Recombinant Prochlorococcus marinus subsp. pastoris 50S ribosomal protein L22 (rplV)

Advanced Research Questions

• What mechanisms regulate rplV expression in Prochlorococcus marinus?

The regulation of rplV expression in P. marinus remains incompletely characterized, but insights can be drawn from cyanobacterial gene regulation studies:

Transcription of ribosomal genes in cyanobacteria differs significantly from E. coli models:

• Cyanobacterial RNA polymerases show lower levels of abortive transcription and less misincorporation

• Transcription is significantly slower in cyanobacteria (164 seconds to complete a transcript compared to 26 seconds in E. coli)

• Cyanobacterial RNAPs pause more frequently and cleave transcripts faster when NTPs are absent

• Cyanobacteria maintain approximately 2 orders of magnitude higher intracellular Mn²⁺ levels than E. coli

These fundamental differences in transcription machinery likely influence rplV expression. Additionally, global regulators such as ppGpp respond to different signals in cyanobacteria (darkness adaptation rather than stringent response) , potentially affecting ribosomal protein gene expression.

To study rplV regulation specifically, researchers should consider:

• Promoter characterization using reporter constructs

• Analysis of transcription factor binding sites

• Examination of potential autoregulation mechanisms common in ribosomal proteins

• How can RNA-binding characteristics of rplV be assessed experimentally?

Based on studies of ribosomal protein L22 in other organisms, experimental approaches for characterizing P. marinus rplV RNA binding should include:

In mouse models, Rpl22 has been shown to bind directly to Rpl22l1 mRNA via a hairpin structure . Similar experiments with P. marinus rplV could reveal whether it has analogous regulatory functions through RNA binding outside the ribosome context.

• What role might rplV play in antibiotic resistance in Prochlorococcus?

Ribosomal protein L22 is a known target of macrolide antibiotics like tylosin , making it potentially important in antibiotic resistance mechanisms:

• L22 mutations can confer resistance to macrolides in many bacteria

• Marine environments may contain natural antibiotics that have driven evolution of resistance

• The small genome of P. marinus (1.66 Mb) may limit dedicated resistance mechanisms, making ribosomal modifications more important

Research methodologies to investigate this include:

• Structural analysis of P. marinus ribosomes with bound antibiotics using cryo-EM (similar to the E. coli 50S subunit structure at 2.2Å resolution )

• Site-directed mutagenesis of rplV residues potentially involved in antibiotic binding

• Comparative growth studies with wild-type and mutant strains in presence of antibiotics

• Molecular dynamics simulations of antibiotic binding to wild-type and mutant ribosomes

These approaches could reveal unique adaptations in P. marinus that might inform broader understanding of antibiotic resistance mechanisms.

• How might rplV function differ between high-light and low-light adapted Prochlorococcus strains?

Prochlorococcus has evolved distinct ecotypes adapted to different light environments, which may influence ribosomal protein function:

The MED4 strain (high-light adapted) inhabits surface waters where light is abundant but nutrient levels may be lower . This ecological context may have selected for ribosomal proteins that function efficiently under these constraints.

To investigate ecotype-specific differences in rplV:

• Compare sequences across multiple HL and LL strains

• Express recombinant rplV from different ecotypes

• Assess functional differences in binding affinity, rate of incorporation into ribosomes, and translation efficiency

• Examine differences in response to environmental stressors

This research could reveal how ribosomal proteins contribute to niche adaptation in these globally important marine cyanobacteria.

• What extra-ribosomal functions might P. marinus rplV perform?

Studies in other organisms have revealed that ribosomal proteins can have functions beyond their structural roles in ribosomes. For rplV in P. marinus:

• RNA regulation: In mice, Rpl22 regulates expression of its paralog Rpl22l1 by binding to a hairpin structure in Rpl22l1 mRNA . Similar regulatory functions could exist in P. marinus.

• Stress response: Ribosomal proteins can be repurposed during stress conditions. P. marinus faces various stresses including light fluctuation, nutrient limitation, and oxidative stress .

• Ecological adaptations: The extreme streamlining of the P. marinus genome (1.66 Mb) may have selected for proteins with multiple functions.

Experimental approaches to identify extra-ribosomal functions include:

• RNA immunoprecipitation followed by sequencing (RIP-seq)

• Differential gene expression analysis comparing wild-type and rplV-depleted cells

• Protein-protein interaction studies using pull-down assays coupled with mass spectrometry

• Localization studies using fluorescently tagged rplV under different conditions

• How does the P. marinus 50S ribosomal subunit assembly pathway incorporate rplV?

Based on research on ribosome assembly in other prokaryotes and limited data on cyanobacterial ribosomes:

• L22 is typically incorporated during later stages of ribosome maturation as an external protein on the 60S ribosomal subunit

• The assembly process likely involves:

  • Initial 23S rRNA folding

  • Sequential incorporation of core ribosomal proteins

  • Addition of peripheral proteins including L22

  • Final conformational adjustments

To study this process specifically in P. marinus:

• In vitro ribosome reconstitution experiments

• Pulse-chase labeling to track protein incorporation timing

• Cryo-EM of assembly intermediates

• Comparison with the high-resolution E. coli 50S structure (2.2Å)

This research would be particularly valuable given the unique adaptations of P. marinus to its marine environment and its streamlined genome.

• What techniques can be used to study the interaction between rplV and other ribosomal components?

Advanced methods for studying P. marinus rplV interactions include:

MRM analysis has been particularly effective for ribosomal proteins, allowing precise quantification of Rpl22 and Rpl22l1 in 60S and 80S subunits with specific peptide targets .

• How can genetic engineering approaches be applied to study rplV function in Prochlorococcus?

Genetic manipulation of Prochlorococcus has been challenging, but recent advances offer promising approaches:

• Transposon mutagenesis: Tn5 transposition has been demonstrated in P. marinus strain MIT9313

• Conjugative transfer: An "agar stab" procedure has been developed that allowed transfer of genetic material to P. marinus

• Reporter systems: The Prochlorococcus Rubisco promoter (PccmK) has been used successfully with fluorescent reporters (GFP/YFP)

• Heterologous expression: Testing P. marinus proteins in more genetically tractable cyanobacteria like Synechococcus

For rplV specifically, approaches could include:

• Conditional expression systems to study depletion effects

• Tagged versions for localization and interaction studies

• Site-directed mutagenesis of key residues

• Complementation studies in other organisms

These genetic tools would significantly advance our understanding of rplV function in this ecologically important marine organism.

• What structural features distinguish P. marinus rplV from its homologs in other bacteria?

While high-resolution structural data specifically for P. marinus rplV is not available in the search results, comparative analysis suggests:

• The P. marinus rplV protein (128 amino acids) maintains the core structural elements needed for ribosomal function

• High-resolution cryo-EM structures of bacterial 50S subunits (like the E. coli structure at 2.2Å) provide templates for structural modeling

• P. marinus adaptation to marine environments may have selected for:

  • Surface residues optimized for high salt conditions

  • Structural stability adaptations for its environmental niche

  • Potential differences in RNA-binding domains reflecting unique rRNA features

Structural studies including X-ray crystallography or cryo-EM of P. marinus ribosomes would provide definitive information on these potential adaptations.

• How can researchers overcome challenges in expressing and studying P. marinus proteins?

Working with P. marinus proteins presents several unique challenges:

For rplV specifically, expressing the protein in multiple systems (as demonstrated by commercial providers ) and comparing their properties can help identify the most native-like preparation for functional studies.