Recombinant Bordetella bronchiseptica 50S ribosomal protein L15 (rplO)

What is the structure and function of 50S ribosomal protein L15 in Bordetella bronchiseptica?

The 50S ribosomal protein L15, encoded by the rplO gene, is a critical component of the large ribosomal subunit in B. bronchiseptica. This approximately 15 kDa protein plays essential roles in ribosome assembly and function. Based on structural studies of homologous proteins in related bacteria, L15 is characterized as a late assembly protein that appears to be required for 5S rRNA incorporation into the ribosome .

L15 interacts with domain II of 23S rRNA in a partially assembled ribosomal particle, forming specific contacts with the region spanning nucleotides 572-654 (based on E. coli numbering) . Importantly, this binding site is not formed in "naked" 23S rRNA but requires a partially assembled particle, indicating the complex structural dependencies in ribosome biogenesis .

The protein contains multiple regions that interact with other ribosomal proteins during 50S assembly, highlighting its role as an architectural component. In the mature ribosome, L15 contributes to the binding site for certain antibiotics, including erythromycin, making it relevant for studies of antimicrobial resistance .

What expression systems are most effective for producing recombinant B. bronchiseptica rplO?

Several expression systems can be employed for the production of recombinant B. bronchiseptica rplO, each with distinct advantages depending on the research goals:

Escherichia coli expression systems remain the most widely used for bacterial ribosomal proteins due to their efficiency, cost-effectiveness, and high yield. The pET expression system with BL21(DE3) or its derivatives offers robust expression for structural and functional studies. Optimal conditions typically include induction with 0.5-1.0 mM IPTG at reduced temperatures (16-25°C) to enhance protein solubility.

For challenging expressions, alternative hosts may be considered. Yeast systems (Pichia pastoris or Saccharomyces cerevisiae) can provide improved protein folding for certain targets . Baculovirus expression in insect cells offers another eukaryotic alternative with sophisticated protein processing capabilities but at higher cost and complexity .

When designing expression constructs, fusion tags can significantly improve yield and solubility. His6-tags facilitate purification via nickel affinity chromatography, while solubility enhancers like MBP (maltose-binding protein) or SUMO can improve folding. Inclusion of a precision protease cleavage site allows tag removal for structural studies.

Codon optimization for the expression host is particularly important for heterologous expression of B. bronchiseptica proteins to avoid translational stalling due to rare codons .

What purification strategies yield the highest purity recombinant B. bronchiseptica rplO protein?

Purification of recombinant B. bronchiseptica rplO requires a multi-step approach to achieve high purity while maintaining native structure and function. Based on established protocols for ribosomal proteins, the following optimized strategy is recommended:

Initial capture is typically performed using immobilized metal affinity chromatography (IMAC) for His-tagged rplO. Buffer optimization is critical at this stage—inclusion of 300-500 mM NaCl and 5-10% glycerol helps prevent non-specific interactions and protein aggregation. Low concentrations of imidazole (10-20 mM) in the binding buffer reduce non-specific binding, while elution with an imidazole gradient (50-300 mM) provides better separation.

Following IMAC, ion-exchange chromatography (typically cation exchange due to rplO's basic properties) significantly improves purity. A linear gradient of NaCl (0-1 M) in a low pH buffer (pH 6.0-6.5) effectively separates rplO from remaining contaminants.

Size-exclusion chromatography as a polishing step not only removes aggregates but also confirms the protein's oligomeric state. For functional studies, this step should be performed in a physiologically relevant buffer.

Nucleic acid contamination is a common challenge when purifying ribosomal proteins due to their natural RNA-binding properties. Treatment with a high-salt wash (1-2 M NaCl) or limited RNase digestion before the ion-exchange step can significantly reduce nucleic acid content .

How can researchers verify the structural integrity and activity of purified recombinant rplO?

Verification of structural integrity and functional activity of recombinant B. bronchiseptica rplO requires a combination of biophysical and biochemical approaches:

Structural integrity can be assessed through circular dichroism (CD) spectroscopy, which provides information about secondary structure elements. Properly folded rplO should display a characteristic spectrum with distinct minima at 208 and 222 nm, reflecting its α-helical content. Thermal denaturation monitored by CD (thermal shift assay) yields the melting temperature (Tm), a valuable parameter for assessing stability under different buffer conditions.

Intrinsic tryptophan fluorescence spectroscopy offers insights into tertiary structure, as the emission maximum shifts to shorter wavelengths in the folded state compared to denatured protein. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirms the monomeric state and absence of aggregates.

Functional activity assessment should focus on RNA binding capacity, which can be measured through:

• Electrophoretic mobility shift assays (EMSA) with labeled 23S rRNA fragments corresponding to domain II (nucleotides 572-654 based on E. coli numbering)

• Fluorescence anisotropy with fluorescently labeled RNA fragments

• Surface plasmon resonance to determine binding kinetics (kon and koff rates)

A comprehensive validation approach would include ribosome reconstitution assays, where recombinant rplO is incorporated into partially assembled 50S particles derived from B. bronchiseptica or related species. The reconstituted particles can then be tested for their ability to form 70S ribosomes and participate in translation .

How does the structure of B. bronchiseptica rplO compare to its homologs in other bacterial species?

The structure of B. bronchiseptica rplO, while not directly determined in the available literature, can be inferred through comparative analysis with homologs from related bacterial species. This comparison provides insights into conserved features and species-specific adaptations.

Ribosomal proteins are generally highly conserved across bacterial species due to their fundamental role in protein synthesis. Sequence alignment of B. bronchiseptica rplO with homologs from E. coli and other bacteria typically reveals >60% identity in the core domains, with variations mainly in surface-exposed regions. These conserved regions include RNA-binding motifs and interfaces with other ribosomal proteins.

The tertiary structure likely follows the canonical fold observed in other bacterial L15 proteins: a globular domain with mixed α/β topology, featuring a characteristic RNA-binding surface rich in positively charged residues. This conservation reflects functional constraints imposed by the need to maintain proper ribosome assembly and function.

Comparative structural analysis can reveal subtle differences that might influence species-specific ribosome properties:

For detailed structural characterization, researchers should employ X-ray crystallography or cryo-electron microscopy of recombinant B. bronchiseptica rplO, ideally in complex with its rRNA binding partners. Homology modeling based on the available L15 structures provides a valuable starting point for structure-function studies and the design of targeted mutations .

What methods can be used to study the impact of rplO mutations on B. bronchiseptica virulence?

Investigating the impact of rplO mutations on B. bronchiseptica virulence requires a multi-faceted approach combining genetic, biochemical, and infection model techniques:

Genetic manipulation strategies should focus on creating defined mutations rather than complete knockouts, as rplO is likely essential. Site-directed mutagenesis targeting specific functional domains (RNA-binding regions, protein-protein interaction interfaces) can be implemented using allelic exchange techniques. For more precise control, CRISPR-Cas9 genome editing allows for nucleotide-level modifications without leaving scars in the genome.

Once mutant strains are generated, in vitro characterization should assess:

• Growth kinetics under various conditions (standard media, iron limitation, stress conditions)

• Expression and secretion of key virulence factors using proteomic approaches

• Ribosome profile analysis to evaluate the impact on translation

• Global gene expression changes via RNA-seq, particularly focusing on BvgAS-regulated genes

In vivo infection models provide the most relevant assessment of virulence:

The mouse respiratory model is particularly valuable as studies have shown that B. bronchiseptica can persist within infected mouse lungs . Assessment should include bacterial load determination, histopathological examination, and analysis of host immune responses (cytokine profiles, cellular infiltration).

Cell-based assays should examine the impact on key virulence mechanisms such as cytotoxicity (as measured for BspR and BcrH proteins ) and hemolytic activity, which are indicative of type III secretion system function.

How can CRISPR-Cas9 technology be applied to study rplO function in B. bronchiseptica?

CRISPR-Cas9 technology offers powerful approaches for precise genetic manipulation to investigate rplO function in B. bronchiseptica. This cutting-edge methodology allows for targeted genetic modifications that traditional techniques cannot achieve as efficiently.

For essential genes like rplO, where complete knockouts may be lethal, CRISPR-based strategies must be carefully designed:

• Conditional Knockdown Systems
  CRISPR interference (CRISPRi) using a catalytically inactive Cas9 (dCas9) can be employed to create titratable repression of rplO. This system allows researchers to reduce expression to various levels and observe the resulting phenotypes. The dCas9 protein can be placed under an inducible promoter (e.g., tet-responsive) for temporal control of repression.

• Point Mutation Generation
  CRISPR-Cas9 coupled with homology-directed repair (HDR) enables precise introduction of specific mutations to investigate structure-function relationships. Target residues should include those involved in rRNA binding, interaction with other ribosomal proteins, or potential antibiotic binding sites.

• Domain Tagging
  CRISPR-Cas9 can facilitate in-frame insertion of epitope or fluorescent tags for tracking rplO localization and dynamics within living bacteria. C-terminal tags are typically less disruptive to function than N-terminal modifications for ribosomal proteins.

• Gene Replacement
  Swapping the native rplO with homologs from related species (e.g., B. pertussis, E. coli) can reveal species-specific adaptations. This approach is particularly valuable for understanding the basis of differential antibiotic susceptibility between Bordetella species.

Technical considerations for CRISPR-Cas9 application in B. bronchiseptica include:

• Optimizing guide RNA design for high specificity and efficiency

• Developing efficient transformation protocols for HDR template delivery

• Establishing screening methods to identify successful editing events

• Validating edited strains through sequencing and phenotypic characterization

CRISPR-based approaches should be complemented with comprehensive phenotypic analysis, including virulence assessment in appropriate infection models .

How does the interaction between rplO and the Type III Secretion System (T3SS) affect B. bronchiseptica pathogenesis?

The potential interaction between rplO and the Type III Secretion System (T3SS) represents an intriguing connection between the translational machinery and virulence mechanisms in B. bronchiseptica. While direct studies linking rplO specifically to T3SS are not explicitly described in the search results, several pieces of evidence suggest possible connections.

The T3SS is a critical virulence determinant in Bordetella species, delivering effector proteins directly into host cells during infection . Search result #4 identifies BspR as a novel type III secreted protein that acts as a regulator of virulence genes in B. bronchiseptica. Similarly, search result #14 describes BcrH proteins as specific chaperones for T3SS components that affect hemolytic activity and cytotoxicity.

Potential mechanisms by which rplO could influence T3SS function include:

• Translational regulation: As a ribosomal protein, rplO may affect the translation efficiency of T3SS components. Different ribosomal protein compositions can create specialized ribosomes that preferentially translate specific mRNAs.

• Coordinated regulation: Both ribosomal proteins and T3SS components respond to environmental cues. Search result #1 shows that mutations affecting ribosomal protein operons can cause global dysregulation of gene expression, including virulence factors.

• Direct protein interactions: Some ribosomal proteins have extraribosomal functions. rplO might interact directly with T3SS regulatory proteins or chaperones.

To investigate these interactions, researchers should:

• Analyze T3SS protein expression and secretion in strains with altered rplO expression

• Examine whether T3SS-dependent phenotypes (hemolytic activity, cytotoxicity) are affected by rplO mutations

• Determine if rplO expression changes in response to conditions that induce T3SS expression

The iron-responsive regulation of T3SS mentioned in search result #4 is particularly relevant, as ribosomal protein expression may also respond to iron availability, potentially creating a coordinated regulatory network affecting both translation and virulence.

What techniques can be used to study the rplO-associated ribosome assembly pathway in B. bronchiseptica?

Investigating the rplO-associated ribosome assembly pathway in B. bronchiseptica requires sophisticated techniques to capture the complex, hierarchical process of ribosome biogenesis. Based on approaches used for other bacterial systems (particularly result #2), the following methodologies are recommended:

In vitro reconstitution experiments provide direct insights into assembly pathways. This approach involves:

• Purification of 23S rRNA and individual ribosomal proteins from B. bronchiseptica

• Sequential addition of proteins to rRNA in different orders

• Analysis of intermediate particles using sucrose gradient centrifugation

• Determination of the stage at which rplO incorporation occurs and its dependencies

A critical experiment would be reconstituting 50S particles with and without rplO to determine its precise role in assembly, similar to the approaches described for E. coli L15 .

Time-resolved cryo-electron microscopy (cryo-EM) offers visualization of assembly intermediates:

Pulse-chase experiments with MS analysis track the kinetics of assembly:

• Grow B. bronchiseptica in medium containing stable isotope-labeled amino acids

• Perform a chase with unlabeled medium

• Isolate ribosomes at different time points

• Analyze the incorporation of labeled proteins using mass spectrometry

• Determine the temporal sequence of rplO incorporation relative to other proteins

Conditional depletion studies in vivo reveal the consequences of rplO absence:

• Create a conditional expression system for rplO where the native gene is deleted and replaced with an inducible copy

• Deplete rplO by removing the inducer

• Isolate ribosomes and analyze them using sucrose gradients and quantitative mass spectrometry

• Identify accumulated assembly intermediates that require rplO for further maturation

Combined with structural information from homologous systems, these approaches would provide a comprehensive understanding of how rplO contributes to ribosome assembly in B. bronchiseptica and identify any pathogen-specific features of this process .

How can rplO be used as a target for developing novel antibiotics against B. bronchiseptica infections?

The exploration of rplO as a target for novel antibiotics against B. bronchiseptica offers promising avenues for addressing the challenges of treating infections caused by this pathogen. The rationale for targeting rplO stems from several key considerations:

Ribosomal proteins represent validated antibiotic targets, with many clinically successful antibiotics binding to components of the bacterial ribosome. Given that B. bronchiseptica shows resistance to several conventional antibiotics including macrolides and cephalosporins , targeting specific ribosomal proteins like rplO could provide new treatment options.

Several characteristics make rplO an attractive target:

• Essential function in ribosome assembly and protein synthesis

• Surface accessibility in the assembled ribosome

• Structural differences between bacterial and mammalian homologs

• Involvement in antibiotic binding sites (particularly for macrolides)

A structure-guided drug discovery approach would involve:

Potential drug development strategies include:

• Small molecules targeting the rRNA-binding interface of rplO to disrupt ribosome assembly

• Peptide mimetics that interfere with rplO-protein interactions during assembly

• Allosteric modulators that induce conformational changes affecting function

• Compounds that stabilize non-productive interactions between rplO and other components

Efficacy testing should include assessment of:

• Minimum inhibitory concentration (MIC) against B. bronchiseptica isolates

• Activity against biofilms, which are relevant to persistent infections

• Efficacy in animal models of respiratory infection

• Lack of toxicity to mammalian cells

• Ability to overcome existing resistance mechanisms

Given that B. bronchiseptica causes infections in various animal species and occasionally humans , successful therapeutics targeting rplO could have broad veterinary and potentially human applications.

How do environmental conditions affect rplO expression and function in the context of B. bronchiseptica's lifecycle?

The expression and function of rplO in B. bronchiseptica are likely influenced by the diverse environmental conditions the bacterium encounters during its complex lifecycle. B. bronchiseptica transitions between host respiratory tissues and environmental reservoirs, requiring sophisticated adaptive mechanisms.

The BvgAS two-component system serves as the master regulator of this transition, controlling expression patterns in response to environmental signals . While specific regulation of rplO has not been directly characterized in the search results, the following environmental factors likely influence its expression and function:

Temperature fluctuations represent a critical signal for B. bronchiseptica's lifestyle transition. At host temperature (37°C), the BvgAS system activates virulence genes (Bvg+ phase), while at lower environmental temperatures (≤25°C), it shifts to the Bvg- phase associated with survival outside the host . Ribosomal protein expression patterns may align with these phases to optimize translation of phase-specific proteins.

Iron availability serves as another important environmental signal. Search result #4 indicates that BspR is involved in iron-responsive regulation of virulence genes. Ribosomal proteins may be similarly regulated by iron availability, creating coordinated responses to this critical nutrient.

Interaction with environmental predators such as amoebae represents a unique aspect of B. bronchiseptica's lifecycle. Search result #7 demonstrates that B. bronchiseptica can survive within and disseminate via the amoeba Dictyostelium discoideum. This process is regulated by Bvg- phase genes, suggesting potential involvement of specific ribosomal composition in adaptation to this environment.