Recombinant Photobacterium profundum Ribonuclease PH (rph)

Basic Research Questions

• What is Ribonuclease PH from Photobacterium profundum and what is its basic function?

  Ribonuclease PH (RNase PH) from Photobacterium profundum is a phosphate-dependent 3'-5' exoribonuclease that plays a critical role in RNA processing, particularly in tRNA maturation. It functions by removing 3' nucleotides from precursor tRNAs . The enzyme is encoded by the rph gene, which shares significant homology with other bacterial rph genes. In P. profundum, RNase PH is particularly noteworthy due to its adaptation to function under high hydrostatic pressure conditions typically found in deep-sea environments .

• How does P. profundum's growth environment relate to its molecular adaptations?

  P. profundum is a deep-sea bacterium originally isolated from the Sulu Sea, capable of growth at temperatures from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa depending on the strain . The type strain SS9 grows optimally at 15°C and 28 MPa, making it both a psychrophile and a piezophile . These extreme environmental conditions have driven specific adaptations in the organism's proteins, including RNase PH, to maintain functionality. The molecular machinery of P. profundum, including its RNA processing enzymes, has evolved to function optimally under high-pressure, low-temperature conditions that would typically inhibit the activity of homologous enzymes from surface-dwelling organisms .

• What is the genomic context of the rph gene in P. profundum?

  The rph gene in P. profundum is located within its genome, which consists of two circular chromosomes and a plasmid . Based on the available data, the gene appears in STRING database listings (protein ID network) . Similar to other bacterial species, the rph gene likely plays a role in RNA metabolism pathways and may be part of operons involved in RNA processing or tRNA maturation. In other bacteria like B. subtilis, the rph gene is located adjacent to specific genes (e.g., gerM at 251 degrees on the B. subtilis genetic map) , suggesting possible co-regulation with other cellular processes.

Advanced Research Questions

• How can recombinant P. profundum RNase PH be expressed and purified for research applications?

  Expression and purification of recombinant P. profundum RNase PH can be achieved through:

  1. Cloning Strategy: The rph gene can be PCR-amplified from P. profundum genomic DNA using primers designed based on the available sequence data. Similar to other recombinant ribonucleases, the gene can be cloned into an expression vector with an appropriate tag (His-tag/GST) for purification .

  2. Expression System: Based on protocols for other recombinant ribonucleases, expression in E. coli BL21(DE3) or similar strains is recommended, with induction using IPTG at lower temperatures (15-20°C) to promote proper folding .

  3. Purification Protocol:

     • Cell lysis using buffer containing 20 mM HEPES-KOH (pH 7.5-8.0), 100-300 mM NaCl, 1 mM DTT

     • Affinity chromatography using the fusion tag

     • Ion exchange chromatography for further purification

     • Size exclusion chromatography for final polishing

     • Addition of RNase inhibitor (1 U/μL) during purification to prevent contamination with other RNases

  4. Quality Control: Assess purity using SDS-PAGE and activity using standard RNase assays with model RNA substrates .

• What experimental approaches can be used to measure RNase PH activity under high-pressure conditions?

  To assess RNase PH activity under high pressure:

  1. High-Pressure Reaction Vessels: Utilize specialized high-pressure equipment similar to those used for P. profundum growth studies . These can maintain pressures of 0.1-40 MPa at controlled temperatures.

  2. Enzyme Activity Assay:

     • Prepare reaction mixtures containing purified RNase PH, substrate RNA, and buffer (typically containing 20 mM HEPES, pH 7.5, 100 mM KCl, and 1 mM MgCl₂)

     • Seal reaction mixtures in pressure-resistant containers excluding air to ensure even pressure distribution

     • Incubate at varying pressures (0.1, 28, and 45 MPa) and temperatures (15°C optimal)

     • After decompression, analyze reaction products using gel electrophoresis, HPLC, or mass spectrometry

  3. Comparative Analysis: Compare activity profiles against RNase PH from non-piezophilic organisms under identical conditions to identify pressure-specific adaptations .

• How does pressure affect the expression and activity of RNase PH in P. profundum?

  Pressure significantly influences gene expression and protein function in P. profundum:

  1. Expression Regulation: While specific data for rph is limited, genome-wide studies show that P. profundum differentially regulates many genes in response to pressure changes . For instance, some genes are upregulated at high pressure (28 MPa) compared to atmospheric pressure (0.1 MPa) .

  2. Activity Profile: The enzymatic activity of P. profundum proteins, including RNases, often displays pressure optima that correspond to the organism's native environment. Similar to observed phenomena with other P. profundum enzymes:

     • Activity likely increases from 0.1 MPa up to around 28 MPa

     • Activity may decrease at pressures significantly above 28 MPa

     • Temperature interactions may exist, with different pressure optima at different temperatures

  3. Structural Considerations: P. profundum proteins like RNase PH likely contain specific structural adaptations (amino acid substitutions, additional domains) that allow function under high pressure, similar to those identified in other P. profundum proteins like malate dehydrogenase and RecD .

• What role might RNase PH play in the cold and pressure adaptation mechanisms of P. profundum?

  RNase PH may contribute significantly to P. profundum's adaptation to its deep-sea environment:

  1. RNA Quality Control: In cold, high-pressure environments, RNA secondary structures may be stabilized, potentially leading to processing challenges. RNase PH likely helps maintain RNA turnover and quality control under these conditions .

  2. tRNA Maturation: Proper tRNA processing is critical for efficient translation, and RNase PH's role in tRNA 3' end maturation may be particularly important for maintaining translation efficiency under pressure stress .

  3. Genetic Evidence: Transposon mutagenesis studies in P. profundum have identified several genes involved in RNA processing and ribosome assembly as being essential for growth under high pressure and low temperature conditions . While rph was not specifically mentioned, proteins with similar functions in translation and RNA processing were identified as pressure-sensitive loci.

  4. Functional Complementation: Based on complementation studies with other genes, it's reasonable to hypothesize that P. profundum rph might complement rph mutations in mesophilic bacteria and restore growth at low temperatures, similar to how B. subtilis rph suppresses cold-sensitive mutations in E. coli .

• How does P. profundum RNase PH activity compare to RNase PH from non-piezophilic bacteria?

  Comparing P. profundum RNase PH with mesophilic counterparts reveals potential adaptations:

  1. Pressure and Temperature Optima: P. profundum RNase PH likely maintains higher activity under elevated pressure (28 MPa) and lower temperature (15°C) compared to homologs from surface bacteria, which typically have activity optima around atmospheric pressure and mesophilic temperatures .

  2. Structural Adaptations: Based on studies of other P. profundum proteins, RNase PH may contain:

     • Specific amino acid substitutions at key residues involved in catalysis or substrate binding

     • Potential additional domains or motifs that confer pressure resistance

     • Modifications that increase structural flexibility to counteract the compression effects of high pressure

  3. Kinetic Parameters: Enzyme kinetics (Km, kcat) likely differ between piezophilic and non-piezophilic RNase PH, with P. profundum RNase PH possibly showing lower Km values for substrates under high pressure conditions, indicating greater substrate affinity .

  4. Functional Conservation: Despite adaptations, P. profundum RNase PH likely maintains the same core function of removing 3' nucleotides from precursor tRNAs, similar to its homologs in E. coli and B. subtilis .

• What factors affect the inhibition and activation of recombinant P. profundum RNase PH?

  Several factors regulate RNase PH activity:

  1. Divalent Cations: Based on data from other RNases:

     • Mg²⁺ is likely required for optimal activity of RNase PH

     • The enzyme is likely strongly inhibited by Cu²⁺, Zn²⁺, and Hg²⁺, similar to RNase T2

     • EDTA may stimulate activity by chelating inhibitory divalent cations

  2. pH Effects:

     • Optimal activity is likely in the range of pH 7.0-8.0

     • Activity decreases significantly outside this range

  3. Pressure Effects:

     • Activity increases with pressure up to approximately 28 MPa

     • Higher pressures may denature the enzyme or alter substrate binding

  4. Temperature Effects:

     • Optimal activity around 15°C, corresponding to P. profundum's growth optimum

     • Lower activity at higher temperatures (>25°C) due to the psychrophilic nature of the enzyme

  5. Product Inhibition:

     • Mononucleotides and digestion products may act as competitive inhibitors, similar to other ribonucleases

• What methodologies can be employed to study the structural adaptations of P. profundum RNase PH?

  To investigate structural adaptations:

  1. Comparative Sequence Analysis:

     • Align RNase PH sequences from P. profundum with homologs from non-piezophilic bacteria

     • Identify unique substitutions, insertions, or deletions in the P. profundum enzyme

     • Use evolutionary and phylogenetic analysis to identify positively selected residues

  2. Structural Biology Approaches:

     • X-ray crystallography of P. profundum RNase PH at various pressures

     • Cryo-electron microscopy for structural determination

     • NMR studies to examine dynamic properties under different pressure conditions

     • High-pressure X-ray crystallography to observe pressure-induced conformational changes

  3. Mutagenesis Studies:

     • Create chimeric enzymes with domains swapped between piezophilic and mesophilic RNase PH

     • Perform site-directed mutagenesis of key residues identified in comparative analysis

     • Test mutant enzymes for altered pressure sensitivity

     • Use pressure-resistant mutants of mesophilic RNase PH to identify critical residues

  4. Molecular Dynamics Simulations:

     • Simulate protein behavior under various pressure conditions

     • Analyze flexibility, compressibility, and water interactions

     • Identify pressure-sensing domains or residues within the structure

• What experimental challenges are associated with working with recombinant P. profundum RNase PH?

  Researchers working with this enzyme face several challenges:

  1. Expression and Solubility:

     • Cold-adapted enzymes often show reduced stability at common laboratory temperatures

     • Expression in mesophilic hosts (E. coli) may result in inclusion bodies or misfolded protein

     • Lower expression temperatures (15°C) may be required for proper folding, reducing yield

  2. High-Pressure Equipment Requirements:

     • Specialized pressure vessels are needed to characterize enzyme behavior under native conditions

     • Challenges in designing real-time assays under high pressure

     • Limited commercial availability of high-pressure systems for enzyme assays

  3. RNase Contamination:

     • Ribonucleases are ubiquitous in laboratory environments

     • Special precautions needed to prevent contamination with RNases from expression host

     • RNase inhibitors may affect RNase PH activity measurements

  4. Stability Issues:

     • Enzymes adapted to high pressure may show reduced stability at atmospheric pressure

     • Storage and handling may require special conditions to maintain native structure

     • Repeated freeze-thaw cycles likely to reduce activity

  5. Activity Assessment:

     • Standard RNase assays may not accurately reflect the enzyme's activity under native conditions

     • Need for specialized assays that can function at high pressure and low temperature

Research Applications

• What are the potential applications of recombinant P. profundum RNase PH in RNA biology research?

  Recombinant P. profundum RNase PH offers unique applications:

  1. Cold-Adapted RNA Processing:

     • RNA structure analysis at low temperatures where conventional enzymes have reduced activity

     • Processing of RNA with strong secondary structures that resist digestion by mesophilic RNases

     • Selective 3' end processing of structured RNAs

  2. tRNA Maturation Studies:

     • Investigation of pressure effects on tRNA processing

     • Studies on the role of 3' processing in tRNA folding and function

     • Comparative analysis of tRNA maturation pathways in extremophiles

  3. Circular RNA Research:

     • Similar to RNase R applications, potential use in circular RNA enrichment protocols

     • Selective degradation of linear RNAs while preserving circular RNA structures

     • Tool for studying circular RNA biology under various conditions

  4. Extremozyme Biotechnology:

     • Model system for understanding enzyme adaptation to extreme conditions

     • Template for engineering pressure-stable variants of other ribonucleases

     • Development of RNA processing tools functional under non-standard conditions

• How can research on P. profundum RNase PH contribute to understanding deep-sea microbial adaptation?

  This research provides valuable insights into deep-sea adaptation:

  1. Molecular Basis of Piezophily:

     • RNase PH serves as a model for how essential RNA processing pathways adapt to high pressure

     • Identification of specific amino acid substitutions that confer pressure resistance

     • Understanding how key cellular processes maintain functionality under extreme conditions

  2. Evolutionary Adaptations:

     • Comparative analysis of RNase PH across bacteria from different depths can reveal convergent or divergent evolutionary strategies

     • Identification of common molecular adaptations to deep-sea environments

     • Understanding of the timeline for adaptation to high-pressure environments

  3. Environmental Microbiology:

     • Insights into how essential cellular processes function in the largest habitat on Earth (deep ocean)

     • Better understanding of biogeochemical cycling in deep-sea environments

     • Development of molecular markers for adaptation to specific depths or pressures

  4. Cold and Pressure Interaction:

     • Understanding how adaptations to high pressure interact with cold adaptation

     • Insights into the molecular trade-offs between different environmental stressors

     • Development of models for predicting how deep-sea microbes might respond to environmental changes

Experimental Data and Protocols

• What protocols are recommended for measuring activity of recombinant P. profundum RNase PH?

  Standard protocol recommendations:

  1. Basic Activity Assay:

     • Substrate: Pre-tRNA or synthetic RNA with defined 3' end

     • Buffer: 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 1 mM DTT

     • Incubation: 15°C for 30 minutes at various pressures (0.1, 28, 45 MPa)

     • Detection: Gel electrophoresis, HPLC, or mass spectrometry analysis of reaction products

  2. Spectrophotometric Assay:

     • Similar to RNase A unit definition: measure increase in absorbance at 260 nm due to RNA hydrolysis

     • One unit causes an increase in absorbance of 1.0 at 260 nm at 15°C, pH 7.5 in 15 minutes

  3. High-Pressure Activity Measurement:

     • Prepare reaction mixtures in pressure-resistant containers

     • Incubate at desired pressure in specialized pressure vessels

     • After decompression, analyze reaction products

     • Compare to activity at atmospheric pressure

  4. Pressure-Temperature Matrix:

     • Perform activity assays at combinations of different pressures and temperatures

     • Generate 3D activity profiles to identify optimal conditions

     • Determine pressure-temperature relationships for enzyme activity

• How do P. profundum growth conditions affect the expression and properties of RNase PH?

  Growth conditions significantly impact enzyme properties:

  1. Growth Pressure Effects:

     • P. profundum grown at high pressure (28 MPa) versus atmospheric pressure (0.1 MPa) may express RNase PH with different post-translational modifications

     • Enzyme extracted from cells grown under native conditions may show higher activity or stability

     • Growth pressure influences global gene expression, potentially affecting RNase PH expression levels

  2. Temperature Interactions:

     • Growth at 15°C (optimal) versus other temperatures can affect enzyme folding and stability

     • Cold growth conditions may induce co-expression of chaperones that assist in proper RNase PH folding

     • Temperature-pressure interactions during growth may yield enzyme with different properties

  3. Growth Phase Considerations:

     • Expression levels of RNase PH may vary between exponential and stationary phases

     • For recombinant expression, harvest timing should be optimized

     • Native enzyme properties may differ depending on the growth stage of source cells

  4. Media Composition:

     • P. profundum growth in marine broth supplemented with glucose and HEPES buffer (pH 7.5) is standard for laboratory cultivation

     • Nutrient availability influences gene expression patterns and may affect enzyme properties

     • For recombinant expression, medium optimization is crucial for high yield of active enzyme