Recombinant Bartonella henselae 50S ribosomal protein L9 (rplI)

What is the structure and role of Bartonella henselae 50S ribosomal protein L9?

Bartonella henselae 50S ribosomal protein L9 is a critical component of the bacterial ribosome with a distinctive architecture featuring two globular RNA-binding domains (N-terminal and C-terminal) connected by an elongated α-helix. This unusual structure functions as a "molecular strut" that plays essential roles in:

The L9 protein typically has a molecular mass of approximately 21.4 kDa and contains roughly 150-200 amino acids, varying slightly between Bartonella species . Nuclear magnetic resonance (NMR) and circular dichroism studies have shown that each of the two RNA-binding domains contains a highly stable core, while the central connecting helix, though helical in solution, is not entirely rigid .

What are the optimal conditions for expression and purification of recombinant B. henselae L9 protein?

Successful expression and purification of recombinant B. henselae L9 protein requires careful optimization of several parameters:

• Expression System Selection: The E. coli BL21(DE3) strain with pET vector systems (such as pET200D/TOPO) has proven effective for rplI expression .

• Expression Conditions:

  • Induction with 0.5-1.0 mM IPTG

  • Post-induction temperature of 30°C (rather than 37°C) to enhance soluble protein yield

  • Expression period of 4-6 hours or overnight at 18°C for difficult constructs

• Purification Strategy:

  • Initial capture using His-tag affinity chromatography with imidazole gradient elution

  • Secondary polishing via size exclusion chromatography

  • Optional ion exchange chromatography for removal of nucleic acid contamination

• Quality Assessment:

  • SDS-PAGE and Western blot analysis to confirm purity and integrity

  • Mass spectrometry for molecular weight verification

  • Circular dichroism to assess secondary structure

When expressing full-length rplI versus specific domains (N-terminal, middle, or C-terminal), different optimization may be required. Research has shown that the middle domain can be particularly challenging to express as a soluble protein compared to N-terminal and C-terminal domains .

How do the different domains of B. henselae L9 contribute to its function?

The unique two-domain architecture of L9 with its connecting α-helix has significant functional implications:

• N-terminal Domain:

  • Contains a conserved glycine and lysine-rich loop that is surprisingly stable and ordered

  • Mutations in this domain (e.g., G24D in Salmonella) can affect translational fidelity, particularly frameshifting

  • Serves as the primary anchor to the ribosome

• Connecting α-helix:

  • Acts as a molecular ruler or strut between domains

  • Disruption of this helix (e.g., A59P mutation) has been shown to induce +1 frameshifting, indicating its crucial role in maintaining reading frame fidelity

  • Provides flexibility while maintaining proper domain spacing

• C-terminal Domain:

  • More dynamic structure correlating with its RNA-binding function

  • Mutations (I94S, A102D, G126V, F132S) can affect ribosomal function and translational fidelity

  • May serve as a recognition platform for other factors

Disruption of either globular domain or the connecting α-helix can lead to significant alterations in translational fidelity, particularly with respect to frameshifting events, suggesting that L9's structure-function relationship is highly dependent on maintaining proper geometric relationships between its domains .

How can recombinant B. henselae L9 protein be utilized in diagnostic assays for bartonellosis?

While traditional B. henselae diagnostics focus on proteins like Pap31, the potential of L9 as a diagnostic target requires evaluation. Research with recombinant Pap31 has demonstrated the challenges in developing serological tests for Bartonella:

• ELISA Development Considerations:

  • When evaluating recombinant B. henselae proteins for diagnostic applications, optimal cutoff values must be determined using ROC analysis to maximize both sensitivity and specificity

  • For human bartonellosis, rPap31 demonstrated 72% sensitivity and 61% specificity at a cutoff value of 0.215

  • Similar approaches could be applied to rplI testing

• Comparative Domain Analysis:

  • When evaluating diagnostic potential, testing individual domains separately may yield different performance metrics

  • For example, in Pap31 studies, the N-terminal domain showed 89% sensitivity but only 56% specificity, while adjusting cutoff values could improve specificity at the cost of sensitivity

  • Such domain-specific analysis would be valuable for L9 protein

• Limitations to Consider:

  • Cross-reactivity with other bacterial species must be evaluated

  • Background seropositivity in healthy populations needs assessment

  • The persistence of antibodies after treatment may limit usefulness for monitoring therapeutic response

Current diagnostic methods for B. henselae infections include serology with IFA having sensitivity and specificity of 100% and 96.8% respectively . Any new L9-based assay would need to demonstrate comparable or superior performance metrics.

What experimental approaches can be used to study interactions between B. henselae L9 and ribosomal RNA?

Several complementary methods can provide insights into L9-rRNA interactions:

• RNA Binding Assays:

  • Electrophoretic mobility shift assays (EMSA) using purified recombinant L9 and in vitro transcribed rRNA fragments

  • Filter-binding assays to determine binding constants

  • Surface plasmon resonance for real-time binding kinetics

• Structural Analysis:

  • X-ray crystallography of L9-RNA complexes

  • Cryo-electron microscopy of ribosomal assemblies

  • NMR studies of domain-specific RNA interactions

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Crosslinking Approaches:

  • UV-induced crosslinking followed by mass spectrometry

  • Site-specific incorporation of photo-activatable crosslinkers

  • CLIP-seq (crosslinking immunoprecipitation-sequencing) for in vivo RNA binding sites

• Mutagenesis Studies:

  • Alanine scanning of conserved residues

  • Domain deletion or exchange experiments

  • Creation of chimeric proteins with L9 from related species

Research has shown that the most dynamic parts of the L9 protein are the regions containing the RNA-binding residues in each domain , suggesting these regions adapt their conformation upon RNA binding.

How conserved is the L9 protein structure across Bartonella species and related bacteria?

The L9 ribosomal protein shows significant conservation across bacterial species while maintaining critical structural features:

• Sequence Homology:

  • High sequence conservation exists in the core domains across bacterial species

  • The L9 protein from cyanobacteria shows homology to those from E. coli and chloroplasts of Arabidopsis and pea

  • Bartonella species typically show >90% amino acid identity in L9 protein

• Structural Conservation:

  • The two-domain architecture with connecting α-helix is preserved across diverse bacterial species

  • Conservation is typically higher in the N-terminal domain compared to the C-terminal domain

  • RNA-binding motifs show the highest degree of conservation

• Functional Implications:

  • Conserved residues typically correspond to functionally critical positions

  • Variable regions may reflect adaptation to specific ribosomal environments or regulatory mechanisms

  • Understanding conservation patterns can guide mutagenesis studies

Comparative sequence analysis between B. henselae and B. quintana L9 proteins reveals high similarity, with the B. quintana protein consisting of 193 amino acids . This conservation reflects the essential nature of L9's function in ribosomal architecture and protein synthesis.

How can site-directed mutagenesis be used to identify critical residues in B. henselae L9 protein?

Site-directed mutagenesis offers powerful insights into L9 protein function:

• Strategic Target Selection:

  • Focus on conserved residues identified through multiple sequence alignments

  • Target residues in predicted RNA-binding regions

  • Examine the connecting α-helix which is critical for maintaining domain spacing

  • Create mutations analogous to those identified in other bacteria that affect translational fidelity

• Specific Mutation Strategies:

  • Alanine scanning to neutralize side chain effects

  • Conservative substitutions to assess specific chemical properties

  • Radical substitutions to disrupt function

  • Introduction of known frameshifting mutations (e.g., G24D, I94S, A102D, G126V, F132S, A59P) identified in other species

• Functional Assays:

  • In vitro translation assays to assess effects on protein synthesis

  • Frameshifting reporter systems to evaluate translational fidelity

  • RNA binding assays to assess domain-specific interactions

  • Growth complementation in L9-deficient strains

Previous studies with Salmonella have shown that mutations in either the N-terminal domain (G24D) or C-terminal domain (I94S, A102D, G126V, F132S) can affect frameshifting, while the A59P mutation in the connecting α-helix induces +1 frameshifting . These findings provide a foundation for similar studies with B. henselae L9.

Is there evidence that L9 protein contributes to B. henselae pathogenesis or host-pathogen interactions?

While direct evidence linking L9 protein to B. henselae pathogenesis is limited, several considerations merit investigation:

• Potential Pathogenesis Connections:

  • Ribosomal proteins can have moonlighting functions beyond protein synthesis

  • Surface exposure of ribosomal proteins has been documented in some bacteria

  • Potential role in stress response during host colonization

  • Possible immunomodulatory effects if recognized by host immune system

• Host-Pathogen Context:

  • B. henselae causes cat scratch disease with 5-15% of affected patients developing ocular involvement

  • B. henselae can incite various forms of intraocular inflammation

  • Any contribution of L9 to bacterial fitness in vivo could influence pathogenesis

• Research Approaches:

  • Comparative proteomics between virulent and avirulent strains

  • Evaluation of L9 expression under host-mimicking conditions

  • Assessment of L9 mutants for virulence in cellular or animal models

  • Investigation of potential L9 interactions with host proteins

The possibility of L9 as an antigenic target during infection also warrants investigation, particularly given the challenges in diagnosing Bartonella infections and the need for improved diagnostic markers .

What approaches can be used to determine the three-dimensional structure of B. henselae L9 protein?

Determining the structure of B. henselae L9 protein requires a multi-technique approach:

• X-ray Crystallography:

  • Requires high-purity protein (>95%) at concentrations of 5-20 mg/ml

  • Screening of multiple crystallization conditions

  • Molecular replacement using L9 structures from related species as search models

  • Potential co-crystallization with RNA fragments

• Nuclear Magnetic Resonance (NMR):

  • Especially suitable for studying domain dynamics

  • Requires isotopically labeled protein (15N, 13C, 2H)

  • Can provide insights into solution behavior and conformational flexibility

  • Previous NMR studies on L9 from other species provide comparative data

• Cryo-Electron Microscopy:

  • Visualization of L9 in the context of the entire ribosome

  • May provide insights into conformational changes during translation

  • Benefits from reference structures of ribosomes from related species

• Computational Approaches:

  • Homology modeling based on existing L9 structures

  • Molecular dynamics simulations to study domain flexibility

  • Integrative modeling combining experimental data from multiple sources

Previous structural studies on L9 from Bacillus stearothermophilus have shown that each of the two RNA-binding domains contains a highly stable core, with the connecting helix being helical in solution but not entirely rigid . These findings provide a foundation for structural studies of B. henselae L9.

What is known about the immunogenicity of B. henselae L9 protein and its potential as a vaccine component?

The immunological properties of B. henselae L9 require systematic evaluation:

• Antigenicity Assessment:

  • Epitope mapping to identify immunodominant regions

  • B-cell epitope prediction followed by experimental validation

  • Comparative antigenicity analysis of full-length protein versus individual domains

  • Evaluation of cross-reactivity with L9 proteins from other bacterial species

• Immunization Studies:

  • Animal models to assess antibody responses

  • Measurement of protective efficacy against B. henselae challenge

  • Comparison with other B. henselae antigens (e.g., Pap31, which has been explored as a diagnostic target)

  • Evaluation of different adjuvant formulations

• Safety Considerations:

  • Assessment of potential autoimmune responses due to protein conservation

  • Evaluation of inflammatory responses at injection sites

  • Monitoring for adverse events in animal models

Studies with other Bartonella antigens have shown variable sensitivity and specificity in diagnostic applications , suggesting careful optimization would be needed for any L9-based applications. For instance, recombinant Pap31 showed 72% sensitivity and 61% specificity for human bartonellosis diagnoses .

How is L9 protein incorporated into the bacterial ribosome during assembly?

The process of L9 incorporation into ribosomes follows a coordinated assembly pathway:

• Assembly Hierarchy:

  • L9 binds to the 23S rRNA as part of the 50S ribosomal subunit assembly

  • Integration occurs at a specific stage in the ribosome maturation process

  • May require assistance from assembly factors or chaperones

• Binding Dynamics:

  • Initial binding likely occurs through the N-terminal domain

  • The connecting α-helix positions the C-terminal domain for secondary interactions

  • Conformational changes in both protein and rRNA may occur during binding

• Experimental Approaches:

  • In vitro reconstitution of ribosomes with labeled L9

  • Time-resolved cryo-EM to capture assembly intermediates

  • Pulse-chase experiments to monitor integration kinetics

  • Depletion studies to assess assembly defects in absence of L9

The molecular strut hypothesis suggests L9 plays a role in ribosome assembly and/or maintaining the catalytically active conformation of ribosomal RNA , making this an important area for further investigation in B. henselae.

Does B. henselae L9 protein play a role in regulating translational fidelity?

Evidence suggests important roles for L9 in translational quality control:

• Frameshifting Effects:

  • Mutations in L9 have been shown to affect frameshifting in various bacteria

  • The geometric relationship between N and C domains appears critical for maintaining reading frame fidelity

  • L9 may prevent slippage during specific translational challenges

• Experimental Evidence:

  • Studies in Salmonella have shown that various L9 mutations can suppress +1 frameshift mutations

  • The A59P mutation, which disrupts the connecting α-helix, induces +1 frameshifting

  • These findings suggest L9 contributes to translational accuracy

• Mechanistic Models:

  • L9 may stabilize specific ribosomal conformations during challenging translational events

  • The extended structure may monitor or influence tRNA positions

  • The protein may facilitate proper mRNA threading through the ribosome

The ability of L9 mutations to affect frameshifting suggests a role in maintaining translational fidelity that likely extends to B. henselae, though specific studies in this organism are needed to confirm this function.

How does L9 expression vary under different growth conditions in B. henselae?

Understanding L9 expression dynamics requires systematic analysis:

• Experimental Approaches:

  • qRT-PCR to measure rplI transcript levels under various conditions

  • Western blot analysis for protein-level quantification

  • Ribosome profiling to assess translation efficiency

  • Promoter-reporter fusions to monitor transcriptional regulation

• Relevant Conditions to Test:

  • Growth phase (log vs. stationary)

  • Stress conditions (oxidative, thermal, pH, nutrient limitation)

  • Host cell infection models

  • Biofilm vs. planktonic growth

• Regulatory Mechanisms:

  • Transcriptional control through specific regulators

  • Post-transcriptional regulation via RNA structures

  • Translational control mechanisms

  • Protein stability and turnover

For B. henselae, which transitions between mammalian hosts and arthropod vectors, expression studies should include conditions mimicking these different environments. The slow-growing nature of B. henselae (cultures typically held for a minimum of 21 days ) should be considered when designing such experiments.

What other ribosomal proteins interact with L9 in the context of the B. henselae ribosome?

Mapping the interaction network of L9 provides insights into its structural and functional roles:

• Experimental Approaches:

  • Crosslinking coupled with mass spectrometry

  • Co-immunoprecipitation followed by protein identification

  • Yeast two-hybrid screening using L9 as bait

  • Proximity labeling in live bacteria

• Expected Interactions:

  • Direct contacts with neighboring ribosomal proteins

  • Potential interactions with translation factors

  • Possible associations with ribosome assembly factors

  • Connections with RNA modification enzymes

• Functional Implications:

  • Interactions may stabilize specific ribosomal conformations

  • Some contacts might only form during specific translation events

  • Regulatory interactions could influence L9's role in translation

The unusual extended structure of L9 suggests it may form long-distance contacts within the ribosome that are not typical of other ribosomal proteins, potentially contributing to its proposed role as a molecular strut .

How does L9 protein sequence and function compare across different Bartonella species?

Comparative analysis across Bartonella species offers evolutionary and functional insights:

• Sequence Comparison:

  • Multiple sequence alignment of L9 proteins from different Bartonella species

  • Identification of conserved and variable regions

  • Phylogenetic analysis to track L9 evolution within the genus

• Functional Conservation:

  • Cross-complementation studies in L9-deficient strains

  • Comparative RNA binding studies

  • Assessment of species-specific properties

• Structural Implications:

  • Modeling of species-specific variations on the conserved L9 fold

  • Prediction of how sequence differences might affect domain interactions

  • Evaluation of surface properties that might influence function

The available sequence data for B. quintana L9 protein (193 amino acids ) provides a starting point for such comparative analyses. Understanding these differences may provide insights into species-specific aspects of ribosome function in Bartonella.