Recombinant Photobacterium profundum 50S ribosomal protein L2 (rplB)

What is Photobacterium profundum and why is it important as a model organism?

Photobacterium profundum SS9 is a Gram-negative bacterium originally isolated from the Sulu Sea at a depth of 2.5 km. It belongs to the Photobacterium subgroup of the Vibrionaceae family and is closely related to Vibrio species. Its genome consists of two chromosomes and an 80 kb plasmid. P. profundum is both a piezophile (thrives under high pressure) and a psychrophile (thrives under cold conditions), with optimal growth occurring at 28 MPa and 15°C .

What makes P. profundum particularly valuable as a model organism is its ability to grow across a wide pressure range (0.1 MPa to 90 MPa), allowing for easy genetic manipulation and culture at atmospheric pressure while still exhibiting clear pressure-dependent adaptations. This characteristic has established it as a key model organism for studying piezophily (high-pressure adaptation) .

What is the 50S ribosomal protein L2 (rplB) and what are its primary functions?

The 50S ribosomal protein L2 (rplB) is a highly conserved component of the large ribosomal subunit in bacteria. It plays critical roles in:

• Ribosome assembly and structural integrity

• Formation of the peptidyltransferase center

• Interaction with rRNA and other ribosomal proteins

• Possibly contributing to high-pressure adaptation in piezophiles

Beyond its structural role in ribosomes, research with E. coli has demonstrated that ribosomal protein L2 (RPL2) can directly interact with RNA polymerase α subunit (RNAPα) and influence transcription regulation . This interaction was confirmed both in vivo and in vitro, and functional assays showed that RPL2 could increase β-galactosidase expression specifically with ribosomal promoters, whereas other ribosomal proteins (L1, L3, L20, and L27) did not have this effect .

How does high pressure affect protein expression in P. profundum?

Proteomic analysis has revealed that P. profundum significantly alters its protein expression profile in response to pressure changes. When comparing growth at high pressure (28 MPa) versus atmospheric pressure (0.1 MPa), several patterns emerge:

• Proteins involved in glycolysis/gluconeogenesis pathways are up-regulated at high pressure

• Conversely, proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure

• Nutrient transport and assimilation proteins show pressure-dependent regulation

• ABC transporters for phosphate and other nutrients exhibit differential expression at varying pressures

These adjustments likely represent adaptations to the distinct nutrient limitations and biochemical challenges present at different ocean depths and pressure regimes.

What methodologies are recommended for expressing recombinant P. profundum rplB?

Based on research protocols for similar piezophilic proteins, the following methodology is recommended:

Expression System Selection:

• E. coli BL21(DE3) is typically suitable for initial expression attempts

• Consider cold-adapted expression systems for better folding of psychrophilic proteins

• For pressure-sensitive proteins, expression at reduced temperatures (15-20°C) often improves solubility

Expression Protocol:

• Clone the P. profundum rplB gene into a vector with a compatible promoter (T7 or tac)

• Transform into the chosen expression strain

• Grow cultures at 20-25°C to mid-log phase

• Induce with reduced IPTG concentration (0.1-0.5 mM) to prevent inclusion body formation

• Continue expression at 15-17°C for 16-24 hours to mimic the natural temperature preference of P. profundum

Purification Considerations:

• Include stabilizing agents (glycerol, certain salts) in buffers to maintain native conformation

• Consider immobilized metal affinity chromatography (IMAC) followed by ion-exchange chromatography

• For functional studies, verify that the recombinant protein maintains its expected interactions with RNA and other proteins

How can researchers investigate the potential transcriptional regulation role of P. profundum rplB?

Building on findings that E. coli RPL2 interacts with RNA polymerase and influences transcription , researchers can apply similar methodologies to investigate P. profundum rplB:

In vitro Transcription Assays:

• Purify recombinant P. profundum rplB and RNA polymerase

• Conduct transcription assays using various promoters (ribosomal and non-ribosomal)

• Measure transcription rates in the presence and absence of rplB

• Compare results with those using E. coli components to identify piezophile-specific effects

Protein-Protein Interaction Studies:

• Perform bacterial two-hybrid assays to confirm interaction between rplB and RNA polymerase subunits

• Use coimmunoprecipitation to validate interactions in cell extracts

• Apply crosslinking mass spectrometry to map interaction interfaces

Reporter Gene Assays:

• Construct β-galactosidase reporter systems with various promoters

• Express rplB in trans and measure effects on reporter expression

• Compare results using promoters from genes known to be pressure-regulated

What is known about the pressure adaptation mechanisms in P. profundum proteins?

Proteomic studies of P. profundum have revealed several mechanisms of pressure adaptation:

• Metabolic Shifts: Under high pressure, P. profundum shows up-regulation of glycolysis/gluconeogenesis pathway proteins and alcohol dehydrogenase (PBPRA2519), suggesting a shift toward fermentative metabolism at high pressure .

• Membrane Transport Modifications: ABC transporters involved in phosphate, ion, sugar, and amino acid transport show pressure-dependent regulation, likely reflecting both functional adaptations to pressure and responses to different nutrient availability at varying depths .

• DNA Recombination and Repair: The RecD protein, involved in DNA recombination and repair, is essential for high-pressure growth in P. profundum. Mutations in recD result in pressure-sensitive growth phenotypes .

• Protein Structural Adaptations: While not specifically documented for rplB, piezophilic proteins often feature structural adaptations such as increased flexibility, reduced hydrophobic cores, and modified surface charge distributions to maintain function under pressure.

How can researchers assess the functionality of rplB under varying pressure conditions?

To evaluate how pressure affects rplB function, researchers should consider these experimental approaches:

High-Pressure Biochemical Assays:

• Utilize specialized high-pressure chambers capable of maintaining experimental conditions at varying pressures

• Measure binding kinetics between rplB and its interaction partners (rRNA, other ribosomal proteins, RNA polymerase) at different pressures

• Assess conformational changes using fluorescence probes or FRET pairs introduced at strategic positions

Comparative Expression Studies:

• Grow P. profundum cultures at different pressures (0.1 MPa, 28 MPa, 40+ MPa)

• Harvest cells and measure rplB expression levels using RT-qPCR and Western blotting

• Analyze ribosome assembly and composition across pressure conditions

Functional Complementation:

• Create rplB mutants or deletions in P. profundum

• Assess growth characteristics across pressure ranges

• Complement with wild-type or modified rplB to identify pressure-critical domains

What structural aspects of rplB may contribute to pressure adaptation?

While specific structural data for P. profundum rplB is not available in the provided literature, general principles of protein pressure adaptation suggest investigating:

• Electrostatic Interactions: Examine charge distribution patterns that might enhance stability under pressure

• Hydrophobic Core Packing: Analyze the composition and arrangement of hydrophobic residues

• Flexibility Features: Identify regions with increased flexibility that might accommodate volume changes under pressure

• Surface Cavities: Assess the presence and size of internal cavities that could be compressed under high pressure

Recommended Analytical Approaches:

• Homology modeling based on known bacterial L2 structures

• Molecular dynamics simulations under varying pressure conditions

• Hydrogen-deuterium exchange mass spectrometry to map flexibility differences

• Site-directed mutagenesis to test the importance of specific residues

What challenges are associated with studying recombinant P. profundum proteins?

Researchers working with recombinant P. profundum proteins face several technical challenges:

How can researchers investigate potential interactions between rplB and RNA polymerase in P. profundum?

Building on the finding that E. coli RPL2 interacts with RNA polymerase α subunit , researchers can apply these methodologies:

• Two-Hybrid Analysis:

  • Construct bacterial two-hybrid system with P. profundum rplB and RNA polymerase subunits

  • Test interactions at different temperatures and with pressure pre-treatment of components

  • Compare results with E. coli proteins to identify piezophile-specific interaction characteristics

• Co-immunoprecipitation:

  • Generate antibodies against P. profundum rplB or use epitope-tagged versions

  • Perform pull-down experiments from cells grown at different pressures

  • Identify interaction partners using mass spectrometry

  • Validate specific interactions with Western blotting

• Reporter Gene Assays:

  • Construct reporter systems with ribosomal promoters from P. profundum

  • Test the effect of rplB expression on reporter activity

  • Compare results using cells adapted to different pressure conditions

What are promising areas for further investigation of P. profundum rplB?

Several research directions could advance our understanding of P. profundum rplB:

• Dual Functionality Analysis: Further characterize the proposed dual role of rplB in ribosome assembly and transcriptional regulation, especially under pressure conditions.

• Pressure-Responsive Domains: Identify specific domains or residues in rplB that confer pressure adaptation through mutational analysis and functional studies.

• Interactome Mapping: Conduct comprehensive interactome studies to identify all protein partners of rplB under different pressure conditions.

• Comparative Genomics: Compare rplB sequences and structures across bacteria from different depth zones to identify convergent adaptations to pressure.

• Regulatory Networks: Investigate how rplB may function within larger regulatory networks that respond to pressure changes, potentially coordinating ribosome assembly with transcriptional responses.

How might findings from P. profundum rplB research impact broader scientific fields?

Research on P. profundum rplB has implications beyond marine microbiology:

• Extremophile Adaptation: Contributes to our understanding of how proteins adapt to extreme conditions, with applications in biotechnology and astrobiology.

• Protein Engineering: Principles of pressure adaptation could inform the design of pressure-stable enzymes for industrial applications.

• Ribosomal Evolution: Insights into extra-ribosomal functions of ribosomal proteins may reshape our understanding of ribosome evolution and functional expansion.

• Marine Ecology: Better understanding of deep-sea microbial adaptation informs models of marine carbon cycling and ecosystem function.

• Synthetic Biology: Knowledge of pressure-adaptive features could enable the engineering of organisms optimized for high-pressure biotechnology applications.