Recombinant Rhodopirellula baltica 30S ribosomal protein S18 (rpsR)

What is Rhodopirellula baltica and why is it significant as a model organism?

Rhodopirellula baltica is a marine, aerobic, heterotrophic representative of the phylum Planctomycetes that was originally isolated from the water column in the Kiel fjord (Baltic Sea) . It represents a significant model organism for several reasons:

• It plays a key role in aerobic carbohydrate degradation in marine ecosystems, where polysaccharides are dominant components of biomass

• Its genome was one of the largest bacterial genomes sequenced at the time of publication, comprising 7,145 Mb with 7,325 open reading frames

• It possesses unique cellular morphology featuring a polar cell organization and a distinctive reproductive cycle including budding and swarmer cell formation

• It contains an exceptionally high number of sulfatase genes (110), which is currently the highest in any sequenced bacterial genome, suggesting specialized metabolic capabilities

• Planctomycetes, including R. baltica, are considered key players in marine carbohydrate metabolism due to their nutritional specialization and association with marine snow particles

What is the 30S ribosomal protein S18 (rpsR) and what is its function in Rhodopirellula baltica?

The 30S ribosomal protein S18 (rpsR) is a component of the small subunit (30S) of the bacterial ribosome in Rhodopirellula baltica. While the search results don't specifically address this protein's function in R. baltica, research on ribosomal proteins indicates that S18:

• Participates in mRNA binding during translation initiation

• Contributes to the structural integrity of the 30S ribosomal subunit

• May play a role in the fidelity of translation

• Can serve as a useful marker for evolutionary studies due to its conserved nature across bacterial species

The specific expression patterns of this gene may vary throughout R. baltica's life cycle, potentially correlating with the morphological changes observed during different growth phases .

What expression systems are suitable for recombinant production of R. baltica ribosomal proteins?

For recombinant expression of R. baltica ribosomal proteins including S18:

• E. coli-based expression systems (BL21(DE3), Rosetta, or Arctic Express strains) are typically the first choice due to their simplicity and high yield potential

• Expression vectors containing T7 or tac promoters provide controlled induction

• Addition of fusion tags (His6, GST, or MBP) can improve solubility and facilitate purification

• Codon optimization may be necessary as R. baltica has a G+C content of approximately 55%, which differs from E. coli

To optimize expression:

• Test multiple expression temperatures (15-37°C)

• Vary IPTG concentration (0.1-1.0 mM)

• Consider auto-induction media for higher cell density

• Evaluate different cell lysis methods to preserve protein functionality

How should I design primers for cloning the R. baltica rpsR gene?

When designing primers for cloning the R. baltica rpsR gene, follow these methodological steps:

• Obtain the complete genomic sequence of the rpsR gene from R. baltica SH1ᵀ from genomic databases

• Design forward and reverse primers that:

  • Include 18-25 nucleotides complementary to the target sequence

  • Maintain a GC content of 40-60%

  • Have similar melting temperatures (difference <5°C)

  • Include appropriate restriction enzyme sites flanked by 3-6 additional nucleotides

  • For protein expression, ensure in-frame fusion with any tags

• Consider adding sequences for:

  • A ribosome binding site if using a vector without one

  • A Kozak consensus sequence for efficient translation

  • Removal of rare codons that might impede expression

• Validate primers using in silico PCR and sequence analysis tools to prevent unintended amplification or secondary structure formation

What purification strategy provides the highest yield of functional recombinant R. baltica S18 protein?

A multi-step purification strategy optimized for R. baltica S18 ribosomal protein typically includes:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using a His-tag

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution with 250-300 mM imidazole gradient

  • Typical recovery: 70-85% of expressed protein

• Intermediate purification: Ion exchange chromatography

  • S18 has a basic pI, making cation exchange chromatography suitable

  • Buffer: 50 mM HEPES pH 7.0 with 50-500 mM NaCl gradient

  • Removes DNA contamination and misfolded protein variants

• Polishing step: Size exclusion chromatography

  • Buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

  • Separates monomeric protein from aggregates and oligomers

  • Final purity typically >95% as assessed by SDS-PAGE

• Quality control assessments:

  • Circular dichroism to confirm secondary structure

  • Dynamic light scattering for homogeneity analysis

  • Functional binding assays with rRNA

Implementing this strategy typically yields 3-5 mg of purified protein per liter of bacterial culture under optimized conditions.

How can I monitor the expression of recombinant R. baltica S18 protein during different growth phases?

To effectively monitor the expression of recombinant R. baltica S18 protein during different growth phases:

• Growth phase sampling protocol:

  • Collect samples at multiple time points similar to those used in R. baltica transcriptome studies: early exponential (equivalent to 44h), mid-exponential (62h), transition phase, and stationary phase (82h)

  • Normalize sample collection based on optical density measurements

  • Process samples consistently to maintain comparability

• Analytical methods for protein detection:

  • SDS-PAGE with Coomassie staining for visual assessment

  • Western blotting with antibodies against the protein or fusion tag

  • Quantitative mass spectrometry using labeled reference peptides

  • Activity assays if applicable for functional protein

• Expression profile analysis:

  • Plot expression levels against growth curve parameters

  • Compare with known gene expression patterns of R. baltica

  • Correlate expression with morphological changes observed microscopically (swarmer cells, budding cells, rosette formation)

When interpreting results, consider that R. baltica shows distinct transcriptional profiles during different life cycle phases, with many genes being differentially regulated during transition to stationary phase .

How do post-translational modifications of R. baltica S18 protein differ from those in other bacterial species?

The analysis of post-translational modifications (PTMs) in R. baltica S18 protein reveals several distinctive features compared to other bacterial species:

Comparison of Major PTMs in Ribosomal Protein S18 Across Bacterial Species:

R. baltica S18 protein likely exhibits a unique PTM profile that reflects its specialized cellular environment and the organism's adaptation to marine conditions. The analysis methodology includes:

• High-resolution mass spectrometry coupled with enrichment techniques for specific modifications

• Site-directed mutagenesis to establish the functional significance of modified residues

• Comparative analysis with homologous proteins from related organisms

When investigating these modifications, researchers should consider that R. baltica's growth cycle and environmental adaptations may influence the PTM pattern observed , potentially correlating with the transition between different morphotypes during its life cycle.

What are the structural and functional implications of heterologously expressing R. baltica S18 protein?

Heterologous expression of R. baltica S18 protein presents several structural and functional considerations:

• Structural integrity challenges:

  • The protein may adopt different conformations without its natural ribosomal RNA partners

  • Reversed-phase liquid chromatography analysis can help identify structural variants and impurities that might affect functionality

  • Temperature sensitivity may differ from other recombinant proteins due to R. baltica's marine origin

• Functional assessment strategies:

  • RNA binding assays to determine affinity for cognate rRNA sequences

  • In vitro translation systems to evaluate incorporation into functional ribosomal subunits

  • Structural studies using X-ray crystallography or cryo-EM to compare with native conformation

• Comparative expression analysis:

  • Expression in diverse host systems (prokaryotic vs. eukaryotic)

  • Assessment of co-expression requirements with other ribosomal components

  • Evaluation of solubility and stability in different buffer systems

The methodological approach should include careful optimization of expression conditions, as temperature, salt concentration, and co-factors may significantly impact proper folding. Researchers should consider that R. baltica's adaptation to marine environments may require modified expression protocols compared to standard laboratory strains .

How can I integrate transcriptomic and proteomic data to understand the regulation of R. baltica S18 expression during its life cycle?

To successfully integrate transcriptomic and proteomic data for understanding R. baltica S18 regulation:

• Data collection and preparation:

  • Conduct parallel RNA-seq and quantitative proteomics experiments across defined growth stages that reflect R. baltica's life cycle

  • Ensure synchronized sampling based on morphological transitions (swarmer cells, budding cells, rosette formation)

  • Process samples using standardized protocols to minimize technical variation

• Integration methodology:

  • Apply time-series analysis to identify temporal patterns in gene and protein expression

  • Implement correlation analyses between transcript and protein levels

  • Utilize pathway enrichment to contextualize S18 regulation within broader cellular processes

  • Develop predictive models that account for time delays between transcription and translation

• Interpretation framework:

  • Compare S18 expression patterns with other ribosomal proteins

  • Correlate expression changes with morphological transitions in the R. baltica life cycle

  • Analyze putative regulatory elements in the promoter region of the rpsR gene

  • Examine potential post-transcriptional regulation mechanisms

Previous studies of R. baltica have revealed that numerous genes, including those involved in basic cellular processes, show differential expression patterns throughout the growth curve . This provides context for understanding how S18 expression may be regulated in coordination with the organism's distinctive life cycle and adaptations to nutrient availability.

Why might recombinant R. baltica S18 protein show unexpected migration patterns on SDS-PAGE?

Recombinant R. baltica S18 protein may exhibit anomalous migration on SDS-PAGE due to several factors:

• Post-translational modifications:

  • Unexpected PTMs acquired during expression may alter mobility

  • Phosphorylation typically decreases migration speed

  • Glycosylation, if present, significantly affects apparent molecular weight

• Structural characteristics:

  • High basic amino acid content (common in ribosomal proteins) can reduce SDS binding

  • Incomplete denaturation due to stable structural elements

  • Interaction with nucleic acid contaminants

• Experimental validation approaches:

  • Mass spectrometry to confirm actual molecular weight

  • Different gel systems (Tris-glycine vs. Tris-tricine) for improved resolution

  • Western blotting with antibodies against different epitopes

  • Treatment with phosphatases or glycosidases to remove potential modifications

• Technical solutions:

  • Increase denaturation time and temperature

  • Add additional denaturants (urea or guanidine HCl)

  • Modify sample buffer composition to enhance SDS binding

  • Use gradient gels for better resolution

When interpreting results, consider that R. baltica proteins may have evolved unique structural properties due to adaptation to marine environments , potentially affecting their behavior in standard laboratory analyses.

How can I optimize recombinant R. baltica S18 protein solubility when expression yields inclusion bodies?

When facing inclusion body formation with recombinant R. baltica S18 protein:

• Modified expression conditions:

  • Reduce expression temperature to 15-18°C

  • Decrease inducer concentration (0.1-0.2 mM IPTG)

  • Use slower induction methods (auto-induction media)

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

• Fusion partner strategy:

  • Test multiple solubility-enhancing tags (MBP, SUMO, TrxA)

  • Position tags at either N- or C-terminus

  • Include flexible linkers between tag and protein

  • Employ enzymatic or chemical tag removal methods post-purification

• Buffer optimization for extraction:

  • Include compatible solutes (0.5-1 M sorbitol, 0.5 M trehalose)

  • Test detergents (0.1% Triton X-100, 0.5% CHAPS)

  • Add stabilizing agents (10% glycerol, 50-100 mM arginine)

  • Adjust ionic strength (100-500 mM NaCl)

• Inclusion body recovery protocol:

  • Mild solubilization using 2 M urea with 0.5% sodium lauroyl sarcosine

  • Gradual dialysis with decreasing denaturant concentrations

  • On-column refolding during affinity purification

  • Pulse renaturation with dilution method

These approaches are informed by R. baltica's natural marine environment and should consider the salt preferences and physiological conditions of the native organism when designing solubilization strategies.

How might the study of R. baltica S18 protein contribute to understanding ribosomal evolution in the Planctomycetes phylum?

The study of R. baltica S18 protein offers several avenues for understanding ribosomal evolution in Planctomycetes:

• Comparative sequence analysis:

  • Alignment of S18 sequences across Planctomycetes and other bacterial phyla

  • Identification of conserved and divergent regions specific to Planctomycetes

  • Calculation of evolutionary rates to identify selective pressures

  • Reconstruction of phylogenetic relationships based on S18 sequence

• Structural biology approaches:

  • Determination of R. baltica S18 structure through X-ray crystallography or cryo-EM

  • Mapping of Planctomycetes-specific features onto the structural model

  • Analysis of RNA-binding interfaces compared to other bacterial S18 proteins

  • Investigation of potential structural adaptations related to the unique cell biology of Planctomycetes

• Functional evolution studies:

  • Complementation experiments in heterologous systems

  • Assessment of S18 interaction with rRNA and other ribosomal proteins

  • Evaluation of translation efficiency and accuracy with hybrid ribosomes

  • Investigation of potential specialized functions in the context of R. baltica's life cycle

The insights gained from these studies could help explain how Planctomycetes have evolved their distinctive cellular features while maintaining essential ribosomal functions, potentially revealing adaptations linked to their important ecological role in marine carbohydrate degradation .

What research gaps remain in understanding the role of S18 protein in R. baltica's unique life cycle?

Several critical research gaps exist regarding S18 protein's role in R. baltica's life cycle:

• Cell-type specific expression patterns:

  • How S18 expression varies between swarmer cells, budding cells, and rosette formations

  • Whether different isoforms or modifications of S18 exist in different cell types

  • The temporal relationship between S18 expression and morphological transitions

• Regulatory mechanisms:

  • Identification of transcription factors controlling rpsR expression

  • Characterization of potential post-transcriptional regulation mechanisms

  • Integration of S18 regulation with cell cycle control networks

  • Connection to R. baltica's unique cell division mechanism, which lacks traditional bacterial cell division genes like ftsZ

• Functional specialization:

  • Whether S18 has acquired additional functions beyond its ribosomal role

  • Potential involvement in R. baltica's distinctive reproductive cycle

  • Possible adaptations to the marine environment and association with marine snow particles

  • Implications for protein synthesis during different metabolic states

• Methodological challenges:

  • Development of techniques for cell-type specific isolation from R. baltica cultures

  • Establishment of genetic manipulation systems for targeted studies

  • Creation of fluorescent tagging systems for in vivo visualization

Addressing these gaps requires interdisciplinary approaches combining molecular biology, biochemistry, microscopy, and computational modeling to fully elucidate S18's role in R. baltica's complex life cycle.