Recombinant Prochlorococcus marinus subsp. pastoris 30S ribosomal protein S14 (rpsN)

What is the structural and functional role of the 30S ribosomal protein S14 in Prochlorococcus marinus?

The 30S ribosomal protein S14 (rpsN) in Prochlorococcus marinus plays a critical role in ribosomal assembly and function. Similar to other organisms, this protein is involved in the assembly of the small ribosomal subunit. Evidence suggests that S14, along with other proteins like S3, is essential during the assembly process of the 30S subunit but may not be required for function once the subunit has been properly assembled . The protein contributes to the structural integrity of the ribosome and facilitates proper mRNA binding during translation.

How does Prochlorococcus marinus adapt to different environmental conditions, and what role might ribosomal proteins play in this adaptation?

Prochlorococcus marinus, as an obligate marine microorganism, has adapted to specific environmental conditions such as salinity. Research has shown that different strains of Prochlorococcus, such as the low-light adapted strain NATL1A and high-light adapted strain MED4, can acclimate to various salinity ranges with the lowest tolerance being 25 psu and 28 psu, respectively .

Transcriptomic studies reveal that under low salinity stress, Prochlorococcus regulates the expression of ribosomal genes. Specifically, in low salinity acclimated NATL1A strain, genes involved in translation, ribosomal structure, and biogenesis are predominantly downregulated, with several 30S ribosomal proteins showing significant repression . This regulation suggests that ribosomal proteins, potentially including S14, play a role in the organism's adaptation to environmental stressors.

How is Prochlorococcus marinus 30S ribosomal protein S14 purified for research purposes?

The purification of recombinant Prochlorococcus marinus 30S ribosomal protein S14 typically involves:

• Expression in a suitable system (commonly E. coli or Baculovirus expression systems as indicated in commercial products)

• Cell lysis under controlled buffer conditions

• Affinity chromatography, usually utilizing a His-tag if the recombinant protein includes one

• Further purification steps such as ion-exchange or size-exclusion chromatography

• Quality assessment using SDS-PAGE to determine purity (typically >80-85%)

• Storage in an appropriate buffer containing stabilizing agents

The commercial formulations indicate that the purified protein is often supplied in a sterile filtered colorless solution with stabilizing agents such as glycerol . For research-grade quality, purity is typically assessed by SDS-PAGE and should be greater than 85% .

What are the optimal expression systems and conditions for producing functional recombinant Prochlorococcus marinus 30S ribosomal protein S14?

Based on the commercial products and research protocols, two main expression systems are commonly used:

E. coli Expression System:

• Expression vector: pET-based vectors with T7 promoter

• Host strain: BL21(DE3) or similar strains

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: Optimal expression often occurs at lower temperatures (16-25°C) for 16-20 hours to maximize solubility

• Lysis buffer: Typically includes 20mM Tris-HCl buffer (pH 8.0), reducing agents like 1mM DTT, and salt (100mM NaCl)

Baculovirus Expression System:

• Used for proteins requiring post-translational modifications

• Host cells: Sf9 or Hi5 insect cells

• Infection: MOI of 1-5 for 48-72 hours

• Purification: Similar to E. coli but may require different buffer compositions

The choice between these systems depends on research requirements, with E. coli being more cost-effective but Baculovirus potentially providing better folding for complex proteins .

How can researchers effectively characterize the interaction of Prochlorococcus marinus 30S ribosomal protein S14 with other ribosomal components?

To characterize interactions between the S14 protein and other ribosomal components, researchers can employ multiple complementary techniques:

• In vitro Reconstitution Assays:

  • Purified S14 can be added to partially assembled 30S subunits lacking this protein

  • Activity of reconstituted particles can be measured to assess functionality

  • This approach helps determine whether S14 is required for assembly or function

• Pull-down Assays and Co-immunoprecipitation:

  • Using recombinant S14 with an affinity tag to identify interaction partners

  • Verification with reverse pull-downs using antibodies against potential partner proteins

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking of assembled ribosomes followed by MS analysis

  • Identification of proteins in proximity to S14 within the ribosomal structure

• Cryo-EM Structural Analysis:

  • Comparison of structures with and without S14 to determine structural contributions

  • Visualization of conformational changes induced by S14 integration

Research has shown that ribosomal proteins like S3 and S14 are involved in assembly but may not be required for activity once the 30S subunit is properly assembled . This suggests that interaction studies should focus on both assembly intermediates and mature ribosomes.

What approaches can be used to generate and validate antibodies against Prochlorococcus marinus 30S ribosomal protein S14?

Development of specific antibodies against Prochlorococcus marinus S14 requires a systematic approach similar to that used for other ribosomal proteins:

Production Methodology:

• Immunogen preparation: Purified recombinant S14 protein with >85% purity

• Animal selection: Typically rabbits for polyclonal antibodies or mice for monoclonals

• Immunization protocol: Initial immunization followed by 3-4 booster doses

• Serum collection and antibody purification: Using protein A/G affinity chromatography

• Characterization: Western blot, ELISA, and immunofluorescence validation

Validation Methods:

• Western Blot Analysis: Should detect a single band of appropriate molecular weight (~16 kDa for S14)

• Cross-reactivity Testing: Assess specificity against related proteins from other species

• Immunofluorescence: Demonstrate proper cellular localization

• Functional Assays: Confirm ability to immunoprecipitate native protein complexes

Similar approaches have been successfully used for ribosomal protein S14 antibody preparation in other systems, such as the polyclonal antibody against RPS14 in broilers . These antibodies allowed for specific identification of RPS14 in important tissues and determination of its expression levels through Western blotting and immunofluorescence techniques .

How does the expression of Prochlorococcus marinus 30S ribosomal protein S14 change under different environmental stressors?

Transcriptomic studies of Prochlorococcus under various environmental conditions provide insights into S14 regulation:

Under Low Salinity Stress:
Low-light adapted Prochlorococcus strain NATL1A shows significant transcriptional changes when acclimated to low salinity (28 psu compared to normal 34 psu). Under these conditions, genes involved in translation, ribosomal structure, and biogenesis are predominantly downregulated . The table below shows differential expression of selected ribosomal proteins in NATL1A under low salinity stress:

While the specific values for S14 (rpsN) aren't provided in the available search results, the pattern of downregulation of ribosomal proteins suggests that S14 might be similarly affected under salinity stress .

Research Methodology for Stress Response Studies:

• Controlled cultivation under various stress conditions (salinity, temperature, light intensity)

• RNA isolation and global transcriptomic analysis (RNA-seq)

• Protein isolation and quantitative proteomics (LC-MS/MS)

• Validation of expression changes through qRT-PCR and Western blotting

What role does the 30S ribosomal protein S14 play in Prochlorococcus-bacteria interactions in marine ecosystems?

Prochlorococcus-bacteria interactions are crucial in marine ecosystems, and while direct evidence for S14's role is limited, several studies suggest potential implications:

• Bacterial Community Support: Prochlorococcus in co-culture with heterotrophic bacteria shows different growth outcomes, which may involve differential regulation of ribosomal genes, including those encoding proteins like S14 .

• Recycling of Cellular Components: When Prochlorococcus interacts with heterotrophic bacteria, there's evidence of recycling nitrogen compounds. This process may involve regulation of translation machinery, including ribosomal proteins .

• Stress Response Mechanisms: Under environmental stressors, Prochlorococcus regulates ribosomal gene expression differently depending on the strain. In NATL1A, genes involved in translation and ribosomal structure are downregulated under low salinity, while MED4 shows upregulation of these genes . These different stress responses may influence interactions with bacteria in varying environments.

Research approaches to study these interactions include:

• Co-culture experiments with Prochlorococcus and various heterotrophic bacteria

• Transcriptomic and proteomic analyses of both organisms during interaction

• Metabolite exchange studies using isotope labeling

Can Prochlorococcus marinus 30S ribosomal protein S14 be used as a biomarker for environmental monitoring or ecological studies?

The potential use of S14 as a biomarker involves several considerations:

Advantages:

• Ribosomal proteins are relatively conserved but contain species-specific regions

• Expression levels may correlate with environmental conditions or physiological states

• Can be detected using molecular methods like qPCR or proteomics

Methodological Approach:

• Development of Specific Detection Methods:

  • Design of primers/probes targeting S14 gene regions specific to Prochlorococcus marinus

  • Development of antibody-based detection systems

• Validation in Environmental Samples:

  • Correlation of S14 detection with other established Prochlorococcus markers

  • Assessment of sensitivity and specificity in mixed microbial communities

• Environmental Response Profiling:

  • Determination of S14 expression patterns under various environmental conditions

  • Establishing baseline measurements for different oceanic regions

Research has shown that Prochlorococcus has specific salinity tolerance ranges (25-50 psu for MED4 and 26-50 psu for NATL1A) , suggesting that detection of S14 could potentially be used to monitor changes in marine ecosystems, particularly in areas experiencing salinity fluctuations due to climate change.

How does the structure and function of Prochlorococcus marinus 30S ribosomal protein S14 compare to homologous proteins in other cyanobacteria and photosynthetic organisms?

Comparative analysis of S14 across different organisms reveals both conservation and divergence:

Structural Comparisons:

• Sequence Homology: Cyanobacterial S14 proteins share significant sequence homology, typically 70-90% among closely related species

• Functional Domains: The RNA-binding domains of S14 are highly conserved across photosynthetic organisms

• Protein Interactions: The interaction sites with neighboring ribosomal proteins and rRNA show higher conservation than peripheral regions

Functional Implications:

• The core role in ribosome assembly appears consistent across species

• Species-specific variations may relate to environmental adaptations

• Interactions with other ribosomal components may vary, potentially affecting translation efficiency under different conditions

For research purposes, these comparisons can be conducted using:

• Multiple sequence alignments of S14 homologs

• Structural modeling based on available ribosome structures

• Functional complementation studies in heterologous systems

What are the evolutionary implications of variations in 30S ribosomal protein S14 sequences across different Prochlorococcus ecotypes?

Prochlorococcus is known for its diverse ecotypes adapted to different light conditions and oceanic regions. Analysis of S14 sequences across these ecotypes can provide insights into evolutionary adaptations:

Research Approaches:

• Phylogenetic Analysis:

  • Construction of phylogenetic trees based on S14 sequences from different ecotypes

  • Correlation with ecological niches and environmental parameters

• Selection Pressure Analysis:

  • Calculation of Ka/Ks ratios to identify sites under positive or purifying selection

  • Identification of ecotype-specific amino acid substitutions

• Structure-Function Relationships:

  • Mapping of variations onto structural models to identify functionally significant changes

  • Experimental validation through site-directed mutagenesis and functional assays

The different responses of high-light adapted MED4 and low-light adapted NATL1A strains to stress conditions suggest that variations in ribosomal proteins, potentially including S14, may contribute to the ecological specialization of Prochlorococcus ecotypes.

What are the optimal storage and handling conditions for recombinant Prochlorococcus marinus 30S ribosomal protein S14 to maintain stability and functionality?

Based on commercial product information and standard practices for recombinant proteins:

Storage Conditions:

• Temperature: -20°C for long-term storage

• Short-term storage: 4°C for up to one week

• Avoid repeated freeze-thaw cycles

Buffer Composition:

• Typical formulation: 20mM Tris-HCl buffer (pH 8.0), 1mM DTT, 40% glycerol, and 100mM NaCl

• The high glycerol concentration (40-50%) is crucial for stability

Handling Recommendations:

• Thaw on ice and centrifuge briefly before opening

• Aliquot to minimize freeze-thaw cycles

• For working solutions, dilute in appropriate buffers containing reducing agents

• For reconstitution of lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

Shelf Life:

• Liquid form: 6 months at -20°C/-80°C

• Lyophilized form: 12 months at -20°C/-80°C

How can researchers optimize in vitro reconstitution assays to study the role of Prochlorococcus marinus 30S ribosomal protein S14 in ribosome assembly?

In vitro reconstitution assays are valuable tools for studying ribosomal assembly. For S14 specifically:

Protocol Optimization:

• Preparation of Core Components:

  • Isolation of 16S rRNA from Prochlorococcus or expression of recombinant RNA

  • Purification of individual ribosomal proteins or protein complexes lacking S14

  • Expression and purification of recombinant S14 with >85% purity

• Assembly Conditions:

  • Buffer composition: Typically containing Mg²⁺ (10-20 mM), NH₄Cl (100-200 mM), and K⁺ (50-100 mM)

  • Temperature gradient: Initial incubation at low temperature (0-4°C) followed by step-wise increase (30-42°C)

  • Assembly intermediates: Sequential addition of protein groups in a specified order

• Functional Validation:

  • In vitro translation assays using reporter mRNAs

  • tRNA binding capacity measurements

  • Structure analysis by cryo-EM or chemical probing

Similar approaches have demonstrated that proteins S3 and S14 are involved in 30S subunit assembly but may not be required for activity once the subunit has been properly assembled . This suggests that reconstitution assays should include both assembly and functional testing phases.

What considerations should be made when designing experiments to investigate the impact of environmental stressors on Prochlorococcus marinus 30S ribosomal protein S14 expression and function?

Comprehensive experimental design should account for:

Stress Conditions and Controls:

• Salinity Gradients: From 25 psu to 40 psu, based on the known tolerance ranges of Prochlorococcus strains

• Light Intensity: Different levels appropriate for high-light vs. low-light adapted strains

• Nutrient Limitation: Particularly nitrogen and phosphorus

• Temperature Variations: Within the growth range of Prochlorococcus

• Control Conditions: Standard culture conditions (34 psu salinity, appropriate light)

Acclimation Approaches:

• Gradual Acclimation: Step-wise changes in conditions over multiple transfers

• Shock Experiments: Sudden exposure to stress conditions

• Long-term Adaptation: Maintained under stress conditions for multiple generations

Analytical Methods:

• Transcriptional Analysis:

  • qRT-PCR targeting S14 and related genes

  • RNA-Seq for global transcriptional changes

• Protein-level Analysis:

  • Western blotting with specific antibodies

  • Proteomics to quantify changes in S14 abundance

• Functional Assessments:

  • Growth rate measurements

  • Photosynthetic efficiency (Fv/Fm measurements)

  • Ribosome profiling to assess translation efficiency

Research has shown that Prochlorococcus strains exhibit different responses to low salinity, with NATL1A showing downregulation of ribosomal genes while MED4 shows upregulation . This strain-specific response should be considered when designing experiments.

What are the most significant unanswered questions regarding Prochlorococcus marinus 30S ribosomal protein S14, and what methodologies might address these gaps?

Several critical knowledge gaps remain:

• Specific Role in Environmental Adaptation:

  • How does S14 contribute to Prochlorococcus adaptation to different oceanic niches?

  • Methodology: Comparative genomics and transcriptomics of S14 across ecotypes, coupled with site-directed mutagenesis and fitness assays

• Post-translational Modifications:

  • Are there specific PTMs on S14 that regulate its function under stress conditions?

  • Methodology: Mass spectrometry-based PTM profiling under various environmental conditions

• Interactions with Non-ribosomal Components:

  • Does S14 have moonlighting functions outside the ribosome?

  • Methodology: Interactome studies using tagged S14 followed by MS identification

• Role in Translational Regulation:

  • Does S14 differentially affect translation of specific mRNAs?

  • Methodology: Ribosome profiling in strains with modified S14 expression

• Ecological Significance:

  • How does variation in S14 contribute to community dynamics in marine ecosystems?

  • Methodology: Metatranscriptomics and metaproteomics of natural communities