Recombinant Rhodopirellula baltica Ribosomal RNA large subunit methyltransferase N (rlmN)

What is the primary function of Rhodopirellula baltica RlmN?

Rhodopirellula baltica RlmN likely functions as a methyltransferase that catalyzes the formation of 2-methyladenosine (m2A) in ribosomal RNA. Based on homology with E. coli RlmN, it may exhibit dual-specificity, capable of methylating both rRNA and tRNA substrates. This dual-specificity characteristic makes it a particularly interesting enzyme for research into RNA modification mechanisms. The methylation activity plays a crucial role in maintaining translational accuracy by ensuring proper ribosome function during protein synthesis, particularly in the proofreading steps occurring at the peptidyl transferase center .

How does Rhodopirellula baltica RlmN expression change during the organism's life cycle?

Expression of genes in Rhodopirellula baltica, including potential expression of RlmN, varies significantly throughout its growth phases. Transcriptional profiling of R. baltica reveals that many genes are differentially regulated during early exponential, mid-exponential, transition, and stationary phases. While specific RlmN expression data is not directly provided, the organism shows distinct expression patterns for many genes during these transitions. For instance, during the stationary phase, genes involved in stress response, cell wall composition modification, and various biosynthetic pathways show altered expression compared to earlier growth phases .

What is known about the substrate specificity of Rhodopirellula baltica RlmN?

Based on studies of the E. coli homolog, Rhodopirellula baltica RlmN likely recognizes specific 3D structural elements in its RNA substrates rather than simply the primary sequence. In E. coli, RlmN performs m2A synthesis at position 37 of specific tRNAs and position 2503 of 23S rRNA. The enzyme appears to work in late steps during RNA maturation, suggesting it requires properly folded and potentially partially modified substrates for activity. The R. baltica version may have evolved specificity adaptations relevant to its marine environment and unique cellular organization .

What experimental approaches are most effective for characterizing the dual-specificity of Rhodopirellula baltica RlmN?

To characterize the dual-specificity of R. baltica RlmN, a multi-faceted approach is recommended:

• In vitro methylation assays: Using purified recombinant R. baltica RlmN with various RNA substrates, including:

  • Native rRNA isolated from R. baltica

  • In vitro transcribed rRNA fragments

  • tRNA substrates, both naturally occurring and chimeric constructs

• Chimeric tRNA approach: Following the methodology used for E. coli RlmN studies, develop chimeric tRNA constructs that incorporate the anticodon stem-loop (ASL) of potential R. baltica tRNA substrates. This approach has proven useful for characterizing tRNA identity determinants for RlmN and other modification enzymes .

• HPLC analysis: Implement high-performance liquid chromatography to detect and quantify m2A modifications in RNA substrates after in vitro or in vivo methylation reactions. This allows precise determination of modification efficiency under various conditions .

• Genetic complementation studies: Express R. baltica RlmN in E. coli ΔrlmN strains to assess functional complementation, providing insights into conserved and divergent features between the two enzymes.

How can researchers address contradictory findings when working with recombinant Rhodopirellula baltica RlmN?

When confronting contradictory findings in R. baltica RlmN research, implement a structured approach:

• Categorize contradiction types: Determine whether contradictions are:

  • Self-contradictory (inconsistencies within a single study)

  • Contradicting pairs (conflicts between two different studies)

  • Conditional contradictions (where a third factor creates apparent contradictions between otherwise consistent results)

• Experimental validation: Design targeted experiments to directly address the contradictions, focusing on:

  • Precise replication of original methods

  • Systematic variation of key parameters

  • Introduction of appropriate controls

• Statistical analysis: Apply rigorous statistical methods to evaluate the significance of contradictory results.

• Growth condition standardization: Since R. baltica exhibits significant changes in gene expression throughout its growth cycle, ensure all experiments specify and control growth conditions, including:

  • Growth phase (early exponential, mid-exponential, transition, or stationary)

  • Culture conditions (temperature, salinity, media composition)

  • Cell morphology predominance (swarmer, budding, rosette formations)

• Domain expertise collaboration: Engage specialists in RNA modification, structural biology, and Planctomycetes biology to provide diverse perspectives on conflicting results.

What are the key methodological considerations for designing mutation studies of Rhodopirellula baltica RlmN?

When designing mutation studies for R. baltica RlmN, consider:

• Target residue selection:

  • Conserved catalytic residues based on homology with E. coli RlmN

  • Residues potentially involved in substrate recognition

  • Surface residues that might confer organism-specific properties

• Mutation strategy:

  • Conservative substitutions to probe specific interactions

  • Alanine scanning to identify functionally important regions

  • Domain swapping with E. coli RlmN to identify specificity determinants

• Functional assays:

  • In vitro methylation activity with various substrates

  • Complementation of E. coli ΔrlmN phenotypes

  • RNA binding assays to distinguish between catalytic and binding defects

• Expression system considerations:

  • Temperature optimization (R. baltica grows optimally at 28°C)

  • Codon optimization for heterologous expression

  • Addition of solubility tags if aggregation occurs

• Phenotypic analysis:

  • Assays for translation fidelity, particularly stop codon readthrough

  • Growth characteristics under various conditions

  • RNA modification profiles by HPLC or mass spectrometry

How might Rhodopirellula baltica RlmN function differ in the context of the organism's unique cell biology?

Rhodopirellula baltica possesses several unique cellular features that may influence RlmN function:

• Compartmentalized cell structure: As a member of the Planctomycetes, R. baltica has a compartmentalized cell organization that differs from typical Gram-negative bacteria. This may affect the localization and substrate accessibility of RlmN.

• Marine adaptation: R. baltica's salt resistance mechanisms and marine adaptations may influence enzyme stability and activity of RlmN under varying salt concentrations.

• Growth phase-dependent morphology: The organism transitions between different morphological states (swarmer cells, budding cells, and rosette formations) throughout its growth cycle. RlmN expression and activity may be regulated in coordination with these transitions .

• Cell wall composition: R. baltica has distinctive cell wall components, including high proline content. Changes in cell wall composition during different growth phases have been observed, which might correlate with changes in RNA modification patterns .

• Stress response integration: R. baltica upregulates various stress-response genes during stationary phase. RlmN activity might be coordinated with these responses to ensure translational accuracy under stress conditions.

What are the optimal conditions for expressing and purifying recombinant Rhodopirellula baltica RlmN?

For optimal expression and purification of recombinant R. baltica RlmN:

• Expression system selection:

  • E. coli BL21(DE3) or Rosetta strains are recommended for initial attempts

  • Consider R. baltica's growth temperature (28°C) for optimal protein folding

  • Test both N-terminal and C-terminal His-tags for improved solubility

• Expression conditions:

  • Induction at lower temperatures (16-20°C) for 16-20 hours

  • IPTG concentration optimization (typically 0.1-0.5 mM)

  • Supplementation with iron and SAM precursors may improve folding

• Purification protocol:

  • Initial capture by Ni-NTA affinity chromatography

  • Secondary purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography

  • Buffer optimization to include reducing agents (DTT or β-mercaptoethanol)

• Stability considerations:

  • Addition of glycerol (10-20%) to storage buffers

  • Flash freezing in small aliquots to avoid freeze-thaw cycles

  • Storage at -80°C for long-term maintenance of activity

• Quality control:

  • SDS-PAGE and western blot analysis for purity assessment

  • Thermal shift assays to optimize buffer conditions

  • Activity assays with model substrates to confirm functionality

What assays can be used to measure the enzymatic activity of Rhodopirellula baltica RlmN?

Several complementary assays can be employed to measure R. baltica RlmN activity:

• Radiometric SAM-dependent methylation assays:

  • Using [3H] or [14C]-labeled S-adenosylmethionine (SAM)

  • Measurement of incorporated radioactivity into RNA substrates

  • Quantification via liquid scintillation counting

• HPLC-based nucleoside analysis:

  • Enzymatic digestion of RNA to nucleosides

  • Separation and quantification of modified nucleosides

  • Comparison of m2A peak areas between experimental and control samples

• Mass spectrometry approaches:

  • LC-MS/MS for precise identification and quantification of m2A

  • RNA digestion followed by mass analysis of oligonucleotides

  • Comparison against synthetic standards

• In vivo complementation assays:

  • Expression of R. baltica RlmN in E. coli ΔrlmN strains

  • Measurement of mistranslation rates using reporter constructs

  • Evaluation of growth phenotypes under stress conditions

• Structural probing of modified RNA:

  • Chemical and enzymatic probing to detect structural changes

  • Primer extension analysis to identify modification sites

  • SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) analysis

How can researchers establish a reliable tRNA substrate system for studying Rhodopirellula baltica RlmN?

To establish a reliable tRNA substrate system for R. baltica RlmN studies:

• Chimeric tRNA approach:

  • Utilize tRNA scaffold systems similar to those employed for E. coli RlmN studies

  • Replace the anticodon stem-loop (ASL) of a stable tRNA scaffold with the ASL of potential R. baltica tRNA substrates

  • Validate the chimeric construct's stability and modification status by HPLC analysis

• Substrate identification:

  • Analyze R. baltica genomic data to identify tRNAs with adenosine at position 37

  • Prioritize tRNAs that decode codons for glutamine, arginine, and other amino acids whose tRNAs are known substrates for E. coli RlmN

  • Generate multiple chimeric constructs to identify optimal substrates

• Expression system optimization:

  • Test different promoters for efficient tRNA expression

  • Optimize growth conditions to enhance tRNA stability

  • Consider co-expression with other tRNA modification enzymes if needed for RlmN recognition

• Structural requirements determination:

  • Introduce systematic mutations in the tRNA chimera to identify critical recognition elements

  • Focus on variations in positions U35 and G36, which are known identity determinants for E. coli RlmN

  • Investigate the importance of other modified nucleotides, such as pseudouridine at position 38

• Activity validation:

  • Confirm the presence of m2A in successfully modified tRNAs by HPLC analysis

  • Compare modification efficiency between in vitro and in vivo systems

  • Establish quantitative methods for measuring the extent of modification

What analytical techniques are most appropriate for detecting and quantifying m2A modifications introduced by Rhodopirellula baltica RlmN?

For detecting and quantifying m2A modifications by R. baltica RlmN:

• HPLC-based methods:

  • Enzymatic digestion of RNA to nucleosides using nuclease P1 and alkaline phosphatase

  • Reverse-phase HPLC separation with UV detection at 260 nm

  • Comparison of retention times with authentic m2A standards

  • Quantification by peak area integration normalized to unmodified nucleosides

• Mass spectrometry approaches:

  • LC-MS/MS analysis for high-sensitivity detection

  • Multiple reaction monitoring (MRM) for quantitative analysis

  • High-resolution mass spectrometry for unambiguous identification

  • Isotope dilution techniques for absolute quantification

• Next-generation sequencing-based methods:

  • Modification-specific library preparation techniques

  • Analysis of reverse transcription signatures at modified positions

  • Nanopore direct RNA sequencing for detecting m2A modifications

• Antibody-based detection:

  • Development or use of antibodies specific for m2A

  • Immunoprecipitation of modified RNA followed by qPCR or sequencing

  • Dot blot assays for rapid screening

• Radiolabeling approaches:

  • In vitro methylation with [3H] or [14C]-SAM

  • Thin-layer chromatography of digested RNA

  • Autoradiography or scintillation counting for quantification

What structural features of RNA substrates are recognized by Rhodopirellula baltica RlmN?

Based on studies of the E. coli homolog, R. baltica RlmN likely recognizes specific structural features in its RNA substrates:

How does the substrate specificity of Rhodopirellula baltica RlmN compare across different growth conditions?

The substrate specificity of R. baltica RlmN may vary across different growth conditions due to:

• Growth phase-dependent regulation:

  • R. baltica exhibits significant transcriptional changes throughout its growth cycle

  • Gene expression profiles differ dramatically between exponential, transition, and stationary phases

  • These changes may influence both RlmN expression and the availability of RNA substrates

• Environmental adaptation:

  • As a marine organism, R. baltica responds to changes in salinity and nutrient availability

  • Changes in cell wall composition and cellular architecture occur in response to environmental stressors

  • These adaptations may alter RNA structure and accessibility to modification enzymes

• Morphological transitions:

  • R. baltica transitions between different cell morphologies (swarmer cells, budding cells, rosettes)

  • These morphological states predominate at different growth phases and may correlate with changes in RNA modification patterns

  • The formation of rosettes in stationary phase, for example, coincides with significant gene expression changes

• Nutrient limitation responses:

  • During nutrient limitation, R. baltica upregulates various stress-response genes

  • Changes in translation efficiency and accuracy may necessitate altered modification patterns

  • The upregulation of ubiquinone biosynthesis genes in the stationary phase suggests coordinated responses to changing conditions

What techniques can be used to map the interactions between Rhodopirellula baltica RlmN and its RNA substrates?

To map interactions between R. baltica RlmN and its RNA substrates:

• RNA footprinting approaches:

  • Chemical footprinting using reagents like DMS, CMCT, or hydroxyl radicals

  • Enzymatic footprinting with RNases to identify protected regions

  • SHAPE chemistry to probe RNA structural changes upon enzyme binding

• Crosslinking methods:

  • UV crosslinking of protein-RNA complexes

  • Site-specific incorporation of photoreactive groups

  • CLIP-seq (Crosslinking and Immunoprecipitation followed by sequencing) for genome-wide identification of binding sites

• Mutational analysis:

  • Systematic mutagenesis of both protein and RNA

  • Evaluation of binding and catalytic activity for each mutant

  • Combining mutations to identify cooperative interactions

• Structural biology techniques:

  • X-ray crystallography of RlmN-RNA complexes

  • Cryo-electron microscopy for larger assemblies

  • NMR spectroscopy for dynamic interaction studies

• Computational approaches:

  • Molecular docking simulations

  • Molecular dynamics studies of enzyme-substrate complexes

  • Sequence and structure conservation analysis across species

How does the catalytic mechanism of Rhodopirellula baltica RlmN compare to other RNA methyltransferases?

The catalytic mechanism of R. baltica RlmN likely differs from classical methyltransferases:

• Radical SAM mechanism:

  • Based on E. coli RlmN studies, the enzyme likely belongs to the radical SAM enzyme family

  • Uses a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical from S-adenosylmethionine

  • This mechanism allows methylation at unreactive carbon positions (C2 of adenosine)

• Comparison with canonical methyltransferases:

  • Traditional RNA methyltransferases typically methylate nitrogen or oxygen atoms

  • They use direct transfer of the methyl group from SAM without radical intermediates

  • R. baltica RlmN likely requires additional cofactors beyond SAM, such as iron and reducing agents

• Dual-specificity characteristic:

  • Unlike most RNA modification enzymes that target either rRNA or tRNA, RlmN modifies both

  • This dual-specificity is rare among RNA methyltransferases, with RluA being one of the few other examples of enzymes modifying both RNA types

• Substrate recognition differences:

  • R. baltica RlmN likely recognizes specific 3D structures rather than consensus sequences

  • The enzyme appears to function in late stages of RNA maturation

  • This contrasts with many methyltransferases that act on nascent or partially processed RNA

What evolutionary insights can be gained from comparing Rhodopirellula baltica RlmN with homologs from other bacterial phyla?

Comparative analysis of R. baltica RlmN with homologs from other bacteria offers several evolutionary insights:

• Planctomycetes-specific adaptations:

  • R. baltica belongs to a phylum with unique cellular organization

  • Comparing RlmN across Planctomycetes may reveal adaptations specific to their compartmentalized cell structure

  • These adaptations might include changes in substrate recognition or cellular localization

• Marine versus terrestrial adaptations:

  • R. baltica is adapted to marine environments

  • Comparing its RlmN with those from terrestrial bacteria may reveal adaptations to different ionic strengths and environmental conditions

  • Such adaptations could include differences in protein stability, substrate affinity, or regulation

• Functional conservation:

  • The dual-specificity of E. coli RlmN suggests a conserved role in translation quality control

  • Presence or absence of this dual-specificity across bacterial lineages may indicate evolutionary pressure on translation accuracy

  • Conservation of substrate specificity would suggest fundamental importance in bacterial physiology

• Structural divergence:

  • Structural variations in RlmN across bacterial phyla may reflect adaptations to different cellular environments

  • These variations could provide insights into the evolution of RNA modification systems

  • Analysis of positively selected residues could reveal adaptation hotspots

What is the significance of dual-specificity in Rhodopirellula baltica RlmN and how prevalent is this characteristic in RNA modification enzymes?

The dual-specificity of R. baltica RlmN has several significant implications:

• Rarity in RNA modification systems:

  • Dual-specificity methyltransferases that modify both rRNA and tRNA are uncommon

  • In E. coli, only a few enzymes show this characteristic, including RluA (pseudouridine synthase) and RlmN

  • This makes RlmN particularly interesting for understanding enzyme evolution and specialization

• Functional implications:

  • Dual-specificity suggests a coordinated regulation of both translation initiation (tRNA) and elongation/termination (rRNA)

  • This coordination may be particularly important during stress response or adaptation to changing environments

  • Inactivation of RlmN in E. coli results in an error-prone phenotype, highlighting its role in translation accuracy

• Evolutionary perspective:

  • Dual-specificity may represent an ancestral state that has been maintained due to functional importance

  • Alternatively, it could represent a specialized adaptation that evolved to coordinate different aspects of translation

  • Comparative genomics across diverse bacteria could clarify the evolutionary trajectory

• Structural basis:

  • The ability to recognize structurally distinct RNA substrates suggests remarkable plasticity in substrate binding

  • Understanding this plasticity could provide insights for engineering RNA modification enzymes

  • Similar structural features at the target sites in both rRNA and tRNA may be recognized by RlmN

What are the consequences of RlmN deletion on Rhodopirellula baltica growth and physiology?

While specific studies on R. baltica RlmN deletion are not available, inferences can be made from related research:

• Translation accuracy effects:

  • Based on E. coli studies, deletion of rlmN may lead to increased misreading of stop codons

  • This error-prone phenotype suggests a role for RlmN in maintaining translational fidelity

  • Similar effects might be expected in R. baltica, potentially with growth phase-dependent severity

• Growth phase-specific consequences:

  • R. baltica exhibits significant transcriptional changes across growth phases

  • The impact of RlmN deletion may vary depending on growth phase and corresponding morphological state

  • Effects might be most pronounced during stress or stationary phase when translation accuracy becomes critical

• Stress response alterations:

  • R. baltica upregulates stress-response genes during stationary phase

  • RlmN deletion may compromise the organism's ability to adapt to stress conditions

  • This could manifest as reduced survival during nutrient limitation or other stressors

• Cell morphology effects:

  • R. baltica forms distinct morphological structures (rosettes) during different growth phases

  • RlmN deletion might affect the transition between morphological states if translational accuracy impacts the expression of key structural proteins

• Marine adaptation implications:

  • As a marine organism, R. baltica faces unique environmental challenges

  • RlmN deletion might particularly affect adaptation to changing salinity or nutrient availability

  • These effects could be studied by examining growth under various salt concentrations

How does the m2A modification introduced by Rhodopirellula baltica RlmN affect RNA structure and function?

The m2A modification likely influences RNA structure and function in several ways:

• Structural effects:

  • Methylation at the C2 position of adenosine alters the nucleoside's chemical properties

  • This modification may influence base-pairing dynamics and local RNA structure

  • In rRNA, m2A2503 is located in the peptidyl transferase center, suggesting a role in ribosome structure and function

• Translational accuracy impact:

  • In E. coli, loss of m2A from 23S rRNA results in an error-prone phenotype

  • The modification appears to play a crucial role in the proofreading step during translation

  • Similar roles might be expected for the modification in R. baltica rRNA

• tRNA function effects:

  • In tRNAs, m2A at position 37 may influence anticodon loop structure and stability

  • This could affect codon recognition and translation efficiency

  • The modification might be particularly important for specific tRNAs or under certain growth conditions

• Interaction with other RNA modifications:

  • RNA modifications often work synergistically to fine-tune RNA function

  • The m2A modification may cooperate with other modifications, such as pseudouridine at adjacent positions

  • The network of modifications may collectively optimize translation in changing environments

• Species-specific functions:

  • As a marine organism with unique cell biology, R. baltica may have evolved specific roles for the m2A modification

  • These roles might relate to adaptation to fluctuating salinity or other marine-specific stressors

  • Comparative studies with non-marine bacteria could highlight these specialized functions

What role does Rhodopirellula baltica RlmN play in translational fidelity and antibiotic resistance?

Based on studies of RlmN in other bacteria:

What are the most common technical challenges when working with recombinant Rhodopirellula baltica RlmN?

Researchers working with recombinant R. baltica RlmN may encounter several technical challenges:

• Protein solubility issues:

  • As a radical SAM enzyme, RlmN likely contains iron-sulfur clusters that can complicate expression

  • Insoluble protein or inclusion body formation may occur during heterologous expression

  • Expression at lower temperatures (16-20°C) and co-expression with chaperones may help

• Iron-sulfur cluster reconstitution:

  • Proper assembly of the [4Fe-4S] cluster is essential for activity

  • Chemical or enzymatic reconstitution may be necessary after purification

  • Anaerobic conditions may be required for maintaining cluster integrity

• Substrate preparation complexity:

  • Natural RNA substrates with appropriate modifications are challenging to prepare

  • In vitro transcribed RNAs may not be suitable substrates due to lack of other modifications

  • Chimeric tRNA approaches require optimization for stable expression

• Activity assay sensitivity:

  • Detecting m2A modifications requires specialized techniques

  • HPLC analysis may have limited sensitivity for low-level modifications

  • Mass spectrometry approaches require specialized equipment and expertise

• Species-specific optimization:

  • R. baltica's optimal growth temperature (28°C) differs from E. coli

  • Codon usage differences may necessitate optimization for heterologous expression

  • Buffer conditions may need to be adapted to reflect the organism's marine origin

How can researchers troubleshoot low activity or specificity when working with recombinant Rhodopirellula baltica RlmN?

When troubleshooting issues with recombinant R. baltica RlmN:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE and mass spectrometry

  • Assess iron-sulfur cluster content using UV-visible spectroscopy

  • Confirm correct folding using circular dichroism or thermal shift assays

• Cofactor supplementation:

  • Ensure sufficient S-adenosylmethionine (SAM) is present

  • Add reducing agents (DTT, β-mercaptoethanol, or sodium dithionite)

  • Supplement with iron and sulfide for iron-sulfur cluster reconstitution

• Substrate optimization:

  • Test multiple RNA substrates, including chimeric constructs

  • Ensure RNAs have appropriate modifications that may be prerequisites for RlmN activity

  • Consider partial digestion of natural RNA to provide more accessible substrates

• Reaction condition optimization:

  • Vary temperature, pH, and salt concentration

  • Consider R. baltica's natural marine environment when choosing buffer conditions

  • Test longer incubation times for slow reactions

• Analytical method sensitivity:

  • Use multiple detection methods (HPLC, MS, radiometric assays)

  • Include positive controls with known methylation levels

  • Consider enrichment steps to concentrate modified nucleosides

What bioinformatic approaches can help predict potential substrates and functional characteristics of Rhodopirellula baltica RlmN?

Bioinformatic approaches for studying R. baltica RlmN include:

• Sequence-based analyses:

  • Multiple sequence alignment with characterized RlmN enzymes

  • Identification of conserved catalytic residues and substrate-binding regions

  • Phylogenetic analysis to understand evolutionary relationships

• Structural prediction and modeling:

  • Homology modeling based on solved structures of related enzymes

  • Molecular docking simulations with potential RNA substrates

  • Molecular dynamics simulations to study enzyme-substrate interactions

• Transcriptome analysis:

  • Mining existing R. baltica transcriptome data to examine rlmN expression patterns

  • Correlation of expression with growth phases and stress conditions

  • Co-expression analysis to identify functionally related genes

• tRNA and rRNA target prediction:

  • Identification of potential target adenosines in R. baltica rRNA and tRNAs

  • Structural analysis of these sites to identify common features

  • Comparison with known RlmN targets in other organisms

• Genome context analysis:

  • Examination of genes adjacent to rlmN in the R. baltica genome

  • Analysis of conserved gene neighborhoods across related species

  • Identification of potential functional partners through gene proximity

How can researchers differentiate between direct and indirect effects when studying the impact of Rhodopirellula baltica RlmN deletion?

To differentiate between direct and indirect effects of R. baltica RlmN deletion:

• Complementation studies:

  • Reintroduce wild-type or catalytically inactive rlmN to deletion strains

  • Compare phenotypic rescue patterns between different constructs

  • Use controlled expression systems to maintain physiological expression levels

• Time-course analyses:

  • Monitor changes in RNA modification, gene expression, and phenotypes over time after deletion

  • Early effects are more likely to be direct consequences of RlmN absence

  • Late-appearing phenotypes may represent adaptive or compensatory responses

• Targeted RNA modification analysis:

  • Directly measure m2A levels at known target sites in rRNA and tRNA

  • Monitor other RNA modifications to identify compensatory changes

  • Use site-specific methods like primer extension or targeted mass spectrometry

• Global approaches:

  • Compare transcriptome and proteome profiles between wild-type and ΔrlmN strains

  • Identify gene expression changes that correlate with phenotypic effects

  • Use pathway analysis to distinguish primary from secondary responses

• Conditional deletion strategies:

  • Use inducible deletion systems to control the timing of RlmN depletion

  • Monitor acute versus chronic effects of RlmN absence

  • Compare effects under different growth conditions to identify context-dependent roles