Recombinant Bartonella henselae Ribosomal RNA large subunit methyltransferase E (rlmE)

What is Bartonella henselae Ribosomal RNA large subunit methyltransferase E (rlmE) and what is its biological significance?

Bartonella henselae Ribosomal RNA large subunit methyltransferase E (rlmE), also known as ftsJ, rrmJ, or BH01550, is an enzyme (EC 2.1.1.166) that catalyzes the 2'-O-methylation of uridine at position 2552 in bacterial 23S ribosomal RNA . This post-transcriptional modification is critical for proper ribosome assembly, structure, and function.

rlmE belongs to the class of S-adenosylmethionine (SAM)-dependent methyltransferases that transfer methyl groups to specific nucleotides in rRNA. The enzyme's activity directly impacts ribosomal subunit maturation, which in turn affects translation efficiency and fidelity. As B. henselae is a fastidious, gram-negative bacterium responsible for cat scratch disease and other infections, understanding the function of its ribosomal modification enzymes provides insights into bacterial adaptation and potential virulence mechanisms .

Methodologically, researchers identify rlmE through comparative genomic analysis, evaluating sequence conservation across bacterial species, and confirming its function through in vitro enzymatic assays with purified recombinant protein and appropriate RNA substrates.

How is recombinant B. henselae rlmE typically expressed and purified for research applications?

Recombinant B. henselae rlmE is typically expressed using heterologous expression systems. According to available product information, the protein can be expressed in E. coli, yeast, baculovirus, or mammalian cell systems depending on research requirements . The methodological approach follows these key steps:

• Gene cloning: The rlmE gene (BH01550) is PCR-amplified from B. henselae genomic DNA and cloned into an expression vector containing appropriate fusion tags (His-tag, GST, or others) to facilitate purification.

• Expression optimization: Conditions are adjusted to maximize soluble protein yield, typically using lower temperatures (16-20°C) and optimized induction parameters to prevent inclusion body formation.

• Purification: Affinity chromatography (e.g., Ni-NTA for His-tagged proteins) followed by size exclusion chromatography to achieve >90% purity, as typically required for biochemical studies .

• Quality control: Purified protein undergoes verification by SDS-PAGE, Western blotting, and activity assays to confirm identity, purity, and functionality before experimental use.

• Storage: The purified protein is stored in buffer containing glycerol at -20°C for medium-term storage or -80°C for long-term storage, with working aliquots maintained at 4°C for up to one week to minimize freeze-thaw damage .

This systematic approach ensures production of functional recombinant rlmE suitable for subsequent biochemical and structural studies.

What are the optimal buffer conditions for maintaining rlmE stability and activity?

Maintaining stability and activity of recombinant B. henselae rlmE requires careful optimization of buffer conditions. Based on standard practices for similar methyltransferases and specific recommendations for rlmE, the following conditions are typically employed:

Additional stabilizing factors may include:

• Addition of S-adenosylmethionine (SAM) at low concentrations (50-100 μM)

• Presence of divalent cations (1-5 mM Mg²⁺)

• Buffer additives such as trehalose or sucrose (5-10%)

Researchers should empirically determine the optimal conditions for their specific recombinant rlmE preparation through thermal shift assays or activity measurements under varying conditions. Buffer optimization is crucial for ensuring reliable and reproducible results in enzymatic studies.

How can recombinant rlmE be used in studying B. henselae pathogenesis mechanisms?

Recombinant rlmE offers several innovative approaches for investigating B. henselae pathogenesis:

• Ribosome modification studies: As rlmE methylates 23S rRNA, researchers can investigate how this modification influences translation efficiency of virulence factors. Comparisons between wild-type and rlmE-deficient strains can reveal whether ribosomal RNA methylation affects expression of pathogenicity determinants.

• Stress response investigation: Similar to other Bartonella proteins such as heme-binding proteins that protect against oxidative stress during host cell invasion , rlmE-mediated rRNA modifications may contribute to bacterial survival under stress conditions encountered during infection.

• Host cell interaction models: Recombinant rlmE can be used to study whether this enzyme interacts with host cell components beyond its canonical role in ribosome modification. Some bacterial proteins have moonlighting functions during infection processes.

• Antibody development: Purified recombinant rlmE can serve as an antigen for raising specific antibodies to track protein expression during different infection stages or in different tissues, similar to approaches used with other B. henselae antigens like Pap31 .

• Drug target evaluation: As a bacterial-specific enzyme critical for ribosome function, rlmE represents a potential therapeutic target. Recombinant protein enables high-throughput screening for specific inhibitors that could be developed into novel antibiotics.

These applications build upon established research approaches for other B. henselae proteins, such as the heme binding proteins that have been shown to play crucial roles in oxidative stress defense during cell and flea invasion .

What techniques can be used to measure methyltransferase activity of recombinant rlmE?

Multiple complementary techniques can be employed to measure the methyltransferase activity of recombinant B. henselae rlmE:

• Radioisotope-based assays:

  • ³H-SAM or ¹⁴C-SAM incorporation into RNA substrates

  • Filter binding followed by scintillation counting

  • Thin-layer chromatography separation of methylated products

  • Advantages: High sensitivity (picomole detection)

  • Limitations: Requires radioisotope handling facilities

• Mass spectrometry approaches:

  • MALDI-TOF MS to detect mass shifts in RNA oligonucleotides

  • LC-MS/MS for precise mapping of methylation sites

  • RNA digestion followed by analysis of modified nucleosides

  • Advantages: Definitive identification of modification position and chemistry

  • Limitations: Requires specialized equipment and expertise

• Enzyme-coupled assays:

  • Detection of S-adenosylhomocysteine (SAH) produced during methylation

  • Coupling to SAH hydrolase and adenosine deaminase

  • Spectrophotometric or fluorometric detection

  • Advantages: Continuous monitoring, adaptable to high-throughput screening

  • Limitations: Potential interference from coupling enzymes

• RNA structure probing:

  • Differential sensitivity of methylated vs. unmethylated positions to chemical probes

  • Primer extension analysis to identify modification sites

  • Advantages: Can be used with complex RNA substrates

  • Limitations: Indirect measure of methylation

Table: Comparison of Key Parameters for rlmE Activity Assays

For comprehensive characterization, researchers should employ multiple orthogonal techniques to confirm rlmE activity and specificity.

How does rlmE compare functionally with homologous methyltransferases from other bacterial species?

Comparative analysis of B. henselae rlmE with homologous methyltransferases from other bacterial species reveals important evolutionary and functional relationships:

• Sequence conservation analysis:

  • rlmE belongs to the FtsJ/RrmJ family of methyltransferases, which are widely distributed across bacterial species

  • Key catalytic residues in the SAM-binding domain are highly conserved

  • RNA-recognition elements show greater variability, potentially reflecting species-specific substrate preferences

• Substrate specificity comparison:

  • Most bacterial rlmE homologs specifically methylate U2552 in 23S rRNA

  • The local RNA structure recognized by these enzymes is generally conserved

  • Species-specific differences may exist in secondary target sites or methylation efficiency

• Expression and regulation patterns:

  • In B. henselae, expression patterns may be adapted to its unique lifecycle involving mammalian hosts and arthropod vectors

  • Similar to other B. henselae proteins like heme binding proteins, expression might be regulated by environmental factors such as oxygen, temperature, and nutrient availability

• Role in bacterial physiology:

  • The methylation activity of rlmE contributes to ribosome biogenesis and function across bacterial species

  • In B. henselae, this activity may be particularly important during adaptation to different host environments

  • Comparative studies with other bacterial methyltransferases can highlight unique aspects of B. henselae rlmE function

The methodological approach for such comparative studies involves recombinant expression of rlmE from multiple species under identical conditions, side-by-side biochemical characterization, and complementation studies in heterologous systems. This strategy has proven effective for other B. henselae proteins, such as the heme binding proteins, where comparative analysis revealed their roles in oxidative stress response and host cell colonization .

What are the critical factors in designing experiments to characterize rlmE enzyme kinetics?

Designing robust experiments to characterize rlmE enzyme kinetics requires careful consideration of multiple factors:

• Substrate preparation and quality:

  • RNA substrate purity: Must be free of RNase contamination and pre-existing modifications

  • RNA folding: Consistent secondary structure should be verified by native gel electrophoresis

  • SAM quality: Fresh preparation is essential as SAM is unstable; purity should be >95%

  • Concentration ranges: Should span at least 0.2-5× Km for accurate parameter determination

• Reaction condition optimization:

  • Temperature: Typically 25-37°C, matching physiological conditions

  • pH optimization: Series of buffers covering pH 6.5-8.5

  • Ionic strength: Affects RNA structure and enzyme-substrate interactions

  • Time course: Initial velocity measurements require linear reaction progress (<15% substrate conversion)

• Controls and validations:

  • Enzyme concentration dependence: Verify linearity with enzyme concentration

  • Heat-inactivated enzyme controls: Essential for background correction

  • Positive controls: Known methyltransferases with established kinetic parameters

  • Substrate controls: Pre-methylated RNA to verify assay specificity

• Data collection and analysis considerations:

  • Replicates: Minimum of triplicate measurements for each data point

  • Model selection: Michaelis-Menten vs. allosteric models based on data behavior

  • Global fitting approaches for complex kinetic mechanisms

  • Statistical validation: Residual analysis, confidence intervals for parameters

• Physiological relevance:

  • Comparison of in vitro conditions with bacterial intracellular environment

  • Inclusion of potential physiological regulators (ions, nucleotides, proteins)

  • Correlation with in vivo activity when possible

These methodological considerations are particularly important for rlmE, as methyltransferase activity can be influenced by subtle changes in reaction conditions. A systematic approach to enzyme kinetics characterization provides the foundation for understanding rlmE's role in B. henselae biology and potential contributions to pathogenesis.

How can site-directed mutagenesis be applied to study rlmE functional domains?

Site-directed mutagenesis provides powerful insights into rlmE structure-function relationships. A comprehensive experimental approach includes:

• Target selection for mutagenesis:

  • Conserved residues identified through multiple sequence alignment of rlmE homologs

  • Predicted catalytic residues in the SAM-binding domain (typically a G-X-G-X-G motif)

  • RNA substrate recognition residues

  • Structural elements identified through homology modeling

• Systematic mutation strategy:

  • Alanine scanning: Systematic replacement with alanine to identify essential residues

  • Conservative substitutions: Maintaining similar chemical properties to test specific interactions

  • Non-conservative substitutions: Altering chemical properties to disrupt specific functions

  • Domain deletions or swaps to investigate larger functional units

• Expression and purification of mutants:

  • Identical conditions for wild-type and mutant proteins

  • Verification of proper folding through circular dichroism or thermal shift assays

  • Solubility and stability assessment before functional studies

• Functional characterization:

  • Activity assays comparing wild-type and mutant proteins

  • Binding studies with SAM and RNA substrates

  • Structural studies of key mutants when possible

Table: Suggested Priority Mutations for B. henselae rlmE Functional Analysis

This approach has been effectively employed for other enzymes in Bartonella species, such as studies on heme binding proteins that identified key residues involved in heme coordination and protein stability . Similar methodology can reveal the structural basis for rlmE activity and specificity.

What are the best approaches for investigating rlmE substrate specificity?

Determining rlmE substrate specificity requires a multi-faceted approach combining biochemical, structural, and computational methods:

• RNA substrate library screening:

  • Synthetic RNA oligonucleotides with variations around the target site

  • Systematic mutations of key nucleotides in the recognition sequence

  • Testing RNA fragments of different lengths to determine minimal substrate requirements

  • Competition assays between canonical and variant substrates

• Structural approaches:

  • RNA footprinting to identify protected regions upon rlmE binding

  • Chemical probing of RNA-protein complexes

  • Crystallography or cryo-EM analysis of rlmE-RNA complexes

  • Molecular docking and MD simulations to predict binding interactions

• Comparative analysis:

  • Testing rlmE against rRNA from different species

  • Comparing activity on different ribosomal assembly intermediates

  • Analysis of activity on non-ribosomal RNA substrates

  • Cross-species complementation studies

• Specificity validation techniques:

  • Mass spectrometry mapping of methylation sites

  • Next-generation sequencing approaches for genome-wide methylation profiling

  • Single-molecule techniques to observe individual binding events

  • Quantitative binding studies (ITC, SPR) with various RNA substrates

• Biological context validation:

  • Testing substrate recognition in the context of ribosomal subunits

  • In vivo analysis of methylation patterns in wild-type vs. rlmE-deficient strains

  • Correlation of in vitro specificity with cellular methylation profiles

This comprehensive approach provides a thorough characterization of rlmE substrate specificity, which is critical for understanding its biological function in B. henselae. Similar methodologies have been successfully applied to other B. henselae proteins, such as in studies of heme binding proteins that demonstrated specific interactions with host cellular components .

What strategies can researchers employ to overcome solubility issues with recombinant rlmE?

Solubility challenges are common when working with recombinant methyltransferases like rlmE. A systematic troubleshooting approach includes:

• Expression system optimization:

  • Testing multiple expression hosts: E. coli, yeast, baculovirus, or mammalian cells, as suggested for rlmE

  • Evaluating different E. coli strains (BL21, Rosetta, Arctic Express)

  • Adjusting expression temperature (16-30°C) and induction conditions

  • Using specialized strains for codon optimization or disulfide bond formation

• Protein construct engineering:

  • Testing different fusion tags (His, GST, MBP, SUMO)

  • Optimizing tag position (N-terminal vs. C-terminal)

  • Using solubility-enhancing fusion partners (MBP, NusA, TrxA)

  • Creating truncated constructs based on domain predictions

  • Removing flexible regions identified through disorder prediction

• Buffer optimization:

  • Systematic pH screening (typically pH 6.5-8.5)

  • Salt concentration variation (100-500 mM NaCl)

  • Addition of stabilizing agents:

    • Glycerol (10-20%)

    • Arginine/glutamic acid (50-100 mM)

    • Mild detergents (0.01-0.05% Triton X-100)

    • SAM cofactor at low concentrations (50-100 μM)

• Purification strategy adjustment:

  • Rapid processing to prevent degradation

  • Inclusion of protease inhibitors

  • On-column refolding for proteins recovered from inclusion bodies

  • Size exclusion chromatography to remove aggregates

Table: Solubility Enhancement Strategies for Recombinant rlmE

These strategies have proven effective for other challenging B. henselae proteins, such as the heme binding proteins that required careful optimization to obtain functional recombinant protein for structural and functional studies .

How can researchers control for and eliminate potential contaminating enzymatic activities?

Controlling for contaminating enzymatic activities is critical for accurate characterization of rlmE. A comprehensive control strategy includes:

• Protein purification controls:

  • Multi-step purification protocol (affinity, ion exchange, size exclusion)

  • Activity assays on different purification fractions to track specific activity

  • SDS-PAGE and Western blotting to confirm purity

  • Mass spectrometry analysis to identify potential contaminants

• Enzymatic controls:

  • Catalytically inactive rlmE mutants as negative controls

  • Heat-inactivation of enzyme samples (95°C for 10 minutes)

  • Including specific methyltransferase inhibitors

  • Testing buffer-only and substrate-only conditions

• Substrate specificity verification:

  • Using pre-methylated substrates to detect additional methylation activities

  • Testing non-target RNA sequences as negative controls

  • Precise mapping of methylation sites using mass spectrometry

  • Competition assays between genuine and non-specific substrates

• Host-derived contamination controls:

  • Mock purifications from non-transformed expression host

  • Expression in methyltransferase-deficient strains when possible

  • Immunodepletion using antibodies against potential contaminating enzymes

  • Testing activity under conditions that differentially affect rlmE vs. contaminants

• Validation approaches:

  • Correlation of activity with protein concentration

  • Inhibition profile characteristic of the target enzyme

  • Activity reconstitution with purified components

  • Comparison with recombinant enzyme from different expression systems

This systematic approach ensures that observed enzymatic activities can be confidently attributed to rlmE rather than contaminants. Similar control strategies have been successfully employed in studies of other B. henselae enzymes, such as in the characterization of heme binding proteins where specific activities needed to be distinguished from host-derived functions .

What approaches can help researchers interpret conflicting or unexpected results in rlmE studies?

When faced with conflicting or unexpected results in rlmE studies, researchers should implement a systematic troubleshooting and validation approach:

• Data verification and quality control:

  • Repeat experiments with fresh reagents and enzyme preparations

  • Verify RNA substrate integrity and SAM quality

  • Check for instrument calibration issues or detection method artifacts

  • Implement additional controls to identify potential interfering factors

• Methodological cross-validation:

  • Apply multiple orthogonal techniques to measure the same parameter

  • For example, confirm methylation with both radioisotope incorporation and mass spectrometry

  • Compare results from different enzyme activity assays

  • Test under varied but controlled conditions to identify condition-dependent effects

• Biological context consideration:

  • Evaluate whether unexpected results might reflect genuine biological complexity

  • Consider potential regulatory mechanisms affecting rlmE activity

  • Examine if observed effects match known behaviors of homologous enzymes

  • Investigate potential moonlighting functions of rlmE beyond canonical activity

• Systematic hypothesis testing:

  • Develop multiple alternative hypotheses to explain unexpected results

  • Design critical experiments to discriminate between competing explanations

  • Use site-directed mutagenesis to test structure-function hypotheses

  • Consider computational modeling to interpret complex kinetic data

• Literature and collaboration:

  • Review literature on related methyltransferases for similar phenomena

  • Consult with specialists in enzyme kinetics or RNA modifications

  • Consider whether observed results parallel findings with other B. henselae proteins

Research on B. henselae proteins has revealed complex regulatory mechanisms and condition-dependent activities, as demonstrated in studies of heme binding proteins whose functions vary under different environmental conditions . Similarly, unexpected findings with rlmE may reveal novel aspects of its regulation or function in B. henselae biology.

What novel applications of recombinant rlmE could advance B. henselae research and diagnostics?

Recombinant rlmE offers several promising applications for advancing both basic research and diagnostic capabilities:

• Structural biology applications:

  • High-resolution structure determination of B. henselae rlmE

  • Structure-guided design of specific inhibitors as potential therapeutics

  • Comparative structural analysis with homologs from other pathogens

  • Investigation of conformational changes during catalysis

• Diagnostic development:

  • Similar to chimeric proteins developed for B. henselae immunodiagnostics , rlmE or its immunogenic epitopes could be incorporated into diagnostic platforms

  • Development of anti-rlmE antibodies for immunohistochemical detection of B. henselae in tissues

  • Potential inclusion in multiplex serological assays for comprehensive bartonellosis diagnosis

• Vaccine research:

  • Evaluation of rlmE as a potential vaccine antigen

  • Investigation of immune responses to rlmE during natural infection

  • Creation of attenuated strains with modified rlmE activity for vaccine development

• Drug discovery:

  • High-throughput screening for rlmE inhibitors

  • Structure-activity relationship studies of identified inhibitors

  • Development of rlmE-targeted antimicrobials with reduced resistance potential

• Systems biology integration:

  • Incorporation of rlmE function into comprehensive models of B. henselae translation

  • Investigation of rlmE interactions with other cellular components

  • Comparative 'omics' studies between wild-type and rlmE-modified strains

These applications build upon methodologies successfully applied to other B. henselae proteins, such as recombinant chimeric proteins used in ELISA-based diagnostics for feline bartonellosis and the Pap31 protein evaluated for serodiagnosis .

How might research on rlmE contribute to understanding B. henselae pathogenesis mechanisms?

Research on rlmE has the potential to significantly advance our understanding of B. henselae pathogenesis through several mechanisms:

• Ribosome function and stress adaptation:

  • Investigation of how rlmE-mediated rRNA modification affects translation of virulence factors

  • Analysis of whether rlmE activity is modulated during different infection stages

  • Determination if rlmE contributes to bacterial adaptation to host environments

  • Comparison with other stress response mechanisms like those involving heme binding proteins

• Host-pathogen interaction studies:

  • Evaluation of whether rlmE-deficient B. henselae shows altered virulence in infection models

  • Investigation of potential interactions between rlmE and host cellular components

  • Assessment of whether rlmE contributes to immune evasion strategies

  • Comparison with other B. henselae factors known to influence host cell colonization

• Vector transmission biology:

  • Analysis of rlmE expression and activity during arthropod vector stages

  • Determination if rlmE plays a role in bacterial survival within the flea vector, similar to heme binding proteins

  • Evaluation of whether rlmE modification contributes to transmission efficiency

• Evolutionary adaptations:

  • Comparative analysis of rlmE across Bartonella species with different host preferences

  • Investigation of whether rlmE sequence variations correlate with host specificity

  • Assessment of rlmE as part of the core or accessory genome in Bartonella evolution

• Therapeutic target potential:

  • Evaluation of whether targeting rlmE could reduce bacterial colonization or persistence

  • Investigation of synergistic effects between rlmE inhibitors and conventional antibiotics

  • Assessment of resistance development potential against rlmE-targeted therapeutics

This research would complement existing knowledge about B. henselae virulence factors, such as the heme binding proteins that have been shown to play crucial roles in oxidative stress defense, host cell colonization, and vector survival .

What emerging technologies could enhance future research on B. henselae rlmE and related enzymes?

Several cutting-edge technologies offer promising approaches for advancing research on B. henselae rlmE:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for high-resolution structure determination without crystallization

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics

  • Single-particle analysis of rlmE-ribosome complexes

  • Integrative structural biology combining multiple data sources

• Next-generation RNA modification analysis:

  • Nanopore direct RNA sequencing for detecting methylated nucleotides

  • MINT-seq (Methylation Isoform sequencing) for transcriptome-wide methylation mapping

  • Epitranscriptomic profiling to identify all rlmE targets in vivo

  • CRISPR-Cas13 systems for targeted RNA modification detection

• Advanced protein engineering:

  • Directed evolution to generate rlmE variants with enhanced properties

  • Computational design of rlmE with altered specificity or activity

  • Split protein complementation for cellular activity sensors

  • Optogenetic control of rlmE activity for temporal studies

• Single-cell technologies:

  • Single-cell RNA-seq to detect heterogeneity in rlmE expression

  • Time-resolved single-cell proteomics

  • Microfluidic systems for high-throughput single bacterium analysis

  • Intracellular biosensors for monitoring rlmE activity in living cells

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Machine learning for identifying patterns in large-scale datasets

  • Network analysis to position rlmE in bacterial response networks

  • Quantitative modeling of ribosome biogenesis incorporating rlmE activity

These emerging technologies could provide unprecedented insights into rlmE function and regulation, similar to how advanced techniques have revealed complex roles for other B. henselae proteins such as the heme binding proteins in pathogenesis and Pap31 in diagnostics .