Recombinant Treponema denticola Ribosomal RNA large subunit methyltransferase E (rlmE)

What is RlmE and what is its primary function in bacterial ribosome assembly?

RlmE is a methyltransferase responsible for the 2'-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA. This enzyme plays a critical role in the late stages of 50S ribosomal subunit assembly. Unlike many other rRNA modification enzymes whose deletion has minimal effects, deletion of RlmE (ΔrlmE) results in a significant 2-4 fold decrease in bacterial growth rate compared to wild-type cells .

The enzyme's primary function involves promoting interdomain interactions via the association between helices 92 and 71, which is stabilized by the single 2'-O-methylation of Um2552. This interaction, in concert with the incorporation of ribosomal protein L36, triggers late steps of 50S subunit assembly . Without this modification, cells accumulate a 45S precursor particle that represents an incomplete large ribosomal subunit .

How does RlmE differ from other ribosomal RNA modification enzymes?

Unlike most bacterial rRNA modification enzymes that can be knocked out individually with minimal effects on growth rate, RlmE knockout has a notable impact on bacterial growth and ribosome assembly . RlmE typically synthesizes Um2552 late in ribosome biogenesis, whereas most other peptidyl transferase center (PTC) region modifications occur during early or intermediate stages of ribosome assembly .

The Um2552 modification is positioned adjacent to G2553, which is an essential base that anchors the 3' CCA terminus of the A-site tRNA in the peptidyl transferase center . This strategic location likely explains why its absence significantly impacts ribosome function and bacterial growth compared to other modifications.

What experimental evidence supports the role of RlmE in T. denticola virulence?

While direct experimental evidence specifically linking T. denticola RlmE to virulence is limited in the provided literature, several key observations can be made by extrapolation. T. denticola is strongly associated with severe periodontal disease and can stimulate pro-inflammatory responses . The bacterium's ability to invade connective tissue and disseminate throughout the host depends on properly functioning ribosomes for protein synthesis .

Given that ribosome assembly defects significantly compromise bacterial fitness, and that RlmE is critical for proper ribosome assembly, it is reasonable to hypothesize that RlmE plays an indirect but important role in T. denticola virulence. Proper ribosome function would be necessary for the expression of virulence factors such as the Major Surface Protein (MSP), which has been shown to induce the synthesis of pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, and MMP-9 in human monocytes .

How does the molecular mechanism of RlmE-mediated methylation contribute to ribosome assembly?

RlmE catalyzes the 2'-O-methylation of uridine at position 2552 in the 23S rRNA, creating Um2552. This modification has been shown to promote critical interdomain interactions within the ribosomal RNA, specifically between helices 92 and 71 . The methylation serves as a molecular trigger that induces structural changes necessary for the final maturation of the 50S ribosomal subunit.

In the absence of RlmE, cells accumulate a 45S precursor particle. In vitro experiments have demonstrated that this 45S particle is a genuine precursor that can be assembled into the 50S subunit when RlmE-mediated Um2552 formation occurs . This indicates that the methylation event is not merely decorative but functionally significant in promoting the transition from the 45S precursor to the mature 50S subunit.

The relationship between RlmE and ribosomal protein L36 (encoded by rpmJ) is particularly noteworthy. L36 is completely absent from the 45S precursor in ΔrlmE strains, and strong genetic interaction has been observed between rlmE and rpmJ . This suggests a coordinated action between RlmE-mediated methylation and L36 incorporation in the final stages of large subunit assembly.

What are the comparative differences between RlmE in T. denticola and other bacterial species like E. coli?

While the search results do not provide direct comparative data between T. denticola and E. coli RlmE, we can infer potential differences based on the general conservation patterns of rRNA modification enzymes across bacterial species.

In E. coli, RlmE is responsible for Um2552 formation in 23S rRNA and plays a crucial role in ribosome large subunit assembly . The enzyme functions in late stages of ribosome biogenesis, and its deletion leads to accumulation of the 45S precursor particle .

T. denticola, as a spirochete, has evolutionary distinctions from E. coli (a gamma-proteobacterium), which might result in specific adaptations in its ribosome assembly pathway and modification patterns. While the core function of RlmE is likely conserved due to the fundamental importance of ribosome assembly, differences might exist in:

• Protein sequence and structure

• Substrate recognition specificity

• Timing of the modification during ribosome assembly

• Interaction with other ribosome assembly factors

• Regulation of expression in response to environmental conditions

These potential differences could reflect adaptations to T. denticola's unique ecological niche in the oral cavity and its role in periodontal disease.

How does the absence of RlmE affect bacterial response to environmental stressors?

The absence of RlmE has profound effects on bacterial fitness, particularly under stress conditions. In E. coli, ΔrlmE strains exhibit severely compromised growth, especially at lower temperatures . This temperature sensitivity suggests that RlmE-mediated methylation may be particularly important for maintaining ribosome structural integrity and function under cold stress.

The functional redundancy observed between RNA modification enzymes and certain small GTPases is also relevant to stress responses. Research has shown that the severe assembly phenotype of ΔrlmE can be partially restored by overexpression of small GTPases from the Obg and EngA families . This suggests that cells have evolved alternative mechanisms to compensate for the absence of RlmE under certain conditions.

For T. denticola specifically, which resides in the periodontal pocket—an environment characterized by fluctuating oxygen levels, pH changes, and immune surveillance—the proper functioning of ribosomes would be crucial for stress adaptation. Since T. denticola must respond to the host immune system, including neutrophils and macrophages , ribosome integrity maintained by RlmE could be particularly important for survival during infection and stress response.

What are the recommended approaches for expressing and purifying recombinant T. denticola RlmE?

Based on established protocols for similar enzymes, the following methodological approach is recommended for expressing and purifying recombinant T. denticola RlmE:

Expression System Selection:

• E. coli BL21(DE3) or Rosetta strains are suitable hosts for heterologous expression

• Consider codon optimization for T. denticola genes in E. coli

• Use vectors with inducible promoters (T7 or tac) and affinity tags (His6, GST, or MBP)

Expression Conditions:

• Induce at OD600 0.6-0.8 with 0.1-0.5 mM IPTG

• Lower induction temperature (16-25°C) may improve solubility

• Extended expression time (16-20 hours) at lower temperatures

Purification Strategy:

• Affinity chromatography using tag-specific resins

• Ion exchange chromatography for further purification

• Size exclusion chromatography to obtain homogeneous protein

Protein Quality Assessment:

• SDS-PAGE and Western blotting to confirm identity

• Circular dichroism to assess secondary structure

• Thermal shift assays to evaluate stability

• Activity assays using in vitro transcribed 23S rRNA substrates

Since RlmE is a methyltransferase, purification under reducing conditions is advisable to maintain the integrity of any catalytically important cysteine residues.

How can researchers effectively design knockout and complementation studies for T. denticola RlmE?

Knockout Strategy:

• Gene Targeting: Design homologous regions flanking the rlmE gene for targeted deletion

• Selection Markers: Use appropriate antibiotic resistance cassettes (e.g., erythromycin or kanamycin resistance) for T. denticola

• Transformation Method: Electroporation is typically effective for T. denticola gene delivery

• Verification: Confirm gene deletion using PCR, Southern blot, and RT-PCR to verify absence of transcript

Complementation Approach:

• Vector Selection: Use shuttle vectors compatible with both E. coli and T. denticola

• Promoter Choice: Native promoter for physiological expression levels or inducible promoter for controlled expression

• Integration Options: Consider both plasmid-based and chromosomal integration approaches

• Control Constructs: Include catalytically inactive mutants (e.g., point mutations in the SAM-binding domain) to distinguish between enzymatic and structural roles

Phenotypic Analysis:

• Growth curve analysis at various temperatures (30°C, 37°C, 42°C)

• Ribosome profile analysis using sucrose gradient centrifugation

• Assessment of 45S to 50S conversion rates

• rRNA modification analysis by primer extension or mass spectrometry

• Virulence assessment in relevant infection models

This approach, similar to the methodology used for E. coli rlmE knockout studies , would allow for comprehensive characterization of T. denticola RlmE function.

What analytical techniques are most effective for assessing RlmE methyltransferase activity?

Several complementary techniques can be employed to effectively assess RlmE methyltransferase activity:

Biochemical Assays:

• Radioactive Methylation Assay: Using S-adenosyl-L-[methyl-³H]methionine (³H-SAM) as methyl donor, measuring incorporation into rRNA substrates

• MTase-Glo™ Assay: A bioluminescent assay detecting S-adenosylhomocysteine (SAH) production during methylation

• HPLC Analysis: To detect and quantify methylated nucleosides after enzymatic digestion of rRNA

Structural Analysis:

• Primer Extension: To identify methylation sites by reverse transcriptase stops

• Mass Spectrometry: For precise identification and quantification of modified nucleosides

• MALDI-TOF Analysis: For comparison of modified and unmodified oligonucleotides

Functional Assays:

• In vitro Ribosome Assembly: Assessing the ability of RlmE to convert 45S precursors to 50S subunits

• Translation Efficiency Tests: Using in vitro translation systems to measure functional impact of methylation

• Antibiotic Sensitivity Assays: As certain modifications affect antibiotic binding

Comparative Analysis Table for RlmE Activity Methods:

The combination of biochemical activity assays with structural confirmation and functional validation provides the most comprehensive assessment of RlmE methyltransferase activity.

How might RlmE function be connected to T. denticola pathogenesis in periodontal disease?

The connection between RlmE function and T. denticola pathogenesis likely involves several interrelated mechanisms:

Ribosome Function and Virulence Factor Expression:
RlmE is essential for proper ribosome assembly , and functional ribosomes are necessary for the translation of virulence factors. T. denticola expresses several virulence factors including Major Surface Protein (MSP), which has been shown to induce pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and MMP-9 in human monocytes . Compromised ribosome assembly due to RlmE deficiency would likely impair the production of these virulence factors.

Stress Response in the Periodontal Environment:
The periodontal pocket represents a challenging environment with fluctuating oxygen levels, nutrient availability, and host defense mechanisms. T. denticola must adapt to these stressors to persist and cause disease. Given that RlmE deletion results in compromised growth particularly under stress conditions like lower temperatures , it's reasonable to hypothesize that RlmE is important for T. denticola's adaptation to environmental stressors in the periodontal pocket.

Interaction with Host Immune System:
T. denticola interacts with neutrophils and macrophages, stimulating the release of inflammatory mediators such as Oncostatin M (OSM) . Proper ribosome function is necessary for the bacterium to respond to immune pressure, potentially by regulating the expression of immune evasion factors. RlmE's role in ensuring proper ribosome assembly would therefore indirectly influence immune evasion capabilities.

Biofilm Formation and Community Interactions:
T. denticola is part of the dysbiotic microbiome in periodontal disease, often found in association with other pathogens like Porphyromonas gingivalis . The ability to form and maintain biofilms requires the coordinated expression of numerous proteins, which depends on efficient translation by properly assembled ribosomes. RlmE deficiency could potentially impair biofilm formation and interspecies interactions within the periodontal pocket.

What approaches can be used to study the structural impact of RlmE-mediated methylation on ribosome conformation?

Multiple complementary approaches can be employed to study the structural impact of RlmE-mediated methylation:

Cryo-Electron Microscopy (Cryo-EM):

• Allows visualization of ribosomal conformational changes at near-atomic resolution

• Can compare structures of wild-type ribosomes versus those lacking Um2552 modification

• Enables identification of long-range structural effects of the methylation

• Can capture different conformational states during the assembly process

Chemical Probing Techniques:

• SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension)

• DMS (Dimethyl Sulfate) probing

• CMCT (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) modification

• These techniques can identify regions with altered RNA flexibility or accessibility due to Um2552 methylation

Biophysical Approaches:

• Analytical ultracentrifugation to assess conformational stability

• Small-angle X-ray scattering (SAXS) to detect large-scale conformational changes

• Nuclear magnetic resonance (NMR) for detailed analysis of specific rRNA fragments

Molecular Dynamics Simulations:

• In silico modeling of methylated versus unmethylated rRNA structures

• Prediction of conformational changes induced by Um2552

• Identification of potential long-range interaction networks affected by methylation

Structural probing experiments have revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2'-O-methylation of Um2552, in concert with the incorporation of L36 . This suggests that the methyl group affects not only local structure but also influences distant regions of the ribosome through a network of interactions.

What are the current limitations in studying T. denticola RlmE and how might they be overcome?

Current Limitations:

• Genetic Manipulation Challenges:
  T. denticola is more difficult to genetically manipulate than model organisms like E. coli. The efficiency of transformation is generally lower, and fewer genetic tools are available .

• Growth and Culture Considerations:
  T. denticola is an anaerobic bacterium with slow growth rates and specialized media requirements, making experimental work more time-consuming and technically challenging .

• Limited Structural Data:
  There is a lack of high-resolution structural information specifically for T. denticola ribosomes and modification enzymes, complicating structure-function analyses.

• Functional Redundancy:
  Potential redundancy among rRNA modification enzymes or between RlmE and assembly GTPases may mask phenotypes in single-gene knockout studies.

Strategies to Overcome Limitations:

• Improved Genetic Tools:

  • Development of CRISPR-Cas9 systems optimized for T. denticola

  • Creation of more efficient transformation protocols

  • Design of inducible expression systems for controlled gene expression

• Heterologous Expression Systems:

  • Expression of T. denticola RlmE in model organisms for functional studies

  • Development of chimeric enzymes to study domain-specific functions

  • Creation of cell-free systems for studying T. denticola ribosome assembly

• Advanced Structural Approaches:

  • Cryo-EM analysis of T. denticola ribosomes and assembly intermediates

  • Comparative structural analysis with model organisms

  • In silico modeling based on homology to better-characterized RlmE enzymes

• Systems Biology Approaches:

  • Transcriptomics and proteomics to identify RlmE-dependent processes

  • Network analysis to map interactions between RlmE and other cellular components

  • Metabolomics to identify changes in S-adenosylmethionine utilization

How can researchers investigate potential inhibitors of RlmE as novel antimicrobial agents?

A systematic approach to investigating RlmE inhibitors would include:

Target Validation:

• Confirm essentiality or significant growth disadvantage of RlmE deletion in T. denticola

• Establish the structural and biochemical properties of T. denticola RlmE

• Determine conservation across bacterial species and divergence from human homologs

Inhibitor Screening Strategies:

Validation and Characterization:

• Biochemical validation of hit compounds

• Structure-activity relationship studies

• Determination of inhibition mechanism (competitive, non-competitive, etc.)

• Assessment of specificity against other methyltransferases

• Evaluation of antibacterial activity against T. denticola and other oral pathogens

In Vivo Efficacy Testing:

• Minimum inhibitory concentration (MIC) determination

• Assessment of activity in biofilm models

• Evaluation in polymicrobial cultures mimicking periodontal pockets

• Testing in animal models of periodontal disease

Potential Applications:
Given RlmE's importance in ribosome assembly , inhibitors could potentially target T. denticola in periodontal disease. The unique aspects of RlmE compared to other methyltransferases might allow for selective targeting while minimizing effects on commensal bacteria.

What innovative approaches could be used to study the impact of RlmE on T. denticola-host interactions?

Several innovative approaches could advance our understanding of RlmE's role in T. denticola-host interactions:

Advanced Cellular Models:

• 3D Organoid Systems: Development of gingival organoids to study T. denticola-epithelial interactions in a complex 3D environment

• Microfluidic Co-culture Systems: To assess dynamic interactions between T. denticola and host cells under controlled flow conditions

• Immuno-competent Ex Vivo Models: Using gingival explants to preserve tissue architecture and resident immune cells

Genetic and Molecular Approaches:

• Conditional RlmE Expression: Inducible systems to control RlmE expression during different phases of host interaction

• Reporter Strains: T. denticola strains with fluorescent tags to monitor localization and activity during host cell interaction

• Ribosome Profiling: To determine changes in translation patterns in response to host factors

Advanced Imaging Techniques:

• Live Cell Imaging: To visualize T. denticola-host interactions in real-time

• Super-resolution Microscopy: For detailed visualization of bacterial-host interfaces

• Correlative Light and Electron Microscopy (CLEM): To connect functional observations with ultrastructural details

Systems Biology Integration:

• Multi-omics Approach: Combining transcriptomics, proteomics, and metabolomics of both T. denticola and host cells

• Network Analysis: To identify key nodes in host-pathogen interaction networks

• Computational Modeling: Predicting the impact of RlmE on bacterial fitness and virulence during infection

In Vivo Models with Enhanced Resolution:

• Intravital Microscopy: For real-time imaging of T. denticola-host interactions in animal models

• Cell-type Specific Host Responses: Using cell-type specific reporter mice to monitor responses to T. denticola

• Humanized Mouse Models: For more relevant study of human-specific aspects of T. denticola interactions

These approaches would help elucidate how RlmE-dependent processes in T. denticola influence interactions with host cells, potentially revealing new targets for therapeutic intervention in periodontal disease.