Recombinant Rhodopirellula baltica 50S ribosomal protein L25 (rplY)

What is the 50S ribosomal protein L25 in Rhodopirellula baltica and how does it compare to homologs in other bacteria?

The 50S ribosomal protein L25 in R. baltica is a component of the large ribosomal subunit involved in protein synthesis. By comparison with better-characterized bacterial homologs like the one in Escherichia coli, L25 likely functions as an rRNA-binding protein that specifically interacts with 5S rRNA to form a stable complex . In E. coli, L25 works alongside L18 and L5 to form a separate domain within the bacterial ribosome . The R. baltica homolog may have evolved specific adaptations related to the organism's marine environment and unique cellular organization, potentially contributing to its distinctive life cycle and morphological changes during growth phases.

What is known about the expression pattern of the rplY gene during R. baltica's life cycle?

The expression of ribosomal proteins in R. baltica, including potentially the rplY gene, shows distinct patterns throughout its growth cycle. While the search results don't specifically mention rplY expression, general trends indicate that genes associated with translation and ribosomal functions tend to be downregulated during the transition from exponential to stationary phase . During early exponential growth, when the culture is dominated by swarmer and budding cells, ribosomal genes are typically highly expressed to support rapid growth and protein synthesis. As cells transition to stationary phase, forming rosette structures, many of these genes are repressed as metabolic activity decreases in response to nutrient limitation .

What cellular localization and structural features characterize the R. baltica L25 protein?

Based on knowledge of homologous proteins, the R. baltica L25 is likely localized within the large subunit of the ribosome, specifically in the region where it interacts with 5S rRNA. While specific structural data for R. baltica L25 is not directly provided in the search results, it likely shares structural features with other bacterial L25 proteins, including RNA-binding domains that facilitate interaction with 5S rRNA and possibly other ribosomal proteins. The precise three-dimensional structure would require crystallographic or other structural biology techniques to determine, similar to the approaches used for other R. baltica proteins such as the polysaccharide lyase RB5312 .

What are the optimal conditions for recombinant expression of R. baltica 50S ribosomal protein L25?

For recombinant expression of R. baltica L25, researchers can adapt methods similar to those used for other R. baltica proteins. Based on successful approaches with other recombinant proteins from this organism, expression in an E. coli system using vectors like pET or pGEX would be appropriate. The expression protocol should account for the marine origin of R. baltica, potentially incorporating:

• Temperature optimization: 18-25°C induction temperature to ensure proper folding

• Salt concentration: 0.3-0.5M NaCl to mimic marine conditions

• Induction parameters: 0.1-0.5mM IPTG for 4-16 hours

• Host strain selection: E. coli BL21(DE3) or Rosetta strains for rare codon optimization

Expression levels should be monitored via SDS-PAGE at different timepoints post-induction to determine optimal harvest time. For proteins like the R. baltica polysaccharide lyase RB5312, recombinant expression has been successfully achieved, suggesting similar approaches may work for L25 .

What purification strategies yield the highest purity and activity for recombinant R. baltica L25 protein?

A multi-step purification strategy is recommended to obtain high-purity recombinant R. baltica L25:

• Initial capture: Affinity chromatography using histidine or GST tags

• Intermediate purification: Ion exchange chromatography (typically cation exchange for basic ribosomal proteins)

• Polishing: Size exclusion chromatography to separate monomeric protein from aggregates

Tag removal using TEV or thrombin protease should be performed if the tag might interfere with downstream applications. Quality control by mass spectrometry and activity assays (RNA binding) should be conducted to ensure proper folding and function. Similar approaches have been successful for other recombinant proteins from R. baltica .

How can the RNA-binding activity of purified R. baltica L25 be verified?

To verify the RNA-binding activity of purified R. baltica L25:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Incubate purified L25 with in vitro transcribed 5S rRNA

  • Analyze complex formation by native gel electrophoresis

  • Visualize with SYBR Green (RNA) and Coomassie (protein)

• Surface Plasmon Resonance (SPR):

  • Immobilize 5S rRNA on a sensor chip

  • Flow L25 protein at various concentrations

  • Determine association/dissociation constants

• Filter-Binding Assay:

  • Radiolabel 5S rRNA with 32P

  • Incubate with increasing concentrations of L25

  • Filter through nitrocellulose membrane and quantify bound RNA

Based on knowledge of E. coli L25, the R. baltica protein should form a stable complex with 5S rRNA and potentially interact with homologs of L16 and other ribosomal components . Confirmation of these interactions would provide evidence of proper folding and biological activity.

What crystallization conditions have been successful for R. baltica ribosomal proteins?

While specific crystallization conditions for R. baltica L25 are not detailed in the search results, successful crystallization of other R. baltica proteins provides useful guidance. For the polysaccharide lyase RB5312, the hanging-drop vapor-diffusion method was successfully employed . Based on this precedent and general approaches for ribosomal proteins:

• Initial screening:

  • Commercial sparse matrix screens (Hampton Research, Molecular Dimensions)

  • Temperature range: 4-20°C

  • Protein concentration: 5-15 mg/ml

  • Drop ratio: 1:1 (protein:precipitant)

• Optimization parameters:

  • pH range: 6.5-8.5

  • Salt concentration: 0.1-0.5M

  • PEG concentration variations

  • Additives: divalent cations (Mg2+), RNA oligonucleotides

For RB5312, crystals belonging to space group P212121 were obtained, with unit-cell parameters a = 39.05, b = 144.05, c = 153.97 Å, α = β = γ = 90°, diffracting to 1.8 Å resolution . This suggests that R. baltica proteins can be successfully crystallized for high-resolution structural studies.

How does the gene expression of rplY change during different growth phases in R. baltica?

The expression of ribosomal protein genes, potentially including rplY, undergoes significant regulation during R. baltica's growth cycle. While the search results don't specifically track rplY expression, general patterns of ribosomal protein gene expression in R. baltica indicate:

• Early exponential phase (dominated by swarmer and budding cells):

  • High expression of genes for 'DNA replication and recombination'[L]

  • Elevated expression of translation-related genes

• Mid-exponential phase (62h vs. 44h comparison):

  • Downregulation of genes for energy production and conservation

  • Decreased expression of genes for DNA replication and recombination

• Transition to stationary phase:

  • Shift toward stress response

  • Induction of genes for protein folding and stress response proteins

  • Repression of genes for translation control

This pattern suggests that rplY expression likely follows the general trend of ribosomal proteins, with highest expression during early growth phases and downregulation as cells enter stationary phase, when rosette formations dominate the culture.

What post-translational modifications have been identified in R. baltica L25 protein?

The search results don't provide specific information about post-translational modifications (PTMs) in R. baltica L25. To identify potential PTMs:

• Mass spectrometry approaches:

  • Bottom-up proteomics with tryptic digestion

  • Top-down proteomics of intact protein

  • Targeted analysis for specific modifications (phosphorylation, methylation)

• Site-directed mutagenesis:

  • Mutate potential modification sites

  • Assess impact on function and localization

• Modification-specific antibodies:

  • Western blotting with antibodies against common PTMs

  • Immunoprecipitation followed by mass spectrometry

Based on knowledge of bacterial ribosomal proteins, potential modifications could include methylation, acetylation, or phosphorylation, which might regulate ribosome assembly or function during different growth phases or stress conditions.

How can recombinant R. baltica L25 be used to study the organism's unique ribosome assembly?

Recombinant R. baltica L25 provides a valuable tool for investigating ribosome assembly in this organism with its unique cellular compartmentalization:

• In vitro reconstitution studies:

  • Combine purified R. baltica ribosomal components

  • Monitor assembly intermediates by sucrose gradient centrifugation

  • Compare with reconstitution of E. coli ribosomes to identify unique features

• Pull-down assays:

  • Immobilize tagged L25

  • Identify interacting partners from R. baltica lysates

  • Characterize unique interactions not found in model organisms

• Fluorescence-based approaches:

  • FRET studies with labeled L25 and 5S rRNA

  • Real-time monitoring of assembly kinetics

  • Competition assays with other ribosomal proteins

These approaches could reveal how R. baltica's ribosome assembly might be adapted to its marine environment and unique cellular organization, potentially identifying novel interactions that contribute to its distinctive life cycle features described in the search results .

What insights can comparative studies of L25 from R. baltica and other bacteria provide about ribosomal evolution?

Comparative studies of L25 from R. baltica and other bacteria can provide valuable insights into ribosomal evolution:

• Sequence-based analyses:

  • Multiple sequence alignment of L25 from diverse bacterial phyla

  • Identification of conserved and variable regions

  • Phylogenetic tree construction to trace evolutionary relationships

• Structural comparisons:

  • Superimposition of determined or predicted structures

  • Identification of structural adaptations in different environments

  • Correlation with 5S rRNA binding specificity

• Functional complementation:

  • Expression of R. baltica L25 in E. coli L25 deletion strains

  • Assessment of growth phenotypes and ribosome assembly

  • Identification of species-specific functions

These studies could reveal how L25 has evolved in the Planctomycetes phylum, which has unique features such as peptidoglycan-free cell walls and intracellular compartmentalization . Since L25 in E. coli is non-essential , its role might have diverged in R. baltica to accommodate the organism's unique cellular organization and marine lifestyle.

How does the structure-function relationship of R. baltica L25 relate to the organism's adaptation to marine environments?

The structure-function relationship of R. baltica L25 may reflect adaptations to marine environments:

• Salt tolerance mechanisms:

  • Surface charge distribution analysis for halophilic adaptations

  • Ion-binding sites for stabilization in high-salt conditions

  • Comparative analysis with L25 from non-marine bacteria

• Temperature adaptations:

  • Stability analyses at various temperatures

  • Identification of structural features conferring cold tolerance

  • RNA-binding activity assessment under marine-relevant conditions

• Protein-protein interaction network:

  • Yeast two-hybrid or bacterial two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Identification of unique interactions compared to model organisms

R. baltica demonstrates salt resistance and adaptability to marine conditions , which may be reflected in the properties of its ribosomal proteins, including L25. Structural features that enhance stability in fluctuating marine environments could provide insights into how this organism has adapted its translational machinery to its ecological niche.

What genomic context surrounds the rplY gene in R. baltica compared to other bacteria?

The genomic context of the rplY gene can provide insights into its regulation and potential operonic structure:

• Comparative genomic analysis:

  • Identify genes flanking rplY in R. baltica

  • Compare with gene arrangements in other bacteria

  • Identify conserved and divergent patterns

• Transcriptomic data integration:

  • Analyze co-expression patterns of rplY and neighboring genes

  • Identify potential operonic structures

  • Compare with known ribosomal protein operons in model organisms

• Promoter and terminator prediction:

  • Identify regulatory elements upstream of rplY

  • Compare with consensus sequences from other bacteria

  • Predict transcriptional units

While the search results don't provide specific information about the genomic context of rplY in R. baltica, it's worth noting that the genome of R. baltica has relatively few operon structures compared to other bacteria , which might indicate different regulatory mechanisms for ribosomal protein genes in this organism.

How do the RNA-binding properties of R. baltica L25 differ from those of other bacterial homologs?

The RNA-binding properties of R. baltica L25 may differ from other bacterial homologs:

To investigate these differences:

• In vitro binding assays:

  • Compare binding affinities to homologous and heterologous 5S rRNAs

  • Determine specificity using mutated RNA constructs

  • Measure binding kinetics under various salt and temperature conditions

• Structural studies:

  • NMR or X-ray crystallography of L25-RNA complexes

  • Identify specific residues involved in RNA recognition

  • Compare binding interfaces across species

These analyses could reveal adaptations in RNA recognition that reflect R. baltica's evolutionary history and environmental adaptations.

What functional redundancies exist between L25 and other ribosomal proteins in R. baltica?

Functional redundancies between L25 and other ribosomal proteins in R. baltica could be investigated through:

• Computational analyses:

  • Structural homology with other R. baltica ribosomal proteins

  • Identification of proteins with similar RNA-binding domains

  • Prediction of overlapping functions based on structural features

• Genetic approaches:

  • Generation of conditional or complete knockouts

  • Identification of suppressor mutations that compensate for L25 deficiency

  • Transcriptomic analysis to identify compensatory expression changes

• Biochemical studies:

  • Competition assays for 5S rRNA binding

  • Reconstitution experiments with various protein combinations

  • Identification of proteins that can functionally substitute for L25

Based on E. coli studies where L25 is non-essential , R. baltica likely has compensatory mechanisms or functional redundancies that can maintain ribosome assembly and function in the absence of L25. Identifying these mechanisms could provide insights into the flexibility and robustness of the translation machinery in Planctomycetes.

What strategies can overcome solubility issues when expressing recombinant R. baltica L25?

Solubility challenges with recombinant R. baltica L25 can be addressed through:

• Expression optimization:

  • Lower induction temperature (16-18°C)

  • Reduced IPTG concentration (0.1-0.2mM)

  • Co-expression with chaperones (GroEL/ES, DnaK/J)

  • Use of solubility-enhancing fusion tags (SUMO, MBP, TrxA)

• Buffer optimization:

  • Incorporation of stabilizing agents (glycerol, arginine, trehalose)

  • Addition of salt concentrations mimicking marine environment (0.3-0.5M NaCl)

  • Testing different pH ranges (6.5-8.5)

  • Inclusion of reducing agents (DTT, β-mercaptoethanol)

• Refolding approaches:

  • Isolation of inclusion bodies under denaturing conditions

  • Stepwise dialysis for gradual removal of denaturants

  • On-column refolding during affinity purification

  • Pulsed renaturation with redox pairs

Similar approaches have been successfully applied to other R. baltica proteins, such as the polysaccharide lyase RB5312, which was successfully expressed in recombinant form and crystallized .

How can researchers distinguish between functional and non-functional forms of purified R. baltica L25?

To distinguish between functional and non-functional forms of purified R. baltica L25:

• Functional assays:

  • 5S rRNA binding (EMSA, filter binding, SPR)

  • Integration into partial ribosomal subunits

  • Protection of specific nucleotides in 5S rRNA from chemical modification

• Structural characterization:

  • Circular dichroism spectroscopy to assess secondary structure

  • Intrinsic fluorescence to monitor tertiary structure

  • Thermal shift assays to determine stability

  • Limited proteolysis to identify well-folded domains

• Quality control metrics:

  • Size exclusion chromatography to assess aggregation state

  • Dynamic light scattering for homogeneity

  • Mass spectrometry for intact mass and modifications

These approaches help ensure that in vitro studies are conducted with properly folded, biologically relevant protein conformations.

What are common pitfalls in structural studies of R. baltica ribosomal proteins and how can they be avoided?

Common pitfalls in structural studies of R. baltica ribosomal proteins include:

• Sample heterogeneity issues:

  • Pitfall: Multiple conformations or oligomeric states

  • Solution: Rigorous purification (multi-step chromatography)

  • Implementation: SEC-MALS to confirm homogeneity before crystallization attempts

• RNA contamination:

  • Pitfall: Co-purification of endogenous RNA

  • Solution: High-salt washes and nuclease treatment

  • Implementation: Monitor A260/A280 ratio; treat with RNase if necessary

• Crystallization challenges:

  • Pitfall: Poor crystal quality or no crystallization

  • Solution: Crystallization with binding partners (5S rRNA fragments)

  • Implementation: Screen various constructs with different boundaries

• Phase determination:

  • Pitfall: Lack of suitable molecular replacement models

  • Solution: Produce selenomethionine-labeled protein

  • Implementation: SAD/MAD phasing approaches

The successful crystallization of other R. baltica proteins, such as the polysaccharide lyase RB5312 which diffracted to 1.8 Å resolution , demonstrates that high-quality structural data can be obtained for proteins from this organism with proper attention to sample preparation and crystallization conditions.