Recombinant Rhodopirellula baltica 30S ribosomal protein S3 (rpsC)

What is Rhodopirellula baltica and why is it significant for studying ribosomal proteins?

Rhodopirellula baltica (R. baltica) is a member of the globally distributed bacterial phylum Planctomycetes, first isolated from the Kiel Fjord in the Baltic Sea. This organism has gained scientific interest due to its unique cell biology, including peptidoglycan-free proteinaceous cell walls, intracellular compartmentalization, and a distinctive reproductive cycle involving budding that resembles that of Caulobacter crescentus .

The complete genome sequencing of R. baltica has revealed numerous unique features including an abundance of sulfatase genes, specialized carbohydrate-active enzymes, and distinctive C1-metabolism pathways . This bacterium transitions through different morphological states during its life cycle, from motile swarmer cells to sessile cells forming rosettes, making it an excellent model for studying how ribosomal proteins might be differentially regulated during cellular differentiation .

What are the known functions of bacterial 30S ribosomal protein S3?

The 30S ribosomal protein S3 (rpsC) is a multifunctional component of the small ribosomal subunit in bacteria. While its primary role involves participation in protein translation as part of the ribosome, extensive research on ribosomal proteins has revealed numerous extraribosomal functions:

• Translational role: As part of the 30S ribosomal subunit, S3 helps in mRNA binding and contributes to the decoding center functionality.

• DNA repair activity: Similar to its eukaryotic counterpart, bacterial S3 likely exhibits endonuclease activity that can contribute to DNA repair mechanisms, particularly for damaged DNA containing 8-oxoguanine .

• Regulatory functions: Evidence suggests S3 may participate in transcriptional regulation and potentially interact with various cellular signaling pathways .

• Stress response: Ribosomal proteins often play roles in bacterial adaptation to environmental stresses, with altered expression patterns under different growth conditions .

How does R. baltica's genome organization impact its ribosomal protein genes?

R. baltica possesses a distinctive genome organization that affects its ribosomal protein genes. Interestingly, R. baltica has two genes encoding DnaA (RB11579 and RB1706), the protein initiating DNA replication . This genomic feature might have implications for the regulation of ribosomal protein genes, including rpsC, especially during the organism's complex life cycle.

The expression of ribosomal proteins in R. baltica, like rpsC, likely varies across growth phases. Transcriptomic studies have demonstrated that R. baltica significantly reorganizes its gene expression profile when transitioning between exponential growth, transition phase, and stationary phase . These changes affect various cellular processes including energy production, amino acid biosynthesis, signal transduction, and stress response mechanisms that could influence ribosomal protein function.

What methods are optimal for recombinant expression and purification of R. baltica rpsC?

Based on successful approaches with other R. baltica proteins, the following methodology is recommended for recombinant expression and purification of rpsC:

Expression system selection:

• E. coli BL21(DE3) or similar strain is typically appropriate for ribosomal protein expression

• Expression vectors containing T7 promoter (pET series) with appropriate fusion tags (His-tag or GST) facilitate purification

• Codon optimization may be necessary due to potential codon usage differences between R. baltica and E. coli

Expression conditions:

• IPTG induction at lower temperatures (16-20°C) often improves solubility

• Induction at OD600 of 0.6-0.8 with 0.1-0.5 mM IPTG

• Extended expression time (overnight) at reduced temperature may increase yield of properly folded protein

• Inclusion of osmolytes or specific additives may improve protein solubility

Purification strategy:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Ion-exchange chromatography for further purification

• Size-exclusion chromatography as a final polishing step

• Buffer optimization to maintain protein stability (typically including 10-20 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl, and potentially 5-10% glycerol as stabilizer)

The polysaccharide lyase RB5312 from R. baltica has been successfully expressed, purified and crystallized, suggesting that similar approaches might work for other R. baltica proteins including rpsC .

What crystallization approaches have proven successful for R. baltica proteins?

The crystallization of R. baltica proteins has been achieved using the hanging-drop vapor-diffusion method, as demonstrated with the polysaccharide lyase RB5312 . Based on this precedent, the following approaches may be effective for rpsC crystallization:

Crystallization conditions:

• Protein concentration: 10-15 mg/mL in a suitable buffer (typically 20 mM Tris-HCl pH 7.5, 100-150 mM NaCl)

• Temperature: 18-20°C

• Screening methods: Commercial sparse matrix screens (Hampton Research, Molecular Dimensions) for initial condition identification

• Optimization: Fine-tuning of precipitant concentration, pH, and additives for crystal quality improvement

Successful conditions for R. baltica RB5312 that might inform rpsC approaches:

• The crystals of RB5312 belonged to space group P2₁2₁2₁

• Unit-cell parameters: a = 39.05, b = 144 (unit-cell parameters as reported in the literature)

• Resolution of 1.8 Å was achieved, suggesting high-quality diffraction is possible with R. baltica proteins

Optimization strategies:

• Microseeding to improve crystal nucleation and growth

• Additive screening to enhance crystal quality

• Surface entropy reduction through targeted mutations if crystallization proves challenging

How can researchers analyze the post-translational modifications of R. baltica rpsC?

Analysis of post-translational modifications (PTMs) of R. baltica rpsC requires a multi-faceted approach:

Mass spectrometry-based methods:

• Bottom-up proteomics: Digestion of rpsC with proteases followed by LC-MS/MS analysis to identify modified peptides

• Top-down proteomics: Analysis of intact rpsC to determine the total pattern of modifications

• Multiple Reaction Monitoring (MRM): Targeted approach for specific modifications of interest

Expected modifications to investigate:

• Phosphorylation: Based on eukaryotic RPS3 studies, phosphorylation may occur at serine, threonine, and tyrosine residues. For example, eukaryotic RPS3 is phosphorylated at S209 by IKKβ, at T42 by ERK1, at S6 and T221 by PKCδ, and at T70 by Akt .

• Methylation and acetylation: Common modifications in bacterial ribosomal proteins

• Ubiquitination: Less common in bacteria but may be relevant for degradation studies

Modification-specific enrichment strategies:

• Phosphopeptide enrichment using TiO₂ or IMAC

• Antibody-based enrichment for specific modifications

• Chemical labeling approaches for cysteine modifications

Functional validation:

• Site-directed mutagenesis of identified PTM sites

• In vitro modification assays with relevant kinases or other enzymes

• Functional assays to determine the impact of modifications on rpsC activity

How can the extraribosomal functions of R. baltica rpsC be experimentally verified?

Investigating the extraribosomal functions of R. baltica rpsC requires multiple complementary approaches:

DNA repair activity assessment:

• In vitro endonuclease assays:

  • Prepare DNA substrates containing specific damages (8-oxoguanine, AP sites)

  • Incubate with purified recombinant rpsC

  • Analyze cleavage products by gel electrophoresis

• Cellular DNA repair assays:

  • Complement E. coli or other bacterial DNA repair mutants with R. baltica rpsC

  • Measure survival or mutation rates after exposure to DNA damaging agents

Protein-protein interaction studies:

• Pull-down assays: Using tagged recombinant rpsC to identify interacting partners

• Yeast two-hybrid screening: To identify potential interaction partners

• Co-immunoprecipitation: If antibodies against rpsC are available

• Bacterial two-hybrid systems: More appropriate for bacterial protein interactions

Transcriptional regulation analysis:

• Chromatin immunoprecipitation (if applicable): To identify DNA binding sites

• Electrophoretic mobility shift assays: To assess direct DNA binding

• Reporter gene assays: To evaluate the impact on gene expression

Subcellular localization studies:

• Fractionation experiments: To determine rpsC distribution in different cellular compartments

• Fluorescence microscopy: Using fluorescently tagged rpsC to visualize localization

By focusing on these extraribosomal functions, researchers can draw parallels to the known activities of eukaryotic RPS3, which regulates DNA repair, apoptosis, and innate immune responses .

What role might R. baltica rpsC play in the organism's stress response mechanisms?

R. baltica transitions through distinct morphological stages during its life cycle and in response to environmental changes, suggesting a complex stress response network. The role of rpsC in these processes can be investigated through:

Transcriptomic analysis:

• RNA-seq under various stress conditions (nutrient limitation, temperature, salinity, oxidative stress)

• Quantitative RT-PCR to validate expression changes of rpsC

• Correlation of rpsC expression with known stress response genes

R. baltica exhibits significant transcriptional reorganization when transitioning between growth phases, with numerous changes in gene expression related to stress response . During transition to stationary phase, R. baltica activates genes for stress response and protein folding while repressing genes for carbon metabolism and translation .

Proteomic approaches:

• Quantitative proteomics to assess rpsC protein levels under different stresses

• Protein-protein interaction studies under stress conditions

• Post-translational modification analysis during stress response

Phenotypic studies:

• Creation of rpsC mutants or knockdowns (if genetic tools are available for R. baltica)

• Comparison of growth characteristics under stress conditions

• Morphological analysis throughout life cycle stages

Comparative analysis:

• Examination of rpsC function in related Planctomycetes

• Comparison with stress-related functions of RPS3 in other organisms

Based on known adaptations in R. baltica, potential stress-related functions of rpsC might include:

• Adaptation to changing nutrient availability during different growth phases

• Response to oxidative stress (potentially linked to DNA repair functions)

• Roles in cell morphology changes between swarmer cells and rosette formation

• Participation in the reorganization of cellular compartments under stress conditions

How does R. baltica rpsC contribute to the organism's unique cell cycle and morphological transitions?

R. baltica undergoes a complex life cycle involving transitions between motile swarmer cells, budding cells, and sessile rosettes . The potential contributions of rpsC to these processes can be investigated through:

Expression pattern analysis:

• Quantification of rpsC expression throughout the cell cycle

• Correlation with cell cycle-regulated genes

• Comparison with rpsC expression in related organisms with different life cycles

Localization studies:

• Tracking of fluorescently tagged rpsC during cell division and differentiation

• Co-localization with cell cycle regulators or cytoskeletal elements

• Examination of potential rpsC redistribution during budding

Functional genomics approaches:

• Conditional depletion of rpsC to assess effects on cell cycle progression

• Overexpression studies to identify potential gain-of-function phenotypes

• Genetic interaction studies with known cell cycle regulators

Comparative cell biology:

• Analysis of rpsC function in comparison to Caulobacter crescentus, another budding bacterium

• Examination of potential interactions with DnaA proteins (RB11579 and RB1706), which R. baltica uniquely possesses two copies of

The presence of micro-compartment proteins (RB2585 and RB2586) that form primitive organelles in R. baltica may indicate specialized subcellular organization , potentially involving ribosomal proteins like rpsC in unique structural or regulatory roles.

How does R. baltica rpsC differ structurally and functionally from ribosomal protein S3 in other bacterial species?

A comprehensive comparative analysis reveals several key differences and similarities:

Sequence analysis:

• Multiple sequence alignment of rpsC proteins from diverse bacterial phyla

• Identification of R. baltica-specific sequence features

• Conservation analysis of functional domains and motifs

Structural comparisons:

• Homology modeling of R. baltica rpsC based on existing bacterial S3 structures

• Mapping of distinctive residues onto the structural model

• Analysis of potential structural determinants of specialized functions

Functional domain analysis:

• Assessment of the conservation of known functional domains:

  • KH domain (K homology domain) present in eukaryotic RPS3 for nucleic acid binding

  • Endonuclease domain for DNA repair functions

  • Protein-protein interaction interfaces

Phylogenetic reconstruction:

• Construction of phylogenetic trees to place R. baltica rpsC in evolutionary context

• Correlation of sequence/structural changes with taxonomic divergence

• Analysis of selection pressure on different domains

The unique phylogenetic position of Planctomycetes and R. baltica's specialized lifestyle suggest that its rpsC may have evolved distinctive features compared to other bacterial lineages, potentially including specialized extraribosomal functions adapted to its marine environment and complex life cycle.

What insights can genomic context analysis provide about the evolution and function of R. baltica rpsC?

Genomic context analysis offers valuable insights into the evolution and function of R. baltica rpsC:

Operon structure and gene neighborhood analysis:

• Identification of genes co-transcribed with rpsC

• Comparison of rpsC genomic context across bacterial species

• Detection of potential regulatory elements in promoter regions

Regulatory network reconstruction:

• Identification of transcription factors potentially regulating rpsC

• Integration with expression data across growth conditions

• Correlation with other ribosomal protein genes and potential extraribosomal partners

Horizontal gene transfer assessment:

• Analysis of GC content, codon usage, and other sequence characteristics

• Identification of potential gene transfer events in rpsC evolutionary history

• Examination of taxonomically restricted sequence features

Duplication and diversification patterns:

• Investigation of potential gene duplication events

• Analysis of paralogs and their functional diversification

• Correlation with the presence of duplicated genes like DnaA in R. baltica

These genomic analyses may reveal unique adaptations of rpsC within the context of R. baltica's specialized environmental niche and cellular biology, potentially linking to the organism's adaptation to marine environments and unusual cell biology.

How can comparative studies of rpsC contribute to understanding bacterial adaptation to marine environments?

Comparative studies of rpsC across marine bacteria can provide insights into environmental adaptation:

Multi-species comparison:

• Analysis of rpsC sequences from diverse marine bacteria

• Identification of convergent adaptations in marine lineages

• Correlation of sequence features with habitat-specific factors (depth, salinity, temperature)

Environment-specific expression patterns:

• Meta-transcriptomic analysis of rpsC expression in different marine environments

• Correlation with environmental parameters

• Identification of condition-specific regulation patterns

Functional adaptations to marine conditions:

• Analysis of structural adaptations that might enhance protein stability in marine environments

• Investigation of potential salt-bridge formation and hydrophobic interactions

• Comparison with terrestrial bacterial counterparts

Experimental validation:

• Heterologous expression of rpsC variants from different marine bacteria

• Comparative stability and activity assays under varying salt concentrations

• Site-directed mutagenesis to test the significance of marine-specific residues

These analyses may reveal how ribosomal proteins like rpsC have adapted to function optimally in marine environments, potentially contributing to the ecological success of bacteria like R. baltica in oceanic habitats.

How can recombinant R. baltica rpsC be utilized to study ribosomal assembly and function?

Recombinant R. baltica rpsC offers multiple research applications for studying ribosomal assembly and function:

In vitro ribosome assembly studies:

• Incorporation of labeled recombinant rpsC into partial or complete ribosomal assembly reactions

• Time-course analysis of assembly intermediates

• Comparison with assembly pathways in model organisms

Structure-function relationships:

• Site-directed mutagenesis of key residues to assess their roles in ribosome function

• Complementation studies in heterologous systems

• In vitro translation assays with reconstituted ribosomes containing mutant rpsC variants

Ribosomal interaction network mapping:

• Crosslinking studies to identify rpsC interaction partners within the ribosome

• Mass spectrometry analysis of crosslinked complexes

• Validation of interactions through mutagenesis and functional assays

Comparative ribosome biology:

• Assessment of functional interchange between rpsC from R. baltica and other bacterial species

• Analysis of chimeric proteins to map domain-specific functions

• Investigation of potential specializations related to R. baltica's unique biology

What approaches can be used to investigate potential interactions between R. baltica rpsC and non-ribosomal proteins?

Investigating non-ribosomal interactions of R. baltica rpsC requires multiple complementary approaches:

Protein-protein interaction screening:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged rpsC in native or heterologous systems

  • Purify complexes and identify interacting partners by MS

  • Validate interactions through reciprocal pulldowns

• Bacterial two-hybrid assays:

  • Screen for interactions using rpsC as bait

  • Validate positive interactions with secondary assays

  • Map interaction domains through truncation constructs

• Proximity labeling approaches:

  • Express rpsC fused to BioID or APEX2

  • Identify proximal proteins through biotinylation and pulldown

  • Distinguish between stable and transient interactions

Functional validation of interactions:

• Co-expression studies to assess functional relationships

• Competition assays to map binding interfaces

• Mutagenesis of key residues to disrupt specific interactions

Eukaryotic RPS3 interacts with various proteins including NF-κB pathway components (p65 subunit), IKKβ, importin-α, ERK1, PKCδ, Akt, E2F1, and NM23-H1 . While bacterial systems differ significantly, these known interactions provide a framework for investigating potential non-canonical functions of bacterial rpsC.

How can structural biology approaches enhance our understanding of R. baltica rpsC function?

Advanced structural biology approaches offer powerful insights into rpsC function:

X-ray crystallography:

• Crystallization of recombinant R. baltica rpsC alone and in complex with interaction partners

• Structure determination at high resolution

• Comparison with existing ribosomal protein structures

The successful crystallization of the R. baltica polysaccharide lyase RB5312 suggests that crystallographic approaches are viable for R. baltica proteins . The crystals of RB5312 diffracted to 1.8 Å resolution, indicating the potential for high-quality structural data from R. baltica proteins.

Cryo-electron microscopy:

• Structure determination of rpsC within intact R. baltica ribosomes

• Visualization of conformational changes under different conditions

• Analysis of rpsC positioning relative to functional sites in the ribosome

Nuclear Magnetic Resonance (NMR) spectroscopy:

• Structure determination of isolated rpsC domains

• Analysis of protein dynamics in solution

• Investigation of interaction interfaces with binding partners

Integrative structural biology approaches:

• Combination of crystallography, cryo-EM, and computational modeling

• Small-angle X-ray scattering (SAXS) for solution structure analysis

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics and interaction mapping

These structural approaches can reveal the molecular basis for both the canonical ribosomal functions of rpsC and its potential extraribosomal activities, contributing to a comprehensive understanding of this multifunctional protein in R. baltica.