Recombinant Prochlorococcus marinus subsp. pastoris 50S ribosomal protein L13 (rplM)

What are the optimal conditions for expressing and purifying recombinant Prochlorococcus L13?

Based on available product information, recombinant Prochlorococcus marinus L13 can be successfully expressed in both E. coli and mammalian cell systems . Here are the recommended parameters for expression and purification:

Table 1: Expression Systems for Recombinant Prochlorococcus L13

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% recommended) for long-term storage

• Aliquot for storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

When designing expression systems, researchers should consider:

• Codon optimization for the expression host

• Inclusion of appropriate promoters (noting that E. coli promoters often function poorly in cyanobacteria )

• Purification strategy compatible with downstream applications

How can researchers validate the functionality of recombinant L13 protein?

Validation of recombinant L13 functionality requires a multi-faceted approach addressing both structural integrity and functional activity:

Structural Validation:

• SDS-PAGE and Western blotting to confirm size and immunoreactivity

• Circular dichroism (CD) spectroscopy to verify secondary structure elements

• Size exclusion chromatography to ensure proper folding and absence of aggregation

Functional Validation:

• RNA binding assays to confirm interaction with 23S rRNA

• Ribosome incorporation assays to verify the ability to integrate into the 50S subunit

• For regulatory function, translational repression assays using reporter constructs containing the rplM-rpsI 5' UTR

A particularly informative approach would be to create a chromosomal reporter fusion similar to the rplM'-'lacZ system used in E. coli studies , which could demonstrate the protein's ability to repress translation of its own mRNA. This would involve:

• Constructing a fusion between the Prochlorococcus rplM 5' UTR/early coding region and a reporter gene

• Measuring reporter activity with and without co-expression of recombinant L13

• Including appropriate controls with unrelated 5' UTRs to confirm specificity of regulation

What experimental approaches are most effective for studying L13's role in ribosome assembly?

Based on studies of ribosomal proteins in other bacterial systems, the following experimental approaches would be most effective for investigating L13's role in Prochlorococcus ribosome assembly:

• In vivo depletion studies:

  • Create conditional expression systems for L13 in Prochlorococcus or a model cyanobacterium

  • Monitor effects on ribosome profiles, cell growth, and translation efficiency

  • Analyze accumulation of ribosomal assembly intermediates using sucrose gradient centrifugation

• In vitro reconstitution experiments:

  • Purify individual ribosomal components from Prochlorococcus

  • Perform stepwise assembly assays with and without L13

  • Use techniques like light scattering or sedimentation to monitor assembly kinetics

• Structural studies:

  • Employ cryo-electron microscopy to visualize L13's position within the Prochlorococcus ribosome

  • Perform cross-linking studies to identify L13's interaction partners

  • Use chemical probing to map L13-induced structural changes in rRNA

• Assembly factor interactions:

  • Investigate potential assembly factors (like SrmB in E. coli) required for L13 incorporation

  • Perform pull-down assays to identify proteins that interact with L13 during ribosome assembly

  • Test the effects of depleting putative assembly factors on L13 incorporation

When designing such experiments, researchers should consider the unique challenges of working with marine cyanobacteria, including potential difficulties in genetic manipulation and growth conditions that differ significantly from model organisms like E. coli.

What is known about L13's role as an autogenous regulator in bacterial systems?

Based on studies in E. coli, L13 functions as an autogenous repressor of the rplM-rpsI operon, which encodes L13 itself and the ribosomal protein S9 . The key features of this regulatory mechanism include:

• Target specificity: L13 specifically represses translation of the rplM-rpsI operon without affecting unrelated mRNAs. When tested with other ribosomal protein operon reporters (rplY-lacZ or rpsO-lacZ), no change in expression was observed in the presence of excess L13 .

• Level of regulation: The regulation occurs post-transcriptionally at the translational level. RT-PCR analysis showed no alteration in transcript amounts in the presence of excess L13, confirming that the regulation does not occur at the transcriptional level .

• Regulatory target: L13 acts on the rplM translation initiation region. The 5' UTR of the rplM-rpsI mRNA forms a developed secondary structure with conserved sequence and structural features, including an unusual Shine-Dalgarno sequence (GGU) .

• Physiological consequences: Overexpression of L13 slows cell growth, likely due to the downregulation of S9 expression, which is essential for proper ribosomal function. Western blot analysis showed that S9 levels were reduced approximately 1.5-fold in the presence of excess L13 .

This regulatory mechanism represents a feedback loop that helps maintain balanced production of ribosomal components, which is crucial for efficient ribosome assembly and cellular growth.

How should experiments be designed to investigate L13's regulatory function in Prochlorococcus?

To investigate whether the autogenous regulatory function of L13 observed in E. coli is conserved in Prochlorococcus, researchers should design experiments that address both the mechanism and specificity of regulation:

• Reporter gene assays:

  • Construct translational fusions between the Prochlorococcus rplM-rpsI 5' UTR/early coding region and a reporter gene like lacZ or GFP

  • Test reporter activity with and without co-expression of L13

  • Include unrelated 5' UTRs as controls to confirm specificity

  • Create a series of mutant constructs to identify critical regulatory elements

• RNA-protein interaction studies:

  • Perform RNA electrophoretic mobility shift assays (EMSA) with purified L13 and the rplM-rpsI 5' UTR

  • Use techniques like RNA footprinting or SHAPE to map the interaction sites

  • Develop in vitro translation assays to directly measure L13's effect on translation initiation

• Distinguishing regulatory levels:

  • Use RT-PCR or Northern blotting to confirm that regulation occurs at the translational rather than transcriptional level

  • Employ polysome profiling to assess the effect of L13 on ribosome loading onto the rplM-rpsI mRNA

• In vivo studies in Prochlorococcus:

  • If genetic manipulation of Prochlorococcus is challenging, consider using a model cyanobacterium like Synechococcus

  • Create strains with controlled expression of L13 to study the effects on rplM-rpsI expression

  • Analyze the physiological consequences of disrupting this regulatory mechanism

When designing these experiments, researchers should note that gene expression tools developed for E. coli may not function optimally in cyanobacteria. Many E. coli promoters like λPL, λPR, and Plac show little to no detectable expression in cyanobacteria like Synechococcus PCC 6803 , necessitating the use of cyanobacteria-specific expression systems.

How does stringent response affect L13 expression in bacteria?

The stringent response is a bacterial stress response to nutrient limitation, particularly amino acid starvation, mediated by the alarmone ppGpp and its cofactor DksA. Based on studies in E. coli, the rplM-rpsI operon encoding L13 and S9 is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation .

Key aspects of this regulation include:

• Transcriptional control: The stringent response primarily acts at the transcriptional level, reducing synthesis of ribosomal components when amino acids are scarce.

• Coordination with translational regulation: While stringent control affects transcription of the rplM-rpsI operon, L13-mediated autogenous regulation acts at the translational level. These two regulatory mechanisms work in concert to ensure balanced production of ribosomal proteins under varying growth conditions.

• Physiological significance: This dual regulation helps bacteria adjust ribosome production in response to nutrient availability, conserving resources during starvation while maintaining the stoichiometry of ribosomal components.

To study this regulation in Prochlorococcus, researchers could:

• Induce amino acid starvation using inhibitors like serine hydroxamate

• Monitor transcription of the rplM-rpsI operon using RT-qPCR

• Analyze the roles of ppGpp and potential DksA homologs

• Compare stringent response between different Prochlorococcus ecotypes adapted to various oceanic environments

Understanding how the stringent response affects L13 expression in Prochlorococcus would provide insights into how this important marine organism adapts its translation machinery to nutrient fluctuations in oligotrophic environments.

How does prokaryotic L13 compare to its eukaryotic homolog L13a?

The bacterial L13 and its eukaryotic homolog L13a share some structural features but exhibit significant differences in function and essentiality:

Structural similarities:

• Both are surface-exposed ribosomal proteins with minimal contacts to other ribosomal components

• Both interact directly with ribosomal RNA in their respective subunits

Functional differences:

• Essentiality: While prokaryotic L13 is likely essential for ribosome function, human L13a has been shown to be "dispensable for canonical ribosome function" . Silencing of L13a in human monocytic cells did not affect global protein synthesis, biogenesis of 60S ribosomes, translational fidelity, or the assembly of 80S ribosomes and polyribosomes .

• Extra-ribosomal functions: Eukaryotic L13a has evolved specialized regulatory functions outside the ribosome. In human cells, L13a can be released from the ribosome to participate in translational control of specific mRNAs. For example, L13a is involved in IFN-γ-mediated inhibition of Cp mRNA translation .

• Regulatory mechanisms: While bacterial L13 regulates its own synthesis through autogenous regulation, eukaryotic L13a participates in more complex regulatory networks involving extra-ribosomal functions.

This functional divergence between prokaryotic and eukaryotic homologs illustrates how ribosomal proteins can evolve new roles while maintaining their core structural functions in ribosome assembly.

What can we learn about L13 evolution by studying Prochlorococcus ecotypes?

Prochlorococcus is the most abundant photosynthetic organism in the ocean and has evolved into multiple ecotypes adapted to different light and nutrient conditions . Studying L13 across these ecotypes can provide valuable insights into ribosomal protein evolution:

• Adaptive evolution: Comparisons of L13 sequences from different Prochlorococcus ecotypes could reveal signatures of selection related to:

  • Temperature adaptation across ocean depth gradients

  • Functional optimization under different nutrient limitations

  • Co-evolution with interacting ribosomal components

• Conservation vs. variability: Identifying highly conserved regions across ecotypes would highlight functionally critical domains, while variable regions might indicate adaptations to specific ecological niches.

• Regulatory evolution: Comparing the structure and sequence of the rplM-rpsI operon and its regulatory elements across ecotypes could reveal how translational regulation evolves in response to different selective pressures.

• Horizontal gene transfer: Prochlorococcus genomes show evidence of gene acquisition through horizontal gene transfer . Analysis of L13 sequences might reveal whether ribosomal proteins have been subject to such transfer events.

Research approaches could include:

• Comparative genomic analysis of L13 sequences across Prochlorococcus ecotypes

• Functional complementation studies to test interchangeability of L13 proteins

• Analysis of selection pressures using dN/dS ratios

• Structural modeling to identify ecotype-specific adaptations

Such evolutionary studies would contribute to our understanding of how core cellular machinery like the ribosome adapts to different environmental conditions.

How can recombinant L13 be used to study translation regulation in cyanobacteria?

Recombinant Prochlorococcus L13 provides a valuable tool for investigating translation regulation in cyanobacteria:

• Mechanisms of autogenous regulation:

  • Using purified L13 in in vitro translation systems to study repression mechanisms

  • Identifying the RNA structural elements recognized by L13

  • Creating reporter constructs to monitor regulation in vivo

• Ribosome assembly studies:

  • Tracking the incorporation of labeled recombinant L13 into assembling ribosomes

  • Identifying assembly intermediates that accumulate in the absence of L13

  • Studying interactions between L13 and potential assembly factors

• Comparative regulatory studies:

  • Testing whether the regulatory mechanism is conserved across different cyanobacterial species

  • Investigating how regulation responds to environmental conditions relevant to marine ecosystems

  • Comparing regulation between different Prochlorococcus ecotypes

• Tool development:

  • Generating antibodies against recombinant L13 for use in immunoprecipitation or Western blotting

  • Creating tagged versions for tracking L13 localization and interactions

  • Developing L13 depletion systems to study consequences of ribosome deficiency

When designing experiments with recombinant L13, researchers should ensure the protein is properly folded and functional, considering that post-translational modifications or specific folding conditions might be required for activity.

What potential applications exist for L13-based gene expression systems in synthetic biology?

Based on L13's role as a translational regulator, its regulatory mechanisms could be harnessed for synthetic biology applications in cyanobacteria:

• Translational control systems:

  • The L13-responsive elements from the rplM-rpsI 5' UTR could be adapted to create synthetic translational regulators

  • Target genes could be placed under translational control of L13 levels

  • Such systems would provide a means to couple gene expression to the cellular ribosome content

• Metabolic engineering tools:

  • L13-based regulatory systems could help balance expression of introduced metabolic pathways

  • The natural feedback mechanism could prevent excessive resource allocation to heterologous proteins

  • This could be particularly valuable for sustainable bioproduction in cyanobacteria

• Biosensors:

  • Since ribosome synthesis responds to nutrient availability and stress conditions, L13-regulated reporter systems could function as biosensors

  • These could be used to monitor environmental conditions relevant to cyanobacterial growth

• Expression optimization:

  • When designing cyanobacterial expression systems, researchers should note that many promoters from E. coli (like λPL, λPR, Plac) show little to no detectable expression in cyanobacteria

  • Successful gene expression tools in cyanobacteria are typically created by "systematically optimizing parameters (e.g., DNA and protein sequences)"

  • L13-based systems would need similar optimization for different cyanobacterial hosts

Key considerations for developing such systems include:

• Characterizing the minimal RNA elements required for L13 recognition

• Determining the optimal positioning of these elements relative to the translation start site

• Testing the specificity and efficiency of regulation in different cyanobacterial species

• Evaluating whether the regulatory mechanism functions properly in heterologous contexts

How might studies of L13 contribute to understanding Prochlorococcus ecology?

Prochlorococcus is a key primary producer in oceanic ecosystems, responsible for a significant portion of global carbon fixation . Studies of L13 and ribosome regulation can provide insights into Prochlorococcus ecology in several ways:

• Growth regulation in nutrient-limited environments:

  • Prochlorococcus thrives in oligotrophic environments with limited nutrients

  • Understanding how ribosome synthesis is regulated under these conditions helps explain this ecological success

  • The dual regulation of rplM-rpsI by stringent response and autogenous control may be a key adaptation to variable nutrient availability

• Interactions with heterotrophs and phages:

  • Prochlorococcus exists in complex ecological relationships with heterotrophic bacteria and phages

  • Ribosomal components like L13 may be targets for phage manipulation during infection

  • Studying how L13 expression responds to these interactions could reveal mechanisms of ecological adaptation

• Ecotype-specific adaptations:

  • Different Prochlorococcus ecotypes have evolved to occupy specific niches in the water column

  • Variations in L13 and ribosome regulation might contribute to these adaptations

  • Comparative studies could identify ecotype-specific regulatory mechanisms

• Carbon and energy flux:

  • As Prochlorococcus supplies photosynthetically fixed carbon to marine heterotrophs , its translation capacity directly affects ecosystem productivity

  • Understanding ribosome regulation provides insights into factors controlling this carbon flux

  • This knowledge has implications for modeling marine ecosystem responses to environmental change

By integrating studies of L13 and ribosome regulation with ecological observations, researchers can build a more comprehensive understanding of how Prochlorococcus maintains its remarkable abundance and productivity in oceanic ecosystems.