Recombinant Prochlorococcus marinus subsp. pastoris 30S ribosomal protein S10 (rpsJ)

What is Prochlorococcus marinus and why is it significant in microbial oceanography?

Prochlorococcus marinus is a marine cyanobacterium first discovered in 1988 that has since been recognized as potentially the most abundant photosynthetic organism on Earth. Despite its small size, it contributes significantly to global nutrient cycling and oceanic primary production . This organism is unique among cyanobacteria in that it uses divinyl-chlorophyll a and b as its major light-harvesting pigments, and it employs chlorophyll-binding antenna proteins (Pcb proteins) rather than the phycobilisomes used by most cyanobacteria .

Prochlorococcus marinus is found predominantly in low- to mid-latitude oceans, thriving in nutrient-poor waters and at greater depths (up to 135m) than its close relative Synechococcus (which is limited to 95m) . The organism has evolved into distinct ecotypes adapted to different light conditions, commonly classified as high-light (HL) and low-light (LL) adapted strains . This ecological diversification makes it an excellent model organism for studying microbial adaptation to varying environmental conditions.

The genome of Prochlorococcus marinus is relatively small (approximately 1.8 Mb), making it amenable to complete genomic analysis . The strain CCMP1986 (also known as MED4) was isolated from 5m depth in the Mediterranean Sea and has been completely sequenced, serving as a reference genome for studies on marine cyanobacteria .

What is the function of the 30S ribosomal protein S10 in bacterial translation?

The 30S ribosomal protein S10 (encoded by the rpsJ gene) is a component of the small ribosomal subunit in prokaryotes. While the search results don't provide specific details about its function in Prochlorococcus marinus, the general functions of S10 proteins in bacterial translation include:

The S10 protein is highly conserved across bacterial species, indicating its fundamental importance in protein synthesis. In Prochlorococcus marinus, this protein likely plays a critical role in adaptation to the organism's unique ecological niche, potentially contributing to efficient translation under varying light and nutrient conditions.

What are the structural characteristics of Prochlorococcus marinus 30S ribosomal protein S10?

Based on available data, the 30S ribosomal protein S10 from Prochlorococcus marinus has the following structural characteristics:

• Protein length: The full-length protein consists of 106 amino acids .

• Molecular features: Like other bacterial S10 proteins, it likely contains several positively charged residues that facilitate interaction with negatively charged ribosomal RNA.

• Secondary structure: Although not explicitly detailed in the search results, bacterial S10 proteins typically contain a mix of alpha-helical and beta-sheet elements that fold into a compact tertiary structure.

• Functional domains: The protein likely contains specific domains for RNA binding and for interaction with other ribosomal proteins within the 30S subunit.

The specific structure of Prochlorococcus marinus S10 may contain unique adaptations that reflect the organism's evolution in the marine environment, potentially influencing translation efficiency under specific ecological conditions characteristic of its habitat.

What are the optimal storage conditions for recombinant Prochlorococcus marinus 30S ribosomal protein S10?

The optimal storage conditions for recombinant Prochlorococcus marinus 30S ribosomal protein S10 depend on the duration of storage and preparation format:

• Short-term storage (up to one week):

  • Working aliquots should be stored at 4°C

  • Repeated freezing and thawing should be avoided to prevent denaturation and activity loss

• Medium-term storage (2-4 weeks):

  • Store at 4°C if the entire vial will be used within this timeframe

• Long-term storage:

  • For liquid formulations: -20°C/-80°C with an expected shelf life of approximately 6 months

  • For lyophilized formulations: -20°C/-80°C with an expected shelf life of approximately 12 months

• Storage additives:

  • Addition of glycerol (5-50% final concentration) is recommended for long-term storage

  • For enhanced stability, addition of carrier proteins (0.1% HSA or BSA) may be beneficial

These conditions help maintain protein stability and prevent degradation, aggregation, or loss of activity that might otherwise compromise experimental results. The specific requirements may vary slightly depending on the preparation method and any modifications (such as fusion tags) present in the recombinant protein.

What reconstitution protocols are recommended for lyophilized Prochlorococcus marinus 30S ribosomal protein S10?

For optimal reconstitution of lyophilized Prochlorococcus marinus 30S ribosomal protein S10, the following protocol is recommended:

• Pre-reconstitution preparation:

  • Briefly centrifuge the vial prior to opening to bring the contents to the bottom and prevent loss of material

  • Allow the vial to reach room temperature before opening to prevent condensation

• Reconstitution procedure:

  • Add deionized sterile water to achieve a final protein concentration of 0.1-1.0 mg/mL

  • Gently mix by inversion or mild vortexing until completely dissolved

  • Avoid generating foam or bubbles which can lead to protein denaturation

• Post-reconstitution stabilization:

  • Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation)

  • Aliquot the reconstituted protein into smaller volumes to minimize freeze-thaw cycles

• Storage of reconstituted protein:

  • Store aliquots at -20°C/-80°C for long-term storage

  • For immediate use, keep at 4°C and use within one week

This protocol is designed to maintain the structural integrity and activity of the protein while minimizing the risk of contamination or degradation. The addition of glycerol serves as a cryoprotectant, preventing ice crystal formation that could damage the protein during freezing.

What expression systems are suitable for producing recombinant Prochlorococcus marinus ribosomal proteins?

Based on the information from the search results and knowledge of protein expression systems, several options are suitable for producing recombinant Prochlorococcus marinus ribosomal proteins:

• Mammalian cell expression systems:

  • Commercial preparations of Prochlorococcus marinus 30S ribosomal protein S10 have been successfully produced in mammalian cells

  • These systems may provide appropriate post-translational modifications and folding environment

• E. coli expression systems:

  • Bacterial expression is commonly used for ribosomal proteins due to their prokaryotic origin

  • E. coli has been successfully used for expression of other ribosomal proteins, such as human RPS10

  • Codon optimization may be necessary to account for differences in codon usage between Prochlorococcus and E. coli

• Cell-free expression systems:

  • May be advantageous for ribosomal proteins that affect host cell translation

  • Allows for rapid production and easy modification of reaction conditions

The choice of expression system should consider factors such as:

• Required protein yield

• Need for post-translational modifications

• Intended experimental applications

• Potential toxicity to the host organism

• Solubility concerns

For structural studies or applications requiring high purity, mammalian or E. coli systems with appropriate purification tags (His-tag, as used in commercial preparations ) appear to be effective choices based on the available evidence.

How can Prochlorococcus marinus 30S ribosomal protein S10 be used in structural biology studies?

Prochlorococcus marinus 30S ribosomal protein S10 can be utilized in various structural biology applications to understand both its individual characteristics and its role within the ribosomal complex:

These structural studies can provide insights into how this abundant marine cyanobacterium has optimized its translation machinery for survival in oligotrophic ocean environments, potentially revealing adaptations that contribute to its ecological success.

What approaches can be used to study interactions between Prochlorococcus marinus 30S ribosomal protein S10 and ribosomal RNA?

Several methodological approaches can be employed to investigate the interactions between Prochlorococcus marinus 30S ribosomal protein S10 and ribosomal RNA:

• Electrophoretic mobility shift assays (EMSA):

  • Used to detect protein-RNA binding through changes in migration patterns

  • Can determine binding affinity and specificity of S10 for different rRNA fragments

  • Allows competition assays to identify critical binding regions

• RNA footprinting:

  • Chemical or enzymatic probing of rRNA in the presence and absence of S10

  • Reveals specific nucleotides protected by protein binding

  • Can be combined with next-generation sequencing for high-resolution mapping

• Surface plasmon resonance (SPR):

  • Provides real-time quantitative measurements of binding kinetics

  • Can determine association and dissociation rates between S10 and RNA

  • Useful for comparing binding properties under different conditions (temperature, salt, pH)

• Isothermal titration calorimetry (ITC):

  • Measures thermodynamic parameters of S10-RNA interactions

  • Provides information on binding stoichiometry, enthalpy, and entropy

  • Useful for understanding the energetics driving complex formation

• Cross-linking approaches:

  • UV cross-linking can capture direct protein-RNA contacts

  • Cross-linked complexes can be analyzed by mass spectrometry to identify interaction sites

  • Can be performed in vitro with purified components or in vivo

• Computational modeling:

  • Molecular dynamics simulations can predict interaction interfaces

  • Homology modeling based on known ribosome structures can guide experimental design

  • Particularly useful for understanding how sequence variations in Prochlorococcus S10 might affect RNA binding

These approaches can reveal how Prochlorococcus marinus S10 contributes to ribosome assembly and function, potentially identifying adaptations that optimize translation in the organism's specific ecological niche.

How can researchers investigate the role of S10 in Prochlorococcus marinus adaptation to different light conditions?

Investigating the role of 30S ribosomal protein S10 in Prochlorococcus marinus adaptation to different light conditions requires integrative approaches spanning from genetic analysis to physiological studies:

• Comparative genomics and transcriptomics:

  • Compare S10 sequences across high-light and low-light adapted ecotypes

  • Analyze transcription patterns of the rpsJ gene under different light regimes

  • Correlate sequence variations with specific light adaptations

• Proteomics approaches:

  • Quantify S10 protein abundance changes in response to varying light conditions

  • Identify potential post-translational modifications that may regulate S10 function

  • Compare protein-protein interaction networks involving S10 under different light regimes

• Ribosome profiling:

  • Analyze translation efficiency and ribosome occupancy patterns under different light conditions

  • Determine if S10 variations influence translation of specific mRNAs, particularly those encoding photosynthetic proteins

  • Compare translation patterns between high-light and low-light adapted strains

• Genetic manipulation approaches:

  • Heterologous expression of S10 variants from different ecotypes

  • Site-directed mutagenesis of specific residues that differ between ecotypes

  • Complementation studies in S10-depleted backgrounds

• Structural studies:

  • Determine if S10 structure differs between high-light and low-light adapted strains

  • Investigate if light conditions affect S10 interactions with other components

  • Examine potential conformational changes in response to light-dependent signals

• Physiological experiments:

  • Compare growth rates and translation efficiency under different light conditions in strains with different S10 variants

  • Measure photosynthetic performance in relation to S10 sequence variations

  • Assess stress responses and survival under fluctuating light conditions

These approaches could reveal whether S10 contributes to the remarkable ability of Prochlorococcus to adapt to varying light conditions, potentially through effects on the translation of genes involved in photosynthesis and light harvesting.

What quality control methods are essential when working with recombinant Prochlorococcus marinus 30S ribosomal protein S10?

Ensuring the quality and integrity of recombinant Prochlorococcus marinus 30S ribosomal protein S10 is crucial for reliable experimental results. The following quality control methods are essential:

• Purity assessment:

  • SDS-PAGE analysis to verify size and purity (commercial preparations typically achieve >85% purity)

  • Size exclusion chromatography to detect aggregates and degradation products

  • Mass spectrometry for precise molecular weight determination

• Identity confirmation:

  • Western blotting using specific antibodies or tag-specific detection

  • Peptide mass fingerprinting to confirm the protein sequence

  • N-terminal sequencing to verify the correct start site

• Structural integrity evaluation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to evaluate tertiary structure

  • Limited proteolysis to verify proper folding

• Functional verification:

  • RNA binding assays to confirm biological activity

  • Assembly into partial or complete ribosomal complexes

  • Translation activity assays in reconstituted systems

• Stability monitoring:

  • Thermal shift assays to assess stability under various conditions

  • Long-term storage testing at different temperatures

  • Freeze-thaw stability evaluation

• Contaminant testing:

  • Endotoxin testing (particularly for proteins expressed in bacterial systems)

  • Nuclease assays to detect RNA/DNA contamination

  • Protease activity assays to identify potential degradative enzymes

What challenges might researchers encounter when studying ribosome assembly in Prochlorococcus marinus, and how can they be addressed?

Studying ribosome assembly in Prochlorococcus marinus presents several unique challenges due to its ecological niche and specialized adaptations. Here are key challenges and potential solutions:

• Challenge: Limited biomass from culture

  • Solution: Optimize growth conditions specific to Prochlorococcus strains

  • Solution: Develop heterologous expression systems for Prochlorococcus ribosomal components

  • Solution: Utilize targeted approaches requiring minimal material, such as cryo-EM of isolated ribosomes

• Challenge: Unique environmental adaptations affecting assembly

  • Solution: Recreate marine-like conditions (salt concentration, pH, temperature) for in vitro studies

  • Solution: Compare assembly processes between high-light and low-light adapted strains

  • Solution: Investigate the role of specific ions or metabolites present in the marine environment

• Challenge: Lack of genetic tools for Prochlorococcus

  • Solution: Utilize comparative genomics across multiple Prochlorococcus strains

  • Solution: Develop new transformation protocols specific for Prochlorococcus

  • Solution: Use model organisms (e.g., Synechococcus) for initial studies, then validate in Prochlorococcus

• Challenge: Distinguishing strain-specific assembly features

  • Solution: Compare ribosome assembly across different Prochlorococcus ecotypes

  • Solution: Conduct detailed sequence analysis of ribosomal proteins and rRNAs across strains

  • Solution: Develop strain-specific antibodies or RNA probes for assembly intermediates

• Challenge: Understanding assembly under varying light conditions

  • Solution: Monitor assembly processes under controlled light regimes mimicking natural conditions

  • Solution: Develop light-synchronized cultures to study assembly throughout diel cycles

  • Solution: Use pulse-chase approaches to track newly synthesized ribosomal components

• Challenge: Limited knowledge of Prochlorococcus-specific assembly factors

  • Solution: Conduct comparative proteomics to identify proteins co-purifying with assembly intermediates

  • Solution: Perform bioinformatic analysis to identify candidate assembly factors

  • Solution: Test the effect of identified factors in reconstituted assembly systems

Addressing these challenges requires integrating approaches from microbial ecology, structural biology, and molecular biology to understand how this abundant marine cyanobacterium has optimized its ribosome assembly process for its unique ecological niche.

How can researchers overcome issues related to the specificity of antibodies when studying Prochlorococcus marinus ribosomal proteins?

Developing and utilizing specific antibodies for Prochlorococcus marinus ribosomal proteins presents several challenges. Here are effective strategies to overcome these issues:

• Epitope selection and antibody development:

  • Select unique epitopes by comparing S10 sequences across related cyanobacteria

  • Use recombinant protein expression systems to generate pure antigen

  • Consider developing monoclonal antibodies for highest specificity

  • Utilize peptide antigens for regions with highest sequence divergence

• Cross-reactivity testing and validation:

  • Perform Western blot analysis against related cyanobacterial proteins

  • Test antibodies against different Prochlorococcus ecotypes to ensure broad utility

  • Include appropriate positive and negative controls in all immunological assays

  • Conduct pre-absorption studies with related proteins to improve specificity

• Alternative approaches when antibodies are problematic:

  • Epitope tagging of ribosomal proteins (where genetic manipulation is possible)

  • Use of RNA aptamers or nanobodies as alternative binding reagents

  • Label-free detection methods such as mass spectrometry

  • Proximity labeling approaches to identify protein interactions

• Optimization for specific applications:

  • For immunoprecipitation: Test different binding and washing conditions

  • For immunofluorescence: Optimize fixation protocols for marine cyanobacteria

  • For flow cytometry: Develop permeabilization protocols that preserve cellular integrity

  • For chromatin immunoprecipitation: Adapt cross-linking conditions for Prochlorococcus cells

• Validation in complex samples:

  • Use knockout controls or siRNA when possible to verify antibody specificity

  • Implement orthogonal detection methods to confirm results

  • Consider using multiple antibodies targeting different epitopes of the same protein

  • Verify results with immunoprecipitation followed by mass spectrometry

• Resource sharing and standardization:

  • Establish repositories for validated Prochlorococcus-specific antibodies

  • Develop standard operating procedures for antibody validation

  • Share detailed protocols for successful immunological applications

These strategies can help researchers overcome the limitations of antibody-based approaches when studying ribosomal proteins in this ecologically important marine cyanobacterium, enabling more reliable and reproducible research outcomes.

How does Prochlorococcus marinus 30S ribosomal protein S10 compare to homologous proteins in other cyanobacteria?

Comparing the 30S ribosomal protein S10 across cyanobacterial species provides insights into evolutionary conservation and adaptation. While the search results don't provide direct comparative information, general principles of ribosomal protein evolution suggest:

Detailed comparative analysis of S10 across diverse cyanobacteria could provide insights into how this essential ribosomal protein has adapted to various ecological niches while maintaining its fundamental role in protein synthesis.

What insights can genomic comparisons provide about the evolution of ribosomal proteins in Prochlorococcus marinus ecotypes?

Genomic comparisons of ribosomal proteins across Prochlorococcus marinus ecotypes can reveal important evolutionary patterns and adaptations:

• Ecotype-specific adaptations:

  • Comparison between high-light and low-light adapted strains may reveal specific adaptations in ribosomal proteins

  • These adaptations could contribute to optimized translation under different light conditions

  • Sequence variations might correlate with differences in growth rates or metabolic efficiencies

• Genome streamlining patterns:

  • Prochlorococcus has undergone genome reduction during evolution

  • Analysis of ribosomal protein genes can reveal if they've been subject to streamlining

  • Comparison with the core genome (estimated at about 1250 genes) can show the relative conservation of ribosomal components

• Selection pressure analysis:

  • Calculation of dN/dS ratios for ribosomal protein genes across ecotypes

  • Identification of sites under positive or purifying selection

  • Correlation of selection patterns with ecological parameters (depth, temperature, nutrient availability)

• Co-evolution patterns:

  • Analysis of coordinated evolution between ribosomal proteins and rRNA

  • Identification of compensatory mutations that maintain ribosome function

  • Mapping of interaction networks within the ribosome complex

• Horizontal gene transfer assessment:

  • Evaluation of ribosomal protein phylogenies compared to organismal phylogeny

  • Identification of potential horizontal gene transfer events affecting ribosomal components

  • Assessment of the impact of gene transfer on ribosome function and adaptation

• Population genomics insights:

  • Analysis of single nucleotide polymorphisms in ribosomal protein genes within populations

  • Correlation of genetic variation with spatial or temporal environmental gradients

  • Identification of selective sweeps affecting ribosomal components

These genomic comparisons can provide a deeper understanding of how translation machinery has evolved in this ecologically important marine cyanobacterium, potentially revealing mechanisms of adaptation to different oceanic niches.

How might the study of Prochlorococcus marinus ribosomal proteins contribute to understanding photosynthetic adaptation in marine environments?

The study of Prochlorococcus marinus ribosomal proteins, including S10, can provide unique insights into photosynthetic adaptation in marine environments through several research avenues:

• Translation-photosynthesis coordination:

  • Investigation of how ribosomal proteins might be specialized for efficient translation of photosynthetic apparatus components

  • Analysis of translation efficiency for genes encoding divinyl-chlorophyll binding proteins versus phycobiliprotein genes

  • Study of light-dependent regulation of ribosome composition and activity

• Ecotype-specific translation adaptation:

  • Comparison of ribosomal proteins between high-light and low-light adapted Prochlorococcus ecotypes

  • Correlation of ribosomal adaptations with differences in photosynthetic apparatus between ecotypes

  • Investigation of how translation machinery adaptations support distinct photosynthetic strategies

• Nutrient limitation responses:

  • Analysis of how ribosomal components adapt to the nutrient-poor conditions where Prochlorococcus thrives

  • Study of translation efficiency for photosynthetic genes under nutrient limitation

  • Investigation of resource allocation between ribosome biogenesis and photosynthetic apparatus assembly

• Evolutionary trajectory analysis:

  • Comparison of ribosomal adaptations in Prochlorococcus versus other photosynthetic organisms

  • Assessment of how genome streamlining has affected translation of photosynthetic components

  • Investigation of co-evolution between translation machinery and photosynthetic apparatus genes

• Environmental response mechanisms:

  • Study of how ribosomal proteins respond to changing light conditions

  • Analysis of translation regulation during diel cycles in relation to photosynthetic activity

  • Investigation of temperature effects on ribosome function in relation to photosynthetic efficiency

• Vertical distribution adaptations:

  • Comparison of ribosomal components from strains adapted to different depths

  • Correlation with light intensity and spectral quality changes across the water column

  • Analysis of how translation machinery optimizes protein synthesis under depth-specific conditions

This research could reveal how one of the most abundant photosynthetic organisms on Earth has fine-tuned its protein synthesis machinery to support its unique photosynthetic adaptations, potentially informing broader understanding of marine ecosystem function and evolution.