Recombinant Bartonella quintana 30S ribosomal protein S15 (rpsO)

What is the primary function of Bartonella quintana 30S ribosomal protein S15 in bacterial ribosomes?

Bartonella quintana S15, like other bacterial S15 homologs, plays two crucial roles:

• Ribosome Assembly: S15 serves as a key component in the assembly of the central domain of the small ribosomal subunit. It functions as a primary binding protein that orchestrates the assembly of additional ribosomal proteins (including S6, S11, S18, and S21) with the central domain of 16S ribosomal RNA to form the platform of the 30S subunit .

• Subunit Association: S15 participates in forming one of the bridges between the 30S and 50S subunits in the functional 70S ribosome, suggesting involvement in subunit association in addition to its role in assembly .

This dual functionality makes S15 essential for both ribosome biogenesis and translation efficiency in B. quintana.

How does recombinant B. quintana S15 compare structurally to S15 proteins from other bacterial species?

While specific structural data for B. quintana S15 is limited, comparison with well-characterized bacterial S15 homologs reveals:

• The core structure is likely highly conserved across bacterial species, given the essential role in ribosome assembly

• B. quintana S15 likely contains the same RNA-binding motifs as other bacterial S15 proteins that interact with the three-way junction and G-U/G-C motif of 16S rRNA

• Minor sequence variations may impact specific RNA recognition profiles, as observed in homologous S15 proteins from different bacterial species

Experimental determination shows that despite shared RNA binding functions in rRNA, S15 homologs from different bacterial species have distinct RNA recognition profiles, suggesting that B. quintana S15 may have unique binding characteristics compared to other bacterial S15 proteins .

What expression systems are optimal for producing recombinant B. quintana S15 protein?

Based on production methods for similar B. quintana ribosomal proteins and S15 from other species, the following expression systems can be recommended:

For most research applications, E. coli expression systems (such as BL21(DE3)) with appropriate induction conditions would be suitable, though toxicity should be monitored . For specialized applications requiring properly folded protein, yeast or mammalian expression may be preferable .

What purification strategies yield the highest purity recombinant B. quintana S15 protein for functional studies?

A systematic purification approach is recommended:

• Initial Capture: Affinity chromatography using His-tag or other fusion tags determined during the manufacturing process .

• Intermediate Purification: Ion exchange chromatography (typically cation exchange as ribosomal proteins are basic).

• Polishing: Size exclusion chromatography to remove aggregates and achieve >90% purity.

Protocol Optimization Points:

• Maintain reducing conditions throughout purification to prevent disulfide formation

• Use protease inhibitors to prevent degradation

• Consider native vs. denaturing conditions based on solubility

• For highest purity (>95%), combine multiple orthogonal purification techniques

The purified protein should be stored in a buffer containing glycerol (recommended 5-50% final concentration) and aliquoted to avoid repeated freeze-thaw cycles, which can compromise activity .

How can researchers accurately assess the RNA-binding specificity of recombinant B. quintana S15?

RNA-binding specificity can be assessed through multiple complementary approaches:

• Filter-Binding Assays: Determine apparent dissociation constants (Kd) through direct RNA-protein binding experiments. This technique allows measurement of binding affinities between purified recombinant S15 and in vitro transcribed RNA targets .

• Footprinting Analysis: Identify specific nucleotides protected by S15 binding using chemical or enzymatic probing. This approach reveals the precise RNA binding sites, as demonstrated for TtS15 mRNA interaction studies .

• Competition Experiments: Assess relative affinities for different RNA targets through competition assays, which can reveal subtle differences in binding specificity .

Kinetic parameters can be determined by:

• Association rate constants (kon) calculated from the initial rate data

• Dissociation rate constants (koff) from chase experiments

• Predict kon values from the koff/Kd ratio and compare with experimental values

For example, TtS15 binds its mRNA with an apparent rate constant (kon) of 25 × 105/M/s, 16-fold slower than for rRNA. Similar experimental approaches can be applied to B. quintana S15 .

What methods can be used to study the role of B. quintana S15 in ribosome assembly in vitro?

Several complementary approaches can be employed:

• Reconstitution Assays: Assess the ability of purified B. quintana S15 to facilitate assembly of 30S subunits in vitro using purified components. Monitor assembly intermediates through sucrose gradient sedimentation or light scattering techniques .

• Subunit Association Assays: Evaluate the role of S15 in 30S-50S subunit association by comparing complete vs. S15-depleted 30S subunits. The assembly can be monitored through sucrose gradient sedimentation profiles, as shown in studies with other bacterial S15 proteins .

• Cross-linking Studies: Identify specific interactions between S15 and other ribosomal components using chemical cross-linking followed by mass spectrometry analysis.

• Electron Microscopy: Visualize structural changes in ribosomal particles with and without S15 to understand its role in organizing the 30S subunit platform.

Data analysis should compare the efficiency of assembly and the stability of formed complexes between B. quintana S15 and well-characterized S15 proteins from model organisms such as E. coli or T. thermophilus .

Does B. quintana S15 autoregulate its own synthesis similar to S15 proteins in other bacterial species?

Based on comparative analysis with other bacterial S15 proteins:

It is highly likely that B. quintana S15 autoregulates its own translation through a feedback regulatory mechanism, though the specific RNA structure may differ from those characterized in other bacteria. Four distinct mRNA structures that interact with S15 to enable regulation have been identified in different bacterial groups, each appearing to be independently derived .

To experimentally determine if B. quintana S15 autoregulates:

• In vitro Translation Assays: Test whether addition of purified recombinant B. quintana S15 protein inhibits translation of its own mRNA in a cell-free system .

• RNA Structure Prediction and Validation: Use bioinformatic approaches to predict potential regulatory structures in the 5' UTR of B. quintana rpsO mRNA, followed by structure probing techniques.

• Deletion Analysis: Identify the minimal mRNA fragment retaining wild-type-like affinity for S15 binding through systematic deletion studies .

Given the diversity of regulatory RNA structures observed among bacterial species, B. quintana likely has evolved its own unique regulatory mechanism that should be characterized experimentally .

How can researchers investigate the transcriptional regulation of B. quintana rpsO in different environmental conditions?

To investigate transcriptional regulation of B. quintana rpsO:

• RNA-seq Analysis: Compare rpsO transcript levels under different environmental conditions relevant to B. quintana's lifecycle, such as:

  • Human host temperature (37°C) vs. arthropod vector temperature

  • Intracellular vs. extracellular growth conditions

  • Various stress conditions (nutrient limitation, pH changes, oxidative stress)

• Quantitative PCR (qPCR): Utilize qPCR for targeted analysis of rpsO expression levels relative to housekeeping genes .

• Promoter Analysis: Characterize the rpsO promoter region to identify regulatory elements through:

  • Reporter gene fusions

  • DNA-protein interaction studies (EMSA, ChIP)

  • Site-directed mutagenesis of predicted regulatory elements

• Northern Blot Analysis: Determine if rpsO is transcribed as a monocistronic mRNA or as part of an operon, which would influence its regulation .

Previous studies with other Bartonella species have revealed temperature-specific and growth phase-specific transcriptional profiles that could inform experimental design for B. quintana rpsO regulation studies .

How can specific amino acid mutations in B. quintana S15 be designed to alter its RNA binding specificity?

Designing specificity-altering mutations requires:

• Sequence Conservation Analysis: Compare S15 proteins across bacterial species to identify:

  • Highly conserved residues likely essential for core function

  • Variable residues potentially involved in species-specific interactions

• Structure-Based Design: Based on known S15-RNA co-crystal structures from model organisms:

  • Target residues at the RNA-protein interface

  • Focus on amino acids that make direct contacts with RNA-specific structural motifs

• Rational Mutagenesis Approach:

  • Create single amino acid substitutions at key positions

  • Generate double or triple mutations to test combinatorial effects

  • Design chimeric proteins combining domains from different bacterial S15 homologs

Experimental validation should include:

• Binding affinity measurements comparing wild-type and mutant proteins

• Specificity assays testing interaction with different RNA targets

• Functional assays measuring the ability to promote ribosome assembly

Research has demonstrated that minor changes to amino acid sequences can have large impacts on RNA-protein recognition patterns in S15 homologs .

What approaches can be used to investigate potential non-canonical functions of B. quintana S15 in host-pathogen interactions?

To investigate potential non-canonical functions:

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid screening against human host proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • Protein microarray analysis

• Bacterial Genetics Approaches:

  • Generate B. quintana strains with modified S15 (tagged versions, expression-controlled variants)

  • Phenotypic analysis of these strains during host cell infection

• Host Cell Response Analysis:

  • Transcriptomic analysis of host cells exposed to wild-type vs. S15-modified B. quintana

  • Proteomic changes in infected host cells

  • Cytokine profiling to assess immunomodulatory effects

• Localization Studies:

  • Immunofluorescence microscopy to track S15 localization during infection

  • Fractionation studies to determine if S15 is secreted or remains intracellular

Recent research on other ribosomal proteins has revealed unexpected extracellular functions in disease contexts, suggesting similar possibilities for B. quintana S15 . For example, studies have shown that ribosomal protein S15 expression is an independent prognostic factor in certain cancer types, indicating potential roles beyond protein synthesis .

How does RNA binding specificity differ between B. quintana S15 and S15 homologs from other bacterial species?

RNA binding specificity among S15 homologs exhibits remarkable diversity:

• Species-Specific Recognition Patterns:
  Despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles as demonstrated by cross-species binding studies .

• Regulatory RNA Structure Diversity:
  Four distinct mRNA structures interact with S15 to enable regulation in different bacterial groups:

  • E. coli S15 recognizes a pseudoknot structure in its mRNA

  • T. thermophilus S15 binds to an RNA junction that partially mimics 16S rRNA

  • B. stearothermophilus S15 interacts with a three-way junction lacking similarity to either E. coli mRNA or 16S rRNA targets

To experimentally characterize B. quintana S15 specificity:

• Test interaction with diverse RNA structural motifs

• Compare binding affinities across RNA targets from different bacterial species

• Examine whether B. quintana S15 can complement S15 deficiencies in heterologous systems

These experiments would reveal whether B. quintana S15 has evolved unique binding specificities or shares recognition patterns with characterized S15 homologs .

What evolutionary insights can be gained by studying amino acid conservation patterns in B. quintana S15 compared to other bacterial species?

Evolutionary analysis of S15 proteins reveals:

• Core Conservation vs. Variable Regions:

  • Identify amino acids conserved across all bacterial S15 proteins (likely essential for ribosomal function)

  • Map species-specific variations that may reflect adaptation to different ecological niches

• Co-evolutionary Patterns:

  • Analyze co-evolution between S15 protein sequences and their corresponding regulatory RNA structures

  • Identify correlated mutations between protein and RNA binding partners

• Phylogenetic Distribution:

  • Compare S15 sequences within the Bartonella genus to identify genus-specific features

  • Examine differences between S15 from facultative intracellular pathogens versus free-living bacteria

Methodological approach:

• Multiple sequence alignment of S15 proteins across diverse bacterial species

• Calculation of conservation scores at each amino acid position

• Mapping conservation data onto known S15 structures to identify functional surfaces

• Molecular phylogenetic analysis to reconstruct evolutionary relationships

These analyses can provide insights into how S15 proteins have evolved different specificities while maintaining core ribosomal functions, and may reveal how B. quintana S15 has adapted to its particular pathogenic lifestyle .

What strategies can overcome stability issues when working with recombinant B. quintana S15 protein?

Recombinant ribosomal proteins often present stability challenges. Based on protocols for similar ribosomal proteins, the following strategies are recommended:

• Buffer Optimization:

  • Include glycerol (5-50%) in storage buffers to prevent aggregation

  • Add reducing agents (DTT or β-mercaptoethanol) to prevent disulfide formation

  • Optimize salt concentration to maintain solubility while preserving activity

• Storage Considerations:

  • Store as aliquots at -20°C or -80°C to avoid repeated freeze-thaw cycles

  • For short-term use, store working aliquots at 4°C for up to one week

  • Consider flash-freezing in liquid nitrogen before -80°C storage

• Stabilizing Additives:

  • Test protein stabilizers such as trehalose or sucrose

  • Consider carrier proteins for very dilute solutions

  • Evaluate chemical chaperones that may improve folding stability

• Prevention of Proteolytic Degradation:

  • Include protease inhibitor cocktails during purification

  • Minimize handling time and maintain cold temperatures throughout processing

  • Consider engineering protease-resistant variants for difficult applications

• Shelf-Life Guidelines:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

What are the key considerations for designing RNA-protein interaction experiments with B. quintana S15 to ensure reproducible results?

Ensuring reproducible RNA-protein interaction studies requires careful attention to:

• RNA Preparation Quality Control:

  • Verify RNA integrity by denaturing gel electrophoresis

  • Confirm proper folding through structure probing techniques

  • Use consistent transcription and purification methods to minimize batch-to-batch variation

• Protein Quality Assessment:

  • Verify protein activity after each purification batch

  • Test binding to known RNA targets as positive controls

  • Monitor protein stability during storage with repeat binding assays

• Binding Condition Standardization:

  • Maintain consistent buffer composition (pH, ionic strength, Mg²⁺ concentration)

  • Control temperature precisely during binding reactions

  • Standardize protein and RNA concentrations across experiments

• Data Analysis Considerations:

  • Perform multiple independent replicates (minimum n=3)

  • Include appropriate positive and negative controls

  • Use statistical methods appropriate for the specific assay

• Validation Through Multiple Techniques:

  • Confirm key findings using orthogonal methods (e.g., filter binding, EMSA, ITC)

  • Consider in vivo validation of key interactions identified in vitro

  • Address potential artifacts through careful experimental design

These methodological considerations are critical when comparing binding specificities between different RNA targets or when assessing the effects of mutations on interaction parameters .

What potential applications exist for B. quintana S15 in understanding bacterial pathogenesis and developing new antimicrobial strategies?

Several promising research avenues include:

• Pathogenesis Mechanisms:

  • Investigate whether S15 is involved in stress responses during host infection

  • Determine if B. quintana S15 plays roles in modulating virulence gene expression

  • Examine potential interactions with host cellular components during infection

• Drug Target Potential:

  • Explore S15-specific inhibitors that could disrupt ribosome assembly

  • Target the unique RNA-binding properties of B. quintana S15

  • Develop compounds that interfere with S15 autoregulation

• Diagnostic Applications:

  • Evaluate S15 as a biomarker for B. quintana infections

  • Develop antibodies against species-specific S15 epitopes for diagnostic tests

  • Use RNA aptamers that mimic S15 binding sites as diagnostic tools

• Vaccine Development:

  • Assess S15 as a potential vaccine antigen

  • Study immune responses to S15 during natural infections

  • Investigate cross-reactivity with S15 proteins from other bacterial species

• Synthetic Biology Applications:

  • Engineer S15-based biosensors for detecting specific RNA sequences

  • Develop synthetic gene circuits using S15-RNA regulatory interactions

  • Create attenuated bacterial strains with modified S15 function

These applications leverage the essential nature of S15 in bacterial physiology and its species-specific characteristics that could be exploited for targeted interventions against B. quintana infections.

How might structural biology approaches advance our understanding of B. quintana S15 function and interactions?

Advanced structural biology approaches would significantly enhance our understanding of B. quintana S15:

• High-Resolution Structure Determination:

  • X-ray crystallography of B. quintana S15 alone and in complex with RNA targets

  • Cryo-electron microscopy of B. quintana ribosomes to visualize S15 in its native context

  • NMR spectroscopy to examine dynamics of S15-RNA interactions

• Comparative Structural Analysis:

  • Superimpose B. quintana S15 structures with homologs from model organisms

  • Identify structural features that contribute to B. quintana-specific functions

  • Map species-specific amino acid variations onto structural models

• Molecular Dynamics Simulations:

  • Model conformational changes during RNA binding

  • Predict effects of mutations on protein stability and function

  • Simulate the assembly process of the 30S platform with B. quintana components

• In situ Structural Studies:

  • Cryo-electron tomography of B. quintana cells to visualize ribosomes in their cellular context

  • Super-resolution microscopy to track S15 localization during infection

  • Correlative light and electron microscopy to connect function with structure

These structural approaches would provide mechanistic insights into B. quintana S15's dual roles in ribosome assembly and gene regulation, potentially revealing unique features that could be exploited for targeted interventions against this pathogen.