Recombinant Bartonella henselae 50S ribosomal protein L31 (rpmE)

What is the functional significance of ribosomal protein L31 in Bartonella henselae?

Ribosomal protein L31 (bL31) in B. henselae, like in other bacteria, serves dual critical functions in bacterial physiology. Primarily, it acts as an integral component of the 50S ribosomal subunit, playing a crucial role in the formation of the protein-protein intersubunit bridge B1b, which contributes significantly to ribosome dynamics during translation . As a component of this bridge, L31 facilitates communication between the large and small ribosomal subunits, affecting translational efficiency and fidelity.

Beyond its structural role, recent research has demonstrated that L31 functions as an autogenous repressor, regulating its own gene expression by binding to a highly conserved stem-loop structure in the 5′UTR of the rpmE mRNA . This self-regulation mechanism represents an important control point in ribosome biogenesis, allowing the bacterium to maintain appropriate stoichiometric ratios of ribosomal components.

How is the rpmE gene organized and regulated in Bartonella species?

The rpmE gene encoding L31 protein in Bartonella species is subject to sophisticated regulatory control. In model systems, the rpmE gene is transcribed from two promoter regions, generating two distinct mRNA transcripts . Both transcripts are subject to feedback regulation by the L31 protein itself, indicating that the autogenous operator is located within the shorter transcript .

Phylogenetic analyses have revealed a highly conserved stem-loop structure in the rpmE 5′UTR that serves as the translational operator targeted by L31 . This stable operator stem-loop has a unique arrangement where it separates an AU-rich translational enhancer from a Shine-Dalgarno element, creating what researchers describe as a "rare case of a noncontiguous translation initiation region" . This structure likely plays a critical role in the fine-tuning of L31 expression in response to cellular needs.

What are the structural characteristics that enable L31's dual function?

The L31 protein possesses distinct structural features that facilitate its dual roles in ribosome structure and gene regulation. Computational and experimental analyses classify L31 as an RNA-binding protein, consistent with both its ribosomal role and its function as a repressor .

A key structural feature is its unstructured amino-terminal region, which is enriched in lysine residues. Mutational analysis has demonstrated that this N-terminal domain is necessary for the protein's repressor activity . This positively charged region likely interacts with the negatively charged RNA backbone of the operator stem-loop structure in the rpmE 5′UTR.

The protein's ability to bind specific RNA structures allows it to recognize both its ribosomal integration site and the regulatory elements in its own mRNA, making it an efficient dual-function molecule in bacterial cells.

What expression systems are most effective for producing functional recombinant B. henselae L31 protein?

For the expression of recombinant B. henselae L31 protein, E. coli-based systems have proven most effective among bacterial expression platforms. When selecting an expression system, researchers should consider several factors specific to the L31 protein:

• Codon optimization: B. henselae has different codon usage patterns compared to E. coli. Optimizing the coding sequence for E. coli expression can significantly improve yield. This is particularly important for the lysine-rich N-terminal region of L31, as lysine codons (AAA and AAG) show significant bias between species .

• Expression vector selection: pET-based vectors with T7 promoter systems offer strong induction and high-level expression suitable for L31. Including a His-tag for purification is recommended, preferably at the C-terminus to preserve the functionally important N-terminal domain .

• Expression conditions: For optimal expression while maintaining protein solubility, induction at lower temperatures (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM) is advantageous, as this slows protein production and allows proper folding .

• Host strain selection: BL21(DE3) derivatives with enhanced disulfide bond formation capabilities are recommended, as the zinc-binding properties of L31 may involve cysteine residues and proper metal coordination .

A comparison of bacterial and eukaryotic expression systems for B. henselae L31 production shows:

How can the dual functionality of L31 as both a ribosomal component and gene regulator be studied separately?

Investigating the dual functionality of L31 requires sophisticated experimental approaches that can distinguish between its ribosomal structural role and its regulatory function. The following methodological framework enables researchers to dissect these distinct functions:

This integrated approach allows researchers to attribute specific phenotypic effects to either the structural or regulatory function of L31, providing a comprehensive understanding of this multifunctional protein.

What are the implications of L31's RNA-binding properties for understanding bacterial translational regulation?

The RNA-binding capabilities of L31 have significant implications for bacterial translational regulation beyond its direct role in ribosome structure. As computational analyses classify L31 as an RNA-binding protein , its interaction with specific RNA structures provides insight into how bacteria coordinate ribosome assembly with translational activity.

L31's ability to recognize and bind to a highly conserved stem-loop structure in its own mRNA represents a sophisticated feedback mechanism. This stem-loop separates an AU-rich translational enhancer from the Shine-Dalgarno element, creating what researchers describe as "a rare case of a noncontiguous translation initiation region" . This arrangement allows for precise control over L31 synthesis based on the protein's availability in the cell.

The regulatory mechanism employed by L31 may serve as a model for understanding how other ribosomal proteins with RNA-binding properties might moonlight as translational regulators. This dual functionality creates an efficient coupling between ribosome assembly and the expression of ribosomal components, ensuring stoichiometric production of these essential elements.

Moreover, the RNA-binding capabilities of L31 may influence how Bartonella interacts with host cells during infection. As a component of the translation machinery, L31's regulatory activity could potentially be modulated during host-pathogen interactions, affecting bacterial protein synthesis in response to environmental cues within the host.

What purification strategies yield the highest quality recombinant B. henselae L31 protein for functional studies?

Purifying recombinant B. henselae L31 protein with preserved functionality requires a carefully optimized protocol. The following comprehensive strategy addresses the unique characteristics of this ribosomal protein:

• Initial capture: Immobilized metal affinity chromatography (IMAC) with Ni-NTA resin is the preferred first step for His-tagged L31 purification. To preserve protein structure:

  • Use mild lysis conditions with non-ionic detergents (0.1% Triton X-100)

  • Include protease inhibitors and reducing agents (1-5 mM β-mercaptoethanol)

  • Maintain buffers at pH 7.5-8.0 for optimal His-tag binding

• Intermediate purification: Ion exchange chromatography exploits L31's basic properties:

  • Employ cation exchange (SP or CM Sepharose) at pH 6.5-7.0

  • Use a shallow salt gradient (0-500 mM NaCl) for resolution

  • This step effectively separates L31 from remaining E. coli proteins

• Polishing step: Size exclusion chromatography provides final purification:

  • Superdex 75 or similar matrix appropriate for the ~7-8 kDa L31 protein

  • Buffer containing 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 5% glycerol

  • This step removes aggregates and provides information about oligomeric state

• Quality assessment: Multiple analytical techniques ensure protein integrity:

  • SDS-PAGE (>90% purity should be achieved)

  • Western blotting with anti-His antibodies

  • Mass spectrometry to confirm molecular weight and detect modifications

  • Circular dichroism to assess secondary structure content

• Storage considerations:

  • Store in small aliquots at -80°C with 10% glycerol

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be maintained at 4°C for up to one week

Following this protocol typically yields 3-5 mg of >95% pure L31 protein per liter of bacterial culture, suitable for functional and structural studies.

How can site-directed mutagenesis be used to identify critical residues for L31's regulatory function?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationship of B. henselae L31 protein, particularly for identifying residues critical for its regulatory activity. Research has established that the unstructured amino-terminal region enriched in lysine residues is necessary for repressor activity . A systematic mutagenesis strategy should include:

• N-terminal lysine cluster analysis:

  • Create alanine substitutions of each lysine residue in the N-terminal domain

  • Generate multiple mutants with combinations of lysine substitutions

  • Assess the impact on regulatory function using reporter assays (e.g., lacZ fusions)

• Targeting the RNA-binding interface:

  • Identify potential RNA-binding residues through structure prediction and homology modeling

  • Create conservative substitutions that maintain charge but alter side chain geometry

  • Assess both RNA binding capacity (through EMSA) and regulatory function

• Mutagenesis of residues involved in ribosome integration:

  • Target residues predicted to participate in intersubunit bridge B1b formation

  • Evaluate effects on both ribosome assembly and regulatory function

  • This approach helps distinguish residues with dual roles from those specific to one function

• Cysteine residue analysis:

  • If zinc coordination is involved in L31 function, systematically mutate cysteine residues

  • Assess impact on protein stability, RNA binding, and regulatory activity

• Methodology refinements:

  • Employ overlap extension PCR for creating precise mutations

  • Verify all constructs by Sanger sequencing to confirm correct mutations

  • Express mutant proteins under identical conditions for comparative analysis

This comprehensive mutagenesis approach enables the construction of a detailed functional map of the L31 protein, identifying specific amino acids and structural elements required for its regulatory activity versus its ribosomal role.

How can recombinant B. henselae L31 be utilized for developing diagnostic tools for Bartonelloses?

While current research indicates that recombinant Pap31 protein has limitations as a diagnostic target due to either low sensitivity or questionable specificity , the potential of recombinant L31 protein for diagnostic applications warrants investigation. A methodological framework for evaluating L31's diagnostic utility includes:

• Immunoreactivity assessment:

  • Express and purify recombinant B. henselae L31 using optimized protocols

  • Develop ELISA and Western blot assays to test reactivity with sera from confirmed Bartonella-infected humans and dogs

  • Compare sensitivity and specificity with current diagnostic antigens like Pap31

• Epitope mapping:

  • Fragment the L31 protein into distinct domains (similar to the approach used with Pap31's N-terminal, middle, and C-terminal domains)

  • Identify immunodominant regions that generate the strongest and most specific antibody responses

  • Develop refined antigens based on these immunoreactive epitopes

• Multiplex diagnostic platform development:

  • Combine L31-derived antigens with other Bartonella immunoreactive proteins

  • Assess whether a multi-antigen approach improves diagnostic sensitivity without compromising specificity

  • Validate using well-characterized sample panels from both humans and animals

• Species-specific detection:

  • Analyze sequence variability of L31 across Bartonella species

  • Identify species-specific epitopes that could distinguish infections by different Bartonella species

  • Develop tests capable of differentiating B. henselae from other species like B. vinsonii subsp. berkhoffii

Current diagnostic challenges with Bartonelloses include numerous false negative results with existing modalities . Whether L31 can overcome these limitations remains to be determined, but its conserved nature and essential function make it a candidate worth exploring for improved serodiagnostic assays.

What are the technical difficulties in studying B. henselae ribosomal proteins and how can they be overcome?

Research on B. henselae ribosomal proteins, including L31, faces several technical challenges that require specialized approaches to overcome:

• Cultivation challenges:

  • B. henselae is a slow-growing, fastidious organism

  • Methodological solution: Develop optimized media formulations with hemin supplementation and extended incubation periods (7-14 days)

  • Alternative approach: Focus on recombinant protein expression in more tractable systems like E. coli

• Protein solubility and stability issues:

  • Ribosomal proteins often aggregate when expressed recombinantly due to their charged surfaces

  • Solution: Expression with solubility-enhancing tags (MBP, SUMO) and optimization of buffer conditions with stabilizing agents like glycerol and reducing agents

  • Additional approach: Co-expression with chaperones or ribosomal RNA fragments that normally interact with the protein

• Ribosome heterogeneity:

  • B. henselae may produce heterogeneous ribosomes with different protein compositions under varying conditions

  • Solution: Implement gradient-based separation techniques to isolate and characterize distinct ribosome populations

  • Analysis method: Quantitative mass spectrometry to determine stoichiometry of ribosomal proteins in different fractions

• Functional verification challenges:

  • Confirming dual functionality (structural and regulatory) requires sophisticated assays

  • Solution: Develop in vitro transcription-translation systems using B. henselae components

  • Alternative: Create hybrid ribosomes with labeled L31 to track incorporation and functional impact

• Limited genetic tools:

  • Genetic manipulation of B. henselae is challenging compared to model organisms

  • Solution: Adapt CRISPR-Cas9 systems for targeted genome editing in Bartonella

  • Alternative: Develop conditional expression systems to study essential ribosomal proteins

By implementing these technical solutions, researchers can overcome the inherent difficulties in studying B. henselae ribosomal proteins and advance our understanding of their structure, function, and potential applications.

What are promising future research directions for B. henselae L31 protein studies?

Future research on B. henselae ribosomal protein L31 should explore several promising directions that could yield significant insights into bacterial physiology, pathogenesis, and potential therapeutic applications:

• Structural biology approaches:

  • Determine high-resolution structures of L31 both in isolation and within the context of the B. henselae ribosome

  • Apply cryo-EM to visualize the dynamic role of L31 in intersubunit bridge B1b formation during translation

  • Characterize the structural basis of L31's interaction with its mRNA operator sequence

• Ribosome heterogeneity and specialization:

  • Investigate whether B. henselae produces "specialized ribosomes" with altered L31 content under different growth conditions or during infection

  • Determine if L31 composition affects the translation of specific mRNA subsets, particularly virulence factors

  • Explore potential zinc-dependent regulation of L31 incorporation into ribosomes

• Host-pathogen interactions:

  • Examine L31 expression patterns during different stages of host cell infection

  • Investigate whether host factors interact with or modulate L31 function during intracellular growth

  • Determine if L31 contributes to Bartonella's ability to adapt to different mammalian hosts

• Antibiotic susceptibility and resistance:

  • Explore whether L31's role in bridge B1b formation influences sensitivity to antibiotics targeting translation

  • Investigate L31 as a potential novel target for antimicrobial development

  • Characterize how alterations in L31 might contribute to antibiotic tolerance or persistence

• Comparative genomics and evolution:

  • Analyze L31 sequence conservation across Bartonella species and correlate with host range or pathogenicity

  • Investigate horizontal gene transfer events involving rpmE and their potential impact on bacterial adaptation

  • Examine the co-evolution of L31 and its mRNA regulatory elements

These research directions capitalize on emerging technologies while addressing fundamental questions about ribosome function, gene regulation, and bacterial pathogenesis in this clinically important organism.