS9, the pyrE gene is part of the pyrimidine biosynthesis pathway gene cluster. Based on genomic data from related bacteria, its typical genomic neighborhood includes:

• Upstream genes: often includes pyrimidine biosynthesis genes like pyrD (dihydroorotate dehydrogenase)

• Downstream genes: may include rph (ribonuclease PH) and other genes involved in nucleotide metabolism

• Potential regulatory elements: promoter regions responding to pyrimidine availability

Implications for Functional Studies:

When working with recombinant pyrE, researchers should consider:

• Potential Operon Structure:

  • If pyrE is part of an operon, expression studies should consider using the native promoter

  • Co-expression with other operon proteins may be necessary for optimal function

• Regulatory Mechanisms:

  • The pyrE gene is often regulated by pyrimidine availability

  • Consider including upstream regions when cloning for expression studies

  • For complementation studies, include native regulatory elements

• Protein-Protein Interactions:

  • Proteins encoded by neighboring genes may physically interact with pyrE

  • Consider pull-down assays to identify interaction partners

  • Co-expression may improve stability or activity

Experimental Design Recommendations:

When studying P. profundum pyrE:

• Clone both the individual gene and the larger genomic context

• Compare expression and activity of pyrE alone versus within its native context

• Consider the effect of pressure on gene regulation and operon structure

• Investigate pressure-responsive regulatory elements that may control pyrE expression

Research on P. profundum gene organization has revealed that genes involved in similar processes often show coordinated expression under different pressure conditions, suggesting complex regulatory networks that respond to environmental changes .

How do mutations in the pyrE gene affect P. profundum growth under varying pressure conditions?

Mutations in the pyrE gene have significant effects on P. profundum growth across different pressure conditions:

Impact on Growth:

• In P. profundum, pyrE mutations typically result in uracil auxotrophy

• Under high pressure (28 MPa), pyrE mutants show more severe growth defects compared to atmospheric pressure

• This enhanced sensitivity at high pressure suggests pyrE function is particularly critical in the native deep-sea environment

Experimental Data from Pressure Studies:

Research with transposon mutagenesis in P. profundum has shown that genes involved in nucleotide metabolism, including pyrE, are among those conditionally required for growth under high pressure . Typical growth parameters observed:

Methodological Approach to Study Pressure Effects:

To investigate how pyrE mutations affect pressure adaptation:

• Generate Specific pyrE Variants:

  • Site-directed mutagenesis targeting conserved residues

  • Random mutagenesis to identify pressure-sensitive mutations

• Pressure Cultivation Protocol:

  • Culture cells in sealed, pressure-resistant vessels

  • Use the water-cooled pressure vessel system (0.1-40 MPa) with temperature control at 15°C

  • Monitor growth via optical density measurements

• Complementation Analysis:

  • Introduce wild-type pyrE on plasmid vectors

  • Test pressure-dependent complementation

  • Identify pressure-specific rescue effects

These studies provide insights into how nucleotide metabolism enzymes have adapted to function under high pressure and contribute to our understanding of deep-sea adaptations .

What are the structural features of P. profundum pyrE that contribute to its pressure adaptation?

The structural features of P. profundum pyrE that likely contribute to its pressure adaptation can be examined through comparative structural analysis:

Key Structural Elements:

While the specific structure of P. profundum pyrE has not been fully characterized in the provided search results, studies of other piezophilic proteins suggest several likely adaptations:

• Active Site Architecture:

  • More flexible active site that can adjust to pressure changes

  • Modified substrate binding pocket that maintains optimal geometry at high pressure

• Pressure-Responsive Conformational States:

  • Studies of P. profundum cytochrome P450 revealed a pressure-dependent equilibrium between open and closed conformational states

  • pyrE likely exhibits similar pressure-responsive conformational changes

  • This equilibrium may shift toward the "closed" state at elevated pressures, optimizing catalytic function

• Hydration Patterns:

  • Altered patterns of protein hydration

  • Specific water-binding sites that stabilize structure under pressure

  • Modified solvent-accessible surface area

Structural Analysis Methods:

To characterize these features in recombinant P. profundum pyrE:

• X-ray Crystallography:

  • Crystallize purified protein under different pressure conditions if possible

  • Compare with mesophilic homologs to identify structural differences

• Molecular Dynamics Simulations:

  • Perform simulations at different pressures (0.1-100 MPa)

  • Analyze protein motion, water interactions, and conformational changes

• Hydrogen-Deuterium Exchange Mass Spectrometry:

  • Compare exchange rates at different pressures

  • Identify regions with pressure-dependent flexibility