Recombinant Photobacterium profundum 30S ribosomal protein S11 (rpsK)

What is Photobacterium profundum and why is it important for ribosomal protein studies?

Photobacterium profundum is a marine bacterium belonging to the Vibrionaceae family, representing the second largest genus within this family. It is characterized as a piezophilic (pressure-loving) organism that has adapted to life in deep-sea environments with high hydrostatic pressure and low temperatures. This extremophile has become a model organism for studying bacterial adaptation to deep-sea conditions.

The importance of P. profundum for ribosomal protein studies stems from several factors:

• It provides insights into how ribosomes function under extreme environmental conditions

• Its genomic adaptations may reveal specialized ribosomal protein variants

• The genus shows remarkable versatility and heterogeneity, making it valuable for comparative studies

• Its ribosomal components may possess unique properties reflecting environmental adaptations

P. profundum has been the subject of extensive genomic studies, including whole genome sequencing approaches that have uncovered its genetic diversity and ecological adaptation mechanisms . These studies help understand how essential cellular machinery like ribosomes maintain functionality in extreme environments.

What is the 30S ribosomal protein S11 (rpsK) and what is its function in bacterial translation?

The 30S ribosomal protein S11, encoded by the rpsK gene, is a crucial component of the small (30S) subunit of the bacterial ribosome. S11 serves several essential functions:

• Forms part of the E site of the ribosome

• Connects the head of the 30S subunit to the platform

• Participates in the formation of the mRNA exit channel

• Contributes to maintaining translational fidelity

• Influences the ribosome's capacity to bind mRNA

Research has demonstrated that S11 forms a functional interaction with another ribosomal protein, S7, at the E site of the 30S subunit . This interaction is critical for proper ribosomal function. When this interaction is disrupted through mutations, significant effects on translational fidelity are observed, including increased frameshifting, readthrough of nonsense codons, and codon misreading .

Additionally, 30S subunits with mutations in S11 show an enhanced capacity to bind mRNA, as demonstrated through toeprinting and filter-binding assays . This suggests that S11 plays a regulatory role in controlling how the ribosome interacts with its mRNA substrate.

How does the genomic organization of rpsK compare between P. profundum and other bacterial species?

While the specific genomic organization of rpsK in P. profundum is not directly addressed in the available search results, comparative genomic analyses provide insights into ribosomal gene organization patterns in marine bacteria.

In most bacteria, ribosomal protein genes are typically organized in conserved operons. The rpsK gene is generally found within the "spc operon" (S10 operon) in many bacterial species, which contains multiple ribosomal protein genes. This organization facilitates coordinated expression of ribosomal components.

Based on genomic studies of the Photobacterium genus, we can infer that P. profundum likely maintains this conserved organization, though with possible adaptations unique to marine bacteria. Comparative genomic analysis reveals that while core functions like translation are conserved, specific gene arrangements may vary between species.

Analysis using tools like ANI (Average Nucleotide Identity) and in silico DNA-DNA hybridization has been valuable for comparing genomic features across Photobacterium species , which could extend to ribosomal gene organization studies.

What structural features characterize the S11 protein in bacterial ribosomes?

The S11 protein possesses several key structural features that facilitate its function in the bacterial ribosome:

• Location at a critical junction connecting the head of the 30S subunit to the platform

• Strategic positioning in the E site of the ribosome

• Direct involvement in forming the mRNA exit channel

• Specific interaction domains that engage with the S7 protein

• RNA-binding motifs that facilitate interaction with ribosomal RNA

Disruption of this S7-S11 interaction through mutations affects the flexibility of the head of the 30S subunit and can lead to an opening of the mRNA exit channel . These structural changes have functional consequences, altering translational fidelity and mRNA binding capacity.

While the specific structural details of P. profundum S11 are not directly addressed in the search results, it likely shares these core features while potentially containing unique adaptations that facilitate ribosomal function under high-pressure, low-temperature conditions.

What expression systems and protocols are recommended for producing recombinant P. profundum S11 protein?

Based on research with marine bacteria including Photobacterium species, the following expression systems and protocols are recommended for recombinant production of P. profundum S11:

Recommended Expression Workflow:

• Gene Amplification: The rpsK gene can be PCR amplified from P. profundum genomic DNA using primers designed based on available genome sequences. An example primer design approach similar to that used for other P. profundum genes would include:

  • Forward primer incorporating restriction sites (e.g., 5'-GTCGAATTCGTATTCAAGATGGGCACTCA-3')

  • Reverse primer with compatible restriction sites (e.g., 5'-GTCGAATTCTAGTAAGCGAATAGCAGGAC-3')

• Vector Selection: Several vectors have been successfully used with Photobacterium genes:

  • pFL122 has been demonstrated effective for cloning Photobacterium genes

  • Vectors with inducible promoters (such as arabinose-inducible systems) have been successfully used with P. profundum genes

• Host Systems: For heterologous expression of P. profundum proteins:

  • E. coli has been successfully used as a model cell factory for expressing marine bacterial genes

  • Alternative hosts may include S. cerevisiae for proteins that prove challenging to express in bacterial systems

• Purification Strategy:

  • Affinity tags (His-tag, GST) facilitate purification

  • Cold-adapted purification protocols may better preserve native structure

  • Size exclusion chromatography as a final purification step

Research with marine bacterial proteins indicates that codon optimization and growth at reduced temperatures (15-20°C) often enhances successful expression of proteins from deep-sea organisms .

How does the functional interaction between S7 and S11 influence ribosomal performance?

The functional interaction between ribosomal proteins S7 and S11 is critical for proper ribosomal performance, particularly in maintaining translational fidelity. Research has revealed several key aspects of this interaction:

• The S7-S11 interaction connects the head of the 30S subunit to the platform, creating an architecturally sound structure for the mRNA exit channel

• This interaction is located in the E site of the ribosome, a position critical for tRNA and mRNA processing

• Disruption of this interaction through mutations affects translational fidelity in multiple ways

In vivo assays with ribosomes containing mutations in either S7 or S11 demonstrated significant effects on translational accuracy:

These alterations in ribosomal function appear to result from increased flexibility of the 30S subunit head, opening of the mRNA exit channel, and perturbation of the allosteric coupling between the A and E sites of the ribosome .

Importantly, while these mutations affect function, they do not prevent incorporation of the proteins into the 30S subunits , suggesting that the interaction is more critical for functional performance than for basic assembly.

What methods are used to study the effects of S11 mutations on translational fidelity?

Several sophisticated methodological approaches are employed to study how mutations in the S11 protein affect translational fidelity:

In Vivo Assays:

• Reporter constructs containing programmed frameshift sites

• Dual-luciferase reporter systems measuring readthrough of premature stop codons

• Reporter systems with near-cognate codons to assess misreading rates

In Vitro Techniques:

• Toeprinting assays to analyze the interaction between ribosomes and mRNA

• Filter-binding assays to quantitatively measure the binding affinity between 30S subunits and mRNA

• Ribosome assembly analysis to confirm proper incorporation of mutated S11 into 30S subunits

The experimental workflow typically involves:

• Generation of mutations in S11 using site-directed mutagenesis techniques

• Expression and purification of mutant S11 proteins

• Reconstitution of ribosomes with the mutant proteins

• Assessment of translational fidelity parameters using the above techniques

• Correlation of functional changes with structural alterations

Research has demonstrated that mutations affecting the S7-S11 interaction increase the capacity for frameshifting, readthrough of nonsense codons, and codon misreading while also enhancing the capacity of 30S subunits to bind mRNA . These findings suggest that the S7-S11 interaction plays a crucial role in maintaining the translational accuracy of the ribosome.

How do environmental adaptations in P. profundum potentially affect S11 structure and function?

Photobacterium profundum's adaptation to deep-sea environments likely influences the structure and function of its ribosomal proteins, including S11. Several environmental factors and their potential impacts include:

High Hydrostatic Pressure Adaptations:

• Ribosomal proteins may have evolved specific amino acid compositions favoring function under pressure

• The S7-S11 interaction could be specially modified to maintain stability under high-pressure conditions

• Structural flexibility might be calibrated to allow necessary conformational changes while preventing pressure-induced denaturation

Low Temperature Adaptations:

• Cold-adapted versions of S11 might show increased flexibility at key regions

• Binding interfaces could be optimized for function at low temperatures

• Energy barriers for necessary conformational changes might be reduced

Genomic Evidence of Adaptation:

• Comparative genomics studies of the Photobacterium genus reveal considerable genetic diversity and adaptation

• Marine bacterial genomes show adaptations in various functional categories, potentially including translation machinery

While the search results don't directly address S11 adaptations in P. profundum, research on other bacterial adaptations provides insights. For example, P. profundum has demonstrated adaptive responses to ultraviolet radiation, with specific genes involved in photoreactivation . Similarly, ribosomal components likely show adaptations to the organism's specific environmental niche.

The observed functional interaction between S7 and S11 may be particularly important in extreme environments, where maintaining translational fidelity despite environmental stressors is critical for survival.

What site-directed mutagenesis approaches are most effective for studying P. profundum S11 function?

For investigating P. profundum S11 function, several site-directed mutagenesis approaches have proven effective, drawing on methodologies used for other bacterial ribosomal proteins and specific techniques applied to Photobacterium genes:

Target Selection Strategies:

• Focus on residues at the S7-S11 interface based on structural data and sequence conservation

• Target amino acids in regions involved in mRNA channel formation

• Identify residues unique to piezophilic bacteria that may contribute to pressure adaptation

Recommended Mutagenesis Workflow:

• Genomic Template Preparation:

  • Extract high-quality genomic DNA from P. profundum cultures

  • PCR amplify the rpsK region with high-fidelity polymerase

• Mutagenesis Methods:

  • Overlap extension PCR for creating specific mutations

  • QuikChange-style protocols for direct plasmid mutagenesis

  • Gibson Assembly for complex modifications

• Vector Construction:

  • Clone amplified fragments into vectors like pFL122 that have been used successfully with Photobacterium genes

  • Include restriction sites (e.g., XhoI and KpnI) as demonstrated for other P. profundum genes

• Verification:

  • Sequence verification using standard thermal cycle dideoxy sequencing with fluorescently labeled terminators

  • Restriction digestion analysis to confirm correct cloning

Examples of successful genetic manipulation techniques with P. profundum include the creation of deletion constructs and promoter region manipulations , which can be adapted for studying S11 function through targeted mutations.

How do experimental approaches for studying translational fidelity differ between standard bacteria and piezophilic species like P. profundum?

Studying translational fidelity in piezophilic species like P. profundum presents unique challenges and requires specialized approaches compared to standard bacterial models:

Pressure Considerations in Experimental Design:

• High-Pressure Cultivation: P. profundum requires specialized equipment for cultivation under pressure (typically 15-40 MPa), necessitating pressure vessels for growth and maintenance

• Pressure Effects on Assays: Standard biochemical assays must be modified to function under pressure or carefully designed to capture effects that persist after decompression

• Pressure-Resistant Reporter Systems: Translational fidelity reporters must remain stable and functional under high pressure conditions

Comparative Analytical Approaches:

Specialized Techniques for P. profundum:

• Pressure-Cycling Protocols: Assess how transitions between different pressure environments affect translational fidelity

• Pressure-Temperature Matrices: Examine the combined effects of pressure and temperature on ribosomal function

• Hybrid Ribosome Construction: Create chimeric ribosomes with components from P. profundum and mesophilic bacteria to isolate pressure-adaptive elements

Research has demonstrated that mutations in S11 affect translational fidelity through mechanisms including increased frameshifting, nonsense codon readthrough, and codon misreading . For P. profundum, these effects must be studied in the context of pressure adaptation, potentially revealing how this extremophile maintains translational accuracy under challenging environmental conditions.

What structural adaptations in P. profundum S11 potentially contribute to pressure resistance?

While the search results don't directly address structural adaptations in P. profundum S11, several potential pressure-adaptive features can be inferred based on research with other piezophilic proteins and known S11 functions:

Amino Acid Composition Adaptations:

• Reduced Volume Change: Pressure-adapted proteins often feature amino acid substitutions that minimize volume changes during conformational shifts

• Reduced Hydrophobic Core: Pressure-adapted proteins typically have smaller hydrophobic cores to counteract pressure-induced compaction

• Increased Flexibility: Strategic glycine residues may provide necessary flexibility in key regions

Structural Elements Contributing to Pressure Adaptation:

Functional Implications:

The S7-S11 interaction is critical for maintaining translational fidelity , and pressure adaptations in this interaction would be essential for P. profundum's survival in the deep sea. The observed effects of S7-S11 disruption on ribosomal function include increased frameshifting, nonsense codon readthrough, and codon misreading - precisely the kinds of errors that would need to be prevented under high pressure.

Comparative genomic analysis of the Photobacterium genus reveals considerable genetic diversity and adaptation to different ecological niches , suggesting that ribosomal components have likely evolved specialized features for their respective environments.

What are the challenges and solutions in crystallizing recombinant P. profundum S11 for structural studies?

Crystallizing recombinant ribosomal proteins from piezophilic organisms like P. profundum presents unique challenges requiring specialized approaches:

Major Challenges:

• Pressure-Adapted Structural Elements:

  • Proteins adapted to high pressure may adopt non-native conformations at atmospheric pressure

  • Structural features stabilized by pressure may be destabilized during crystallization

• Ribosomal Protein Instability:

  • S11 normally functions within the ribosomal complex and may be unstable in isolation

  • The functional interaction with S7 suggests potential instability when this partner is absent

• Extremophile Expression Hurdles:

  • Codon usage differences between P. profundum and expression hosts

  • Potential toxicity when expressing foreign ribosomal proteins in E. coli

• Crystallization Condition Complexity:

  • Need to screen conditions that might mimic aspects of the high-pressure environment

  • Potential requirement for stabilizing agents specific to pressure-adapted proteins

Strategic Solutions:

Methodological Approaches:

• Modified Expression Strategies:

  • Cold-adapted expression systems (15-20°C) that better accommodate proteins from psychrophilic organisms

  • Cell-free expression systems to avoid toxicity in whole-cell systems

  • Similar approaches have been successful for other marine bacterial proteins

• Advanced Crystallization Techniques:

  • Lipidic cubic phase crystallization for membrane-associated portions

  • Microseeding with crystals of homologous proteins

  • Automated high-throughput screening with specialized deep-sea mimetic conditions

• Alternative Structural Approaches:

  • Cryo-electron microscopy of entire ribosomal complexes

  • NMR studies of specific domains with isotope-labeled protein

  • Hydrogen-deuterium exchange mass spectrometry for dynamics studies

Studies on the functional interaction between S7 and S11 suggest that co-crystallization of these partners may be necessary to capture physiologically relevant conformations.