Recombinant Rhodopirellula baltica 50S ribosomal protein L35 (rpmI)

What is Rhodopirellula baltica ribosomal protein L35 and why is it significant for research?

Rhodopirellula baltica ribosomal protein L35 (rpmI) is a component of the large 50S ribosomal subunit in this marine planctomycete bacterium. Its significance stems from R. baltica's unique cellular features, including compartmentalized structure and proteinaceous cell wall. Similar to other ribosomal proteins, L35 likely plays a critical role in ribosome assembly and protein synthesis. Based on comparative studies with other bacterial L35 proteins, it is believed to be involved in the formation of functional ribosomes and potentially in pre-rRNA processing . R. baltica's distinctive position in bacterial phylogeny makes its ribosomal proteins particularly valuable for evolutionary studies of translation machinery.

What is currently known about the genomic organization of the rpmI gene in R. baltica?

The rpmI gene in R. baltica is part of its 7.15 Mb genome, which contains relatively few operon structures compared to other bacteria . Unlike in many bacteria where ribosomal protein genes are organized in conserved operons, the genomic context of rpmI in R. baltica reflects the organism's unique genomic organization. During stressful conditions or stationary phase, genome rearrangements may occur, potentially affecting the expression and regulation of ribosomal genes, including rpmI . These genomic features should be considered when designing expression systems for recombinant production.

What effects do environmental stressors have on rpmI expression in R. baltica?

Environmental stressors significantly impact ribosomal protein gene expression in R. baltica. Studies have shown that under stress conditions such as nutrient limitation and high cell density during transition and stationary phases, R. baltica cells decrease the expression level of genes belonging to the ribosomal machinery . The organism activates genes associated with stress response, including glutathione peroxidase (RB2244), thioredoxin (RB12160), and universal stress protein (uspE, RB4742), while simultaneously downregulating ribosomal protein genes . This regulatory pattern likely applies to rpmI as well, suggesting that stress conditions are not optimal for recombinant production of L35.

How might the function of L35 in R. baltica differ from that in other bacteria?

Given R. baltica's unique cellular features and phylogenetic position, its L35 protein may exhibit functional adaptations compared to other bacteria. R. baltica possesses intracellular compartmentalization and proteinaceous cell walls instead of peptidoglycan , which may influence ribosome localization and assembly. During different growth phases, especially when forming rosettes in stationary phase, the regulation and function of ribosomal proteins like L35 may be adapted to support the organism's complex life cycle . Additionally, R. baltica's adaptation to marine environments may have selected for specific structural features in L35 that optimize ribosome function under varying salinity conditions.

What expression systems are optimal for recombinant R. baltica L35 production?

For recombinant production of R. baltica L35, E. coli expression systems are generally recommended due to their versatility and high yields. Based on experiences with other R. baltica proteins:

• Vector selection: pET-based vectors with T7 promoter systems offer strong, inducible expression suitable for ribosomal proteins.

• Host strains: BL21(DE3) derivatives, particularly those optimized for rare codon usage (like Rosetta strains), are advisable given R. baltica's different codon preference compared to E. coli.

• Induction conditions: Lower temperatures (16-20°C) after induction with reduced IPTG concentrations (0.1-0.5 mM) may improve solubility, as ribosomal proteins can form inclusion bodies at higher expression rates.

• Media optimization: For standard protein production, LB or TB media are suitable, while defined minimal media should be used for isotopic labeling experiments.

Research has shown that genes from R. baltica can be subject to active genome rearrangements under stress conditions , suggesting careful optimization of expression constructs may be necessary to ensure stable gene integrity during recombinant production.

What purification strategies yield the highest purity recombinant L35?

For purification of recombinant R. baltica L35, a multi-step approach is typically most effective:

• Initial capture: Affinity chromatography using poly-histidine tags (His6) with Ni-NTA resins offers efficient initial purification. Elution should employ an imidazole gradient to minimize co-purification of E. coli proteins.

• Tag removal: If structural or functional studies are planned, consider incorporating a TEV protease cleavage site to remove the affinity tag after initial purification.

• Secondary purification: Ion exchange chromatography exploiting L35's predicted basic properties (common among ribosomal proteins) can effectively separate it from remaining contaminants.

• Final polishing: Size exclusion chromatography to achieve high purity and remove any aggregated protein.

• Buffer optimization: Consider buffers containing moderate salt concentrations (300-500 mM NaCl) to maintain solubility, as R. baltica is adapted to marine environments with higher salt content .

The purification approach should be tailored based on the intended application of the recombinant protein, with more stringent purification required for structural studies versus functional assays.

How can R. baltica L35 be used to investigate evolutionary aspects of ribosome assembly?

R. baltica L35 offers a unique window into ribosome evolution due to the distinct phylogenetic position of Planctomycetes. Research approaches could include:

• Comparative structural analysis: Solving the structure of R. baltica L35 and comparing it with homologs from diverse bacterial phyla can reveal evolutionary adaptations in ribosomal architecture.

• Chimeric ribosome assembly: Replacing L35 in model organisms with R. baltica L35 to assess functional conservation and specificity of ribosomal protein-RNA interactions.

• Phylogenetic reconstruction: Using L35 sequences along with other ribosomal proteins to refine understanding of Planctomycetes' evolutionary relationships with other bacterial groups.

• Functional complementation studies: Testing whether R. baltica L35 can rescue growth defects in L35-deficient strains of model organisms to assess functional conservation.

R. baltica's unique cellular features, including intracellular compartmentalization , make its ribosomal components particularly interesting for understanding how translation machinery adapted during bacterial evolution.

What approaches can be used to study interactions between L35 and other ribosomal components?

To investigate interactions between R. baltica L35 and other ribosomal components, several methodological approaches are recommended:

• In vitro reconstitution assays: Using purified recombinant L35 and in vitro transcribed rRNA segments to study assembly kinetics and binding affinities.

• Crosslinking coupled with mass spectrometry (XL-MS): Applying chemical crosslinking followed by MS analysis to identify proteins and rRNA regions in proximity to L35 within the ribosome.

• Cryo-electron microscopy: For structural determination of R. baltica ribosomes with focus on L35 positioning and interactions.

• Fluorescence resonance energy transfer (FRET): Using fluorescently labeled L35 and potential interacting partners to measure binding dynamics in real-time.

• Surface plasmon resonance (SPR): Quantifying binding kinetics between L35 and other ribosomal components or assembly factors.

The choice of method should be guided by the specific research question and available resources, with consideration for R. baltica's unique ribosomal architecture.

What are the common challenges in working with recombinant R. baltica L35 and how can they be addressed?

Researchers working with recombinant R. baltica L35 may encounter several challenges:

• Solubility issues: Ribosomal proteins often have poor solubility when expressed recombinantly. Solutions include:

  • Using solubility-enhancing fusion tags (SUMO, MBP, or GST)

  • Expressing at lower temperatures (16-18°C)

  • Including stabilizing agents like arginine or trehalose in buffers

• Protein instability: L35 may be unstable outside the ribosomal context. Consider:

  • Adding nucleic acids (RNA oligonucleotides) to mimic natural binding partners

  • Optimizing buffer conditions (pH, salt concentration)

  • Including protease inhibitors throughout purification

• Binding partner co-purification: E. coli ribosomal components may co-purify with R. baltica L35. Address by:

  • Using stringent washing conditions during affinity purification

  • Incorporating additional purification steps like ion exchange chromatography

  • Performing RNA digestion if RNA contamination is observed

• Functional assays: Designing relevant assays can be challenging. Options include:

  • In vitro translation assays with reconstituted ribosomes

  • Pre-rRNA processing assays if L35 is involved in ribosome biogenesis

  • Structural studies to compare with other bacterial L35 proteins

How can researchers assess the functionality of recombinant R. baltica L35?

Assessing the functionality of recombinant R. baltica L35 requires specialized approaches:

• RNA binding assays: Using electrophoretic mobility shift assays (EMSA) or filter binding assays to quantify binding to potential rRNA targets.

• In vitro assembly assays: Testing the ability of L35 to incorporate into partially assembled ribosomal subunits.

• Complementation studies: Examining whether R. baltica L35 can functionally replace L35 in other bacterial systems where genetic manipulation is more established.

• Structural integrity verification: Using circular dichroism (CD) spectroscopy or thermal shift assays to confirm proper protein folding.

• Pre-rRNA processing analysis: If L35 is involved in rRNA processing (as suggested by research on L35 in other organisms ), assessing its ability to promote specific processing steps in vitro.

Each of these approaches provides different insights into L35 functionality and should be selected based on the specific research question being addressed.

What are promising areas for future research involving R. baltica L35?

Several promising research directions for R. baltica L35 include:

• Comparative ribosome biology: Investigating how L35's structure and function in R. baltica compare to those in bacteria with different cell architectures, potentially revealing adaptations related to compartmentalized cell organization.

• Stress response mechanisms: Exploring how L35 expression and modification respond to environmental stressors, building on R. baltica's known transcriptional responses to stress conditions .

• Ribosome heterogeneity: Examining whether L35 variants or modifications contribute to ribosome heterogeneity during different growth phases or environmental conditions.

• Structure-function relationships: Determining the specific residues in L35 critical for its function through systematic mutagenesis studies.

• Biotechnological applications: Exploring whether R. baltica L35 has unique properties that could be exploited in biotechnology, similar to other R. baltica proteins with potential biotechnological value .

These research directions would significantly advance our understanding of ribosome biology in Planctomycetes and potentially reveal novel aspects of translation regulation in bacteria with complex cellular organization.

How might understanding R. baltica L35 contribute to broader knowledge of bacterial ribosome diversity?

Research on R. baltica L35 can provide valuable insights into bacterial ribosome diversity in several ways:

• Evolutionary adaptations: As a member of Planctomycetes with unique cellular features, R. baltica may have evolved specific adaptations in its translation machinery, including L35, that differ from those in model bacteria.

• Environmental specialization: R. baltica's adaptation to marine environments may be reflected in structural or functional specializations of its ribosomal proteins, including L35.

• Life cycle regulation: The complex life cycle of R. baltica involves morphological changes and the formation of rosettes in stationary phase . Understanding how L35 functions throughout these transitions could reveal novel regulatory mechanisms of ribosome assembly.

• Compartmentalized translation: R. baltica's intracellular compartmentalization raises questions about ribosome localization and specialized ribosomes that could be addressed through L35 studies.

• Protein-RNA interactions: The study of L35-rRNA interactions in R. baltica could reveal novel binding motifs or interaction patterns not observed in model organisms.

Such knowledge would expand our understanding of bacterial translation machinery beyond well-studied model organisms and potentially reveal new principles of ribosome assembly and function.