Recombinant Rhodopirellula baltica 30S ribosomal protein S19 (rpsS)

What is the functional role of 30S ribosomal protein S19 in Rhodopirellula baltica?

S19 in R. baltica, like its homologs in other bacteria, primarily functions in rRNA binding and contributes to ribosomal structural integrity. Based on studies of S19 in other organisms, it likely participates in forming intersubunit bridges between the 30S and 50S ribosomal subunits . In E. coli, S19 forms part of bridge B1a, which connects to the 23S rRNA of the 50S subunit . This bridge is dynamically rearranged during different stages of translation.

Given R. baltica's unique lifestyle and marine environment adaptation, its S19 might have evolved specialized functions related to salt resistance. Analysis of gene expression patterns during R. baltica's life cycle suggests that ribosomal proteins may play roles in morphological differentiation between motile swarmer cells and sessile adult cells .

What experimental approaches are recommended for recombinant expression of R. baltica S19?

For successful expression of recombinant R. baltica S19:

• Expression system selection: E. coli BL21(DE3) is typically suitable for expressing prokaryotic ribosomal proteins. Consider using pET vector systems with T7 promoters for high-level expression.

• Codon optimization: Although not explicitly mentioned for S19, expression of other R. baltica proteins has benefited from codon optimization for the host expression system. This is particularly important given the GC-rich genome of Planctomycetes.

• Expression conditions:

  • Temperature: 18-25°C typically yields better soluble protein than 37°C

  • IPTG concentration: 0.1-0.5 mM

  • Post-induction time: 4-16 hours

Similar approaches were successfully used for the expression of the R. baltica polysaccharide lyase RB5312, which yielded sufficient protein for crystallization studies .

How can researchers purify recombinant R. baltica S19?

A systematic purification strategy based on successful approaches with other R. baltica proteins includes:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged S19 protein.

• Intermediate purification: Ion-exchange chromatography (typically Q-Sepharose) to separate based on charge properties.

• Polishing step: Size-exclusion chromatography using Superdex 75 or Superdex 200 columns.

For the R. baltica polysaccharide lyase RB5312, researchers successfully employed affinity chromatography followed by gel filtration to achieve protein of sufficient purity for crystallization . This protocol likely needs optimization for S19, but provides a starting point.

What crystallization methods are most effective for structural studies of R. baltica S19?

Based on successful crystallization of other R. baltica proteins:

• Initial screening: Use commercial sparse-matrix screens (Hampton Research, Molecular Dimensions) at protein concentrations of 5-15 mg/ml.

• Optimization technique: The hanging-drop vapor-diffusion method has proven successful for R. baltica proteins . For RB5312, crystals were obtained using this method with drops containing equal volumes of protein and reservoir solution.

• Crystallization conditions: For R. baltica RB5312, crystals belonged to space group P2₁2₁2₁ and diffracted to 1.8 Å resolution . While specific conditions will differ for S19, this suggests that R. baltica proteins can form well-diffracting crystals under appropriate conditions.

• Data collection strategy: Collection temperature of 100 K with appropriate cryoprotectants (typically 20-25% glycerol or ethylene glycol added to mother liquor).

How is S19, along with other ribosomal proteins, regulated during R. baltica's life cycle?

R. baltica undergoes a complex life cycle that includes motile swarmer cells and sessile adult forms. Gene expression analysis reveals that:

• The early exponential growth phase is dominated by swarmer and budding cells.

• The transition phase shows a shift to single cells, budding cells, and rosette formations.

• The stationary phase is dominated by rosette formations .

While S19-specific regulation isn't detailed in the available data, studies of R. baltica's transcriptome throughout its growth cycle reveal that numerous genes, including those encoding ribosomal proteins, show differential expression patterns corresponding to morphological changes . This suggests that S19 expression may be regulated in concert with other ribosomal proteins to support the changing protein synthesis needs during different life cycle stages.

What role might S19 play in R. baltica's adaptation to salt stress?

While specific information about S19's role in salt adaptation is not available, R. baltica demonstrates remarkable salt resistance , an important feature for marine organisms. In other organisms, ribosomal proteins have been implicated in stress responses:

• Gene expression changes: Studies in rice show that RPS genes exhibit significant expression changes under abiotic stress treatments, including salt stress .

• Regulatory elements: Analysis of promoter regions of RPS genes in rice revealed the presence of stress-responsive elements, including those responsive to salt stress .

• Methodological approach: To investigate S19's role in salt adaptation:

  • Conduct expression analysis of rpsS under varying salt concentrations

  • Perform site-directed mutagenesis to identify salt-responsive domains

  • Use recombinant S19 for in vitro salt stability assays

By analogy, R. baltica S19 might contribute to ribosome stability under varying salt conditions in the marine environment.

How does R. baltica S19 contribute to ribosomal bridge formation?

Based on studies in E. coli:

• S19 likely participates in forming intersubunit bridge B1a, connecting the 30S subunit to the 23S rRNA of the 50S subunit .

• This bridge appears to be dynamic, with the 23S rRNA contact site in bridge B1a modeled to differ in different ribosomal states, alternately contacting S13 or S19 .

• In high-resolution ribosome structures, contacts between ribosomal proteins that form bridges (including S19) confirm the dynamic nature of these interactions .

To study the specific contribution of R. baltica S19 to bridge formation:

• Cryo-electron microscopy of R. baltica ribosomes

• Site-directed mutagenesis of predicted bridge-forming residues

• In vitro ribosome assembly assays with wild-type versus mutant S19

What computational approaches can help predict the structure and interactions of R. baltica S19?

In the absence of an experimental structure, computational methods can provide valuable insights:

• Homology modeling: Using E. coli S19 (PDB: 4V4Q) as a template, build a structural model of R. baltica S19 using SWISS-MODEL or Phyre2.

• Molecular dynamics simulations: Simulate the behavior of R. baltica S19 under various conditions, particularly focusing on salt effects given its marine environment.

• Protein-RNA docking: Predict interactions between S19 and rRNA using tools like HADDOCK or NPDock.

• Evolutionary analysis: Perform multiple sequence alignment of S19 across diverse bacteria to identify conserved residues and R. baltica-specific adaptations.

These computational approaches can guide experimental design by identifying key residues for mutagenesis and potential interaction interfaces.

How can researchers assess S19's role in R. baltica's response to various environmental stresses?

Based on methodologies used for studying stress responses in other organisms:

• Gene expression analysis: Quantify rpsS expression levels under various stress conditions (temperature, pH, oxidative stress, nutrient limitation) using RT-qPCR.

• Promoter analysis: Examine the upstream regulatory region of rpsS for stress-responsive elements similar to those found in rice RPS genes, which contain ABREs, DREs, and other stress-responsive elements .

• Functional complementation: Express R. baltica S19 in E. coli with its native S19 knocked out, and assess growth under various stress conditions.

• Protein interaction changes: Use pull-down assays or crosslinking mass spectrometry to identify stress-induced changes in S19's interaction partners.

What potential post-translational modifications might occur in R. baltica S19?

While specific information about post-translational modifications (PTMs) in R. baltica S19 is not available, ribosomal proteins often undergo various PTMs that affect their function:

• Common ribosomal protein PTMs: Look for methylation, acetylation, phosphorylation, and ubiquitination sites using mass spectrometry.

• Marine-specific modifications: Given R. baltica's marine environment, consider unique modifications that might confer salt stability.

• Methodological approach:

  • Express recombinant S19 in R. baltica itself to preserve native PTMs

  • Analyze using high-resolution mass spectrometry

  • Compare PTM patterns under different growth and stress conditions

How does the gene organization of rpsS in R. baltica compare to other bacteria?

While specific information about the genomic context of rpsS in R. baltica is not provided in the search results, general analyses of ribosomal protein genes can guide research:

• Genomic context analysis: In many bacteria, rpsS is part of the spc operon. Researchers should determine whether R. baltica follows this organization or has a unique arrangement.

• Regulatory element identification: Analyze the promoter region for transcription factor binding sites, particularly those related to growth phase regulation and stress response.

• Comparative genomics approach: Compare the genomic neighborhood of rpsS across multiple Planctomycetes to identify conserved synteny or R. baltica-specific arrangements.

What techniques can be used to study the interaction between R. baltica S19 and rRNA?

To characterize the interaction between recombinant R. baltica S19 and rRNA:

• RNA binding assays:

  • Electrophoretic mobility shift assay (EMSA)

  • Filter binding assay

  • Surface plasmon resonance (SPR)

• Structural approaches:

  • X-ray crystallography of S19-rRNA complex

  • Cryo-electron microscopy

  • Nuclear magnetic resonance (NMR) for dynamics

• Crosslinking methods:

  • UV crosslinking followed by mass spectrometry

  • Chemical crosslinking with specific reagents that target RNA-protein interfaces

• Mutagenesis studies:

  • Site-directed mutagenesis of predicted RNA-binding residues

  • Truncation analysis to identify minimal binding domains

These approaches can reveal the specific nucleotides and amino acids involved in the interaction, as well as the binding kinetics and thermodynamics.

How can researchers develop antibodies against R. baltica S19 for localization studies?

For generating specific antibodies against R. baltica S19:

• Antigen preparation: Use purified recombinant S19 or synthesized peptides corresponding to unique, surface-exposed regions of the protein.

• Antibody production strategy:

  • Polyclonal antibodies: Immunize rabbits with full-length protein

  • Monoclonal antibodies: Use peptide antigens for mouse immunization

  • Recombinant antibodies: Phage display selection against purified S19

• Validation methods:

  • Western blotting against recombinant S19 and R. baltica cell lysates

  • Immunoprecipitation followed by mass spectrometry

  • Preabsorption controls with recombinant protein

• Application in localization studies:

  • Immunofluorescence microscopy to track S19 throughout the R. baltica life cycle

  • Immunoelectron microscopy for precise subcellular localization

  • Chromatin immunoprecipitation (ChIP) if S19 has potential extraribosomal functions

What can functional genomics approaches reveal about S19's role in R. baltica?

Advanced functional genomics strategies can provide comprehensive insights:

• CRISPR-Cas9 genome editing: Generate knockdown or conditional mutants of rpsS to observe phenotypic effects.

• RNA-Seq analysis: Compare transcriptomes with normal versus reduced S19 levels to identify affected pathways.

• Ribosome profiling: Assess how S19 alterations affect translation efficiency and specificity.

• Protein-protein interaction network mapping: Use techniques like BioID or proximity labeling to identify S19 interaction partners beyond the ribosome.

• Comparative genomics: Analyze rpsS sequences across diverse planctomycetes to identify conserved and variable regions that might indicate specialized functions.

These approaches can reveal unexpected roles of S19 beyond its canonical function in translation, potentially including regulatory functions in R. baltica's unique life cycle or stress responses.

How does the amino acid sequence of R. baltica S19 compare to homologs in other bacteria?

While specific sequence data for R. baltica S19 isn't provided in the search results, researchers can:

• Perform multiple sequence alignment of S19 proteins from diverse bacteria, including:

  • Model organisms (E. coli, B. subtilis)

  • Other Planctomycetes

  • Marine bacteria from different phyla

• Identify R. baltica-specific features such as:

  • Unique residues in RNA-binding regions

  • Insertions or deletions relative to other bacteria

  • Amino acid composition changes that might reflect adaptation to marine environments

• Analyze selection pressures using dN/dS ratios to identify regions under purifying or positive selection.

This comparative approach can reveal evolutionary adaptations potentially related to R. baltica's unique cellular biology and environmental niche.

What biophysical methods can assess the stability of R. baltica S19 under marine conditions?

To characterize the physical properties of recombinant S19 under conditions relevant to R. baltica's marine environment:

• Thermal stability assays:

  • Differential scanning calorimetry (DSC)

  • Differential scanning fluorimetry (DSF/Thermofluor)

  • Circular dichroism (CD) thermal melts

• Salt stability analysis:

  • Measure stability across a range of salt concentrations (0-1M NaCl)

  • Compare different salt types (NaCl, KCl, MgCl₂)

  • Assess protein solubility and activity under varying ionic conditions

• Structural characterization:

  • Small-angle X-ray scattering (SAXS) to analyze shape in solution

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe dynamics

  • NMR for atomic-level dynamics in different salt environments

These approaches can reveal molecular adaptations that allow R. baltica S19 to function optimally in its natural marine habitat.