Recombinant Photobacterium profundum 50S ribosomal protein L3 (rplC)

What is the significance of studying Photobacterium profundum ribosomal protein L3?

Ribosomal protein L3 (rplC) is an essential and indispensable component for the formation of the peptidyl transferase center (PTC) in the bacterial ribosome. In P. profundum, L3 plays a critical role in adaptation to high-pressure environments, making it a valuable model for studying how essential cellular machinery adapts to extreme conditions. L3 is one of the first ribosomal proteins to be assembled onto the 23S rRNA and is one of only a few proteins required for peptidyltransferase activity . Furthermore, mutations in L3 have been associated with resistance to antibiotics that target the PTC, including linezolid and tiamulin, making it significant for antibiotic resistance research .

How can I clone and express recombinant P. profundum L3 protein?

For efficient cloning and expression of P. profundum L3, the following methodology has proven effective:

• Gene amplification: PCR-amplify the rplC gene from P. profundum genomic DNA using primers with appropriate restriction sites (e.g., BamHI and SmaI).

• Vector selection: Clone the rplC gene into an expression vector such as pGEX4T-3 for GST-tagged protein expression .

• Host strain: Transform into E. coli BL21-CodonPlus (DE3)-RIL competent cells to address potential codon bias issues .

• Culture conditions: Grow the transformed E. coli in LB medium containing appropriate antibiotics (ampicillin 100 μg/ml, chloramphenicol 25 μg/ml) at 37°C until A600 reaches 0.4, then decrease temperature to 22°C for protein expression .

• Purification: Purify using affinity chromatography followed by gel filtration on a Superdex 200 column in an appropriate buffer such as 10 mM HEPES (pH 7.5) .

This methodology has been validated for expression of functional P. profundum proteins and can be adapted for L3 specifically.

What are the optimal growth conditions for P. profundum to study ribosomal protein expression?

P. profundum SS9 has unique growth requirements reflecting its deep-sea origin:

For ribosomal protein expression studies, compare expression levels between atmospheric pressure (0.1 MPa) and high pressure (28 MPa) conditions, as many ribosomal proteins are differentially expressed based on pressure conditions . Culture cells to early logarithmic phase (OD600 0.1-0.4) for optimal comparison between pressure conditions .

How can I verify the expression and stability of recombinant P. profundum L3?

To verify expression and stability of recombinant L3 protein:

• Western blot analysis: Use antibodies against a conserved 15-amino-acid peptide present in L3 to detect the protein. Include a control protein such as GroEL as a reference band .

• Mass spectrometry validation: For definitive identification, excise the band detected by Western blotting from an SDS-PAGE gel, digest with trypsin, and analyze using peptide mass fingerprinting .

• Functional complementation: If working with L3 mutants, verify functionality through complementation of L3-deficient strains and assessment of growth patterns under varying pressure conditions .

• Size-exclusion chromatography: Assess the oligomeric state and potential aggregation of purified L3 protein using gel filtration chromatography.

Researchers have successfully employed these methods to confirm the expression and stability of ribosomal proteins from P. profundum, ensuring that the recombinant proteins are properly folded and functional .

What are common challenges in working with recombinant P. profundum proteins, and how can they be addressed?

Several challenges may arise when working with recombinant P. profundum proteins:

• Pressure adaptation effects: P. profundum proteins often have pressure-adapted structures that may affect folding at atmospheric pressure. Solution: Use pressurized expression systems or express the protein at reduced temperatures (15-22°C) .

• Codon bias: P. profundum has different codon usage compared to E. coli. Solution: Use E. coli strains optimized for rare codons such as BL21-CodonPlus (DE3)-RIL .

• Protein solubility: Deep-sea adapted proteins may have solubility issues. Solution: Use solubility-enhancing fusion tags (GST, MBP) and optimize buffer conditions (add glycerol, adjust salt concentration) .

• Expression toxicity: Overexpression of ribosomal proteins can be toxic to host cells. Solution: Use tightly regulated expression systems and lower induction temperatures .

• Functional validation: Confirming proper folding and function can be challenging. Solution: Develop specific activity assays or complementation tests in L3-deficient strains .

How do mutations in P. profundum L3 affect antibiotic resistance compared to other bacterial species?

Research on L3 mutations and antibiotic resistance in P. profundum builds on studies in other bacterial species, revealing species-specific differences:

While E. coli studies demonstrated that mutations in L3 loops near the PTC can confer resistance to specific antibiotics, the effects in P. profundum may be unique due to pressure-adapted protein conformations. Research indicates that pressure adaptation in P. profundum proteins may create novel interaction sites for antibiotics or alter existing binding pockets .

To characterize L3 mutation effects in P. profundum:

• Create targeted mutations in the loops of L3 near the PTC using site-directed mutagenesis

• Assess antibiotic susceptibility across a pressure gradient (0.1-28 MPa)

• Perform computational modeling of the impact of L3 mutations on 50S structure and antibiotic binding

• Compare results with equivalent mutations in non-piezophilic bacteria to identify pressure-specific effects

What methodologies are most effective for studying the impact of pressure on L3 protein structure and function?

To effectively study the impact of pressure on L3 structure and function, combine these complementary approaches:

• High-pressure protein crystallography: While technically challenging, this provides direct structural insights into pressure-induced conformational changes in L3. Use specialized high-pressure crystallization cells with beamline facilities equipped for high-pressure data collection.

• Molecular dynamics simulations: Simulate the behavior of L3 under different pressure conditions to predict structural changes and interactions with rRNA and antibiotics.

• High-pressure circular dichroism: Monitor secondary structure changes in purified L3 protein under varying pressure conditions.

• High-pressure NMR spectroscopy: For detailed atomic-level analysis of pressure effects on protein structure.

• Genetic approaches: Create chimeric L3 proteins with domains from pressure-sensitive species to identify pressure-adaptation determinants.

• High-pressure microscopic chambers: Direct observation of cellular processes under pressure, as demonstrated for motility studies in P. profundum .

How does L3 contribute to the assembly of pressure-adapted ribosomes in P. profundum?

L3 plays a critical role in pressure-adapted ribosome assembly in P. profundum through several mechanisms:

• Early assembly factor: As in E. coli, L3 is likely one of the first ribosomal proteins to be assembled onto the 23S rRNA in P. profundum, serving as a nucleation point for 50S subunit formation .

• Pressure-specific conformation: Under high pressure (28 MPa), P. profundum ribosomes show altered composition and structure. Proteomics studies have identified significant up-regulation of ribosomal proteins under pressure, suggesting pressure-specific ribosome assembly pathways .

• rRNA stabilization: P. profundum has an unusually large number of rRNA operons (15, the highest reported in any bacterium) with high variation between operons, suggesting pressure-specific roles for L3-rRNA interactions . L3 likely stabilizes these varied rRNA structures differently under pressure.

• Interaction with other assembly factors: L3 interactions with assembly factors may be pressure-dependent, facilitating efficient 50S assembly under high pressure.

• Central extension: The central extension of L3 protrudes deep into the core of the large subunit and may function as an allosteric switch, potentially adapting differently under pressure conditions .

Research approaches to study this could include:

• Ribosome reconstitution experiments under varying pressure conditions

• Cryo-EM structural analysis of P. profundum ribosomes assembled at different pressures

• In vitro binding assays measuring L3-rRNA interactions under pressure

What are the key structural features of P. profundum L3 that differentiate it from mesophilic bacterial L3 proteins?

The structural features that distinguish P. profundum L3 from mesophilic bacterial L3 proteins are likely adaptations to high pressure environments:

• Amino acid composition: Pressure-adapted proteins often show increased hydrophilicity in core regions and decreased volume change of ionization. Comparative analysis reveals P. profundum L3 likely has:

  • Reduced number of void volumes

  • Increased number of salt bridges

  • Higher glycine content in flexible regions

  • Reduced hydrophobic core packing

• Domains and extensions: The central and W-finger extensions of L3 are critical for function. In P. profundum, these extensions likely show pressure-specific adaptations:

  • The central extension that protrudes near the PTC may have altered flexibility

  • The W-finger domain likely contains modifications for stability under pressure

  • Key residues in these extensions (W-255 equivalent in yeast) may have altered interactions with rRNA under pressure

• Interaction with rRNA: Pressure-adapted L3 might show altered base-stacking interactions with rRNA nucleotides like A2940 (equivalent to A2572 in E. coli) .

• Dynamic range: P. profundum L3 likely maintains functionality across a broader pressure range (0.1-90 MPa) compared to mesophilic L3 proteins.

To characterize these features experimentally:

• Perform comprehensive sequence alignment of L3 proteins across pressure gradients

• Use computational modeling to predict pressure effects on specific residues

• Create chimeric L3 proteins with domain swaps between piezophilic and mesophilic species

How can genetic suppressor analysis be used to study L3 function in P. profundum?

Genetic suppressor analysis provides powerful insights into L3 function in P. profundum by revealing functional relationships between genes. This methodology has been successfully employed with P. profundum for other systems and can be adapted for L3 studies:

Protocol for Suppressor Analysis of L3 Mutations:

• Create primary mutation: Generate L3 mutants in P. profundum through site-directed mutagenesis or random mutagenesis focused on the central extension region.

• Phenotype selection: Screen for pressure-sensitive phenotypes (growth defects at 28 MPa) or antibiotic sensitivity.

• Suppressor generation: Allow spontaneous suppressors to arise or use chemical mutagenesis (EMS treatment) to increase mutation rate.

• Suppressor identification: Use whole-genome sequencing or targeted sequencing of candidate genes to identify suppressor mutations.

• Genetic reconstruction: Re-create suppressor mutations in clean genetic backgrounds to verify their effects.

• Biochemical validation: Purify suppressor-containing ribosomes and test for restored function in in vitro translation assays.

This approach has successfully identified genetic interactions in P. profundum pressure adaptation and fatty acid biosynthesis pathways , and can provide insights into the network of interactions involving L3 in ribosome assembly and function.

What is known about the coordinated expression of L3 with other ribosomal proteins in P. profundum under pressure stress?

The coordinated expression of ribosomal proteins, including L3, under pressure stress in P. profundum reveals sophisticated regulatory mechanisms:

• Proteome-wide coordination: Label-free quantitative proteomic analysis has identified differential expression of ribosomal proteins between atmospheric (0.1 MPa) and high pressure (28 MPa) conditions .

• Ribosomal protein enrichment: A total of 25 significantly up-regulated ribosomal proteins were identified under pressure conditions, representing an enrichment with a p-value of 3×10^-9, one of the highest enrichment factors observed for any protein group .

• Operon structure: In bacteria, the rplC gene encoding L3 is typically located in the S10 operon from the str-spc region of the chromosome, encoding multiple ribosomal proteins . In P. profundum, this organization is likely preserved but with pressure-specific regulatory elements.

• Regulatory mechanisms: The L4 ribosomal protein typically regulates the S10 operon containing the L3 gene . Under pressure, this regulation may be altered to optimize ribosome assembly.

• rRNA coordination: P. profundum has an unusually large number of rRNA operons (15), allowing for pressure-specific ribosome compositions .

To study this coordination:

• Perform RNA-seq analysis comparing transcriptomes at different pressures

• Use qRT-PCR to measure expression of specific ribosomal genes, including rplC (L3)

• Analyze promoter regions for pressure-responsive elements

• Create reporter fusions to monitor operon expression under varying pressure

How does the function of L3 in the peptidyltransferase center change under high pressure conditions?

The function of L3 in the peptidyltransferase center (PTC) under high pressure likely undergoes significant adaptations in P. profundum:

• Altered tRNA binding affinities: Studies of L3 mutants in other systems show that L3 modifications can result in ribosomes having increased affinities for both aminoacyl- and peptidyl-tRNAs . In P. profundum, these affinities are likely pressure-modulated.

• Peptidyltransferase activity modulation: L3 mutations in other organisms correlate with decreased peptidyltransferase activities . P. profundum L3 likely maintains optimal peptidyltransferase activity across a pressure range through structural adaptations.

• Antibiotic binding changes: L3 mutations affect binding of PTC-targeting antibiotics like linezolid and tiamulin . P. profundum L3 may show pressure-dependent antibiotic sensitivity profiles.

• Conformational switching: The central extension of L3 may function as an allosteric switch in coordinating binding of elongation factors . This switching mechanism is likely adapted to function under pressure.

• A-site accommodation corridor: L3 influences the opening of the aa-tRNA accommodation corridor . This function is critical for translation and may be specifically adapted in P. profundum for pressure conditions.

Experimental approaches to investigate these changes include:

• In vitro translation assays under varying pressure conditions

• tRNA binding studies at different pressures

• Antibiotic sensitivity testing across pressure gradients

• Structural studies of the PTC at different pressures

• Molecular dynamics simulations of L3 function under pressure

What are the most promising applications of research on P. profundum L3 for understanding extremophile adaptation?

Research on P. profundum L3 provides unique insights into extremophile adaptation with several promising applications:

• Biotechnology applications: Understanding pressure adaptation mechanisms in L3 could lead to the development of pressure-resistant enzymes and expression systems for industrial biocatalysis under high-pressure conditions.

• Antibiotic development: The unique structure of piezophile L3 and its role in antibiotic resistance could guide the development of new antibiotics effective against deep-sea bacteria or drug-resistant pathogens.

• Fundamental ribosome biology: P. profundum L3 studies illuminate the core principles of ribosome evolution and adaptation to extreme environments, providing insights into the fundamental limits of protein synthesis machinery.

• Astrobiology implications: Understanding how translation machinery adapts to extreme pressure has implications for theories about life in extreme environments, including potential subsurface oceans on icy moons like Europa or Enceladus.

• Synthetic biology tools: Engineered pressure-adapted ribosomes containing modified L3 proteins could enable protein synthesis under non-standard conditions, expanding the toolkit for synthetic biology applications.