Recombinant Mannheimia succiniciproducens Ribosomal RNA large subunit methyltransferase H (rlmH)

What is Mannheimia succiniciproducens and why is it significant for rlmH research?

Mannheimia succiniciproducens MBEL55E is a capnophilic (CO2-loving) gram-negative facultative anaerobic bacterium isolated from bovine rumen. It has gained significant research attention primarily due to its efficient production of succinic acid from various carbon sources including pentose sugars, hexose sugars, and disaccharides . Its genome-scale metabolic network consists of 686 reactions and 519 metabolites, with distinctive features including strong PEP carboxylation, branched TCA cycle, relatively weak pyruvate formation, and lack of glyoxylate shunt . These metabolic characteristics create a unique cellular environment in which rlmH functions, potentially influencing enzyme activity and regulation compared to homologs in other bacterial species.

What is the general role of ribosomal RNA methyltransferases in bacterial systems?

Ribosomal RNA methyltransferases catalyze the transfer of methyl groups to specific positions in ribosomal RNA, using S-adenosylmethionine (SAM) as the methyl donor. Specifically, large subunit methyltransferases like rlmH modify nucleotides in the 23S rRNA, which can significantly influence ribosome assembly, structure, and function. These modifications often occur at functionally critical regions of the ribosome, including the peptidyl transferase center and intersubunit bridges. Methylation of rRNA contributes to ribosomal stability, fine-tunes translation efficiency, and in some cases, provides resistance to certain antibiotics that target the ribosome.

How can researchers design gene constructs for recombinant rlmH expression?

For optimal expression of recombinant rlmH from M. succiniciproducens, researchers should consider the following design principles:

• Codon optimization: Adapt the coding sequence to the preferred codon usage of the expression host.

• Fusion tags selection: Include affinity tags (His6, GST, or MBP) to facilitate purification, with consideration for tag position (N- or C-terminal) based on predicted protein structure.

• Promoter selection: For E. coli expression systems, IPTG-inducible promoters (T7, tac) work effectively, while for expression in M. succiniciproducens itself, promoters compatible with its transcriptional machinery must be selected.

• Vector backbone: Use vectors with appropriate origins of replication and selection markers for the chosen expression host.

• Cloning strategy: Include appropriate restriction sites or use Gibson Assembly for seamless cloning.

• Secretion signals: Consider including secretion signals if extracellular expression is desired.

• TEV or PreScission protease sites: Include if tag removal is necessary for functional studies.

This design should be informed by M. succiniciproducens' genetic characteristics, including its genomic organization where genes for specific functions might not form operons or clusters, as observed with its sucrose metabolism genes .

What are the optimal conditions for expressing recombinant rlmH from M. succiniciproducens?

Based on studies of M. succiniciproducens and other recombinant proteins, the following expression conditions are recommended:

• Expression host: While E. coli BL21(DE3) is commonly used for heterologous expression, homologous expression in M. succiniciproducens may preserve native folding and post-translational modifications.

• Growth medium: For M. succiniciproducens, modified MH5S medium supplemented with an appropriate carbon source (10 g/L) and 10 g/L NaHCO3 to provide CO2 .

• Temperature: Lower temperatures (16-25°C) often improve solubility of recombinant proteins.

• Induction conditions: For IPTG-inducible systems, 0.1-0.5 mM IPTG at mid-log phase (OD600 ~0.6-0.8).

• Growth parameters: Maintain anaerobic or microaerobic conditions with CO2 supplementation for M. succiniciproducens-based expression systems.

• Harvest timing: 4-16 hours post-induction, depending on expression kinetics.

The specific composition of MH5S medium (2.5 g/L yeast extract, 2.5 g/L polypeptone, 1 g/L NaCl, 8.7 g/L K2HPO4, 10 g/L NaHCO3, 0.02 g/L CaCl2·2H2O, and 0.2 g/L MgCl2·6H2O) provides essential nutrients for optimal growth of M. succiniciproducens .

What purification strategies are most effective for recombinant rlmH?

Effective purification of recombinant rlmH from M. succiniciproducens involves:

• Cell lysis optimization:

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Lysis methods: Sonication or French press for E. coli, more gentle methods for M. succiniciproducens

  • Protease inhibitors: PMSF (1 mM) or commercial cocktails

• Chromatographic separation:

  • IMAC (for His-tagged constructs): Binding in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole; elution with 250-300 mM imidazole gradient

  • Ion exchange chromatography: Based on rlmH theoretical pI

  • Size exclusion chromatography: For final polishing and buffer exchange

• Tag removal (if necessary):

  • Proteolytic cleavage using TEV or PreScission protease

  • Re-chromatography to remove the cleaved tag

• Quality control:

  • SDS-PAGE to assess purity (>90% recommended for enzymatic studies)

  • Western blot to confirm identity

  • Mass spectrometry for accurate mass determination

  • Dynamic light scattering to assess aggregation state

• Storage conditions:

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 50% glycerol

  • Temperature: -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles

How can researchers troubleshoot solubility issues with recombinant rlmH?

When encountering solubility challenges with recombinant rlmH, researchers should systematically implement the following strategies:

• Fusion tag modification:

  • Test solubility-enhancing tags (MBP, SUMO, TrxA)

  • Evaluate tag position effects (N- vs C-terminal)

  • Consider dual tagging approaches

• Expression condition optimization:

  • Reduce induction temperature to 16-20°C

  • Decrease inducer concentration

  • Test auto-induction media formulations

  • Implement slower induction strategies

• Co-expression approaches:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Include rare tRNA-encoding plasmids for heterologous expression

• Buffer optimization:

  • Screen various pH conditions (pH 6.5-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Add stabilizing agents (glycerol, sucrose, arginine, glutamic acid)

  • Include potential cofactors (SAM at 50-100 μM)

• Refolding strategies (if inclusion bodies form):

  • On-column refolding during purification

  • Stepwise dialysis to remove denaturants

  • Rapid dilution methods with optimized redox conditions

Each approach should be systematically tested and documented to determine optimal conditions for obtaining soluble, active recombinant rlmH.

What are the recommended methodologies for assaying rlmH methyltransferase activity?

For comprehensive characterization of rlmH methyltransferase activity from M. succiniciproducens, researchers should consider these methodological approaches:

• Radiometric assays:

  • Using [³H]- or [¹⁴C]-labeled SAM (10-50 μM)

  • Reaction conditions: 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 100 mM NH4Cl, 1 mM DTT

  • Filter binding assay with appropriate rRNA substrate (0.1-1 μM)

  • Detection via liquid scintillation counting

• HPLC-based methods:

  • Quantify SAM to SAH conversion using reverse-phase HPLC

  • Monitor methylated nucleosides after enzymatic hydrolysis of rRNA

  • UV detection at 254-260 nm

• Mass spectrometry approaches:

  • LC-MS/MS analysis of methylated nucleosides

  • RNA fragment analysis by MALDI-TOF

  • Intact mass analysis to determine methylation stoichiometry

• Coupled enzyme assays:

  • Link SAH production to adenosine deaminase and xanthine oxidase reactions

  • Monitor absorbance change at 265 nm

• Substrate preparation:

  • In vitro transcription of rRNA fragments containing target sites

  • Isolation of ribosomes from rlmH knockout strains

  • Preparation of defined ribosomal subunit assembly intermediates

Each method offers different advantages in terms of sensitivity, throughput, and information content, allowing researchers to select appropriate techniques based on their specific research questions.

How can researchers determine the substrate specificity of rlmH from M. succiniciproducens?

To determine the substrate specificity of rlmH from M. succiniciproducens, implement the following systematic approach:

• rRNA substrate analysis:

  • Test various rRNA fragments containing potential methylation sites

  • Compare activity with naked rRNA vs. partially assembled ribosomal particles

  • Assess sequence context requirements using mutated substrates

  • Determine minimum substrate length for activity

• Kinetic characterization:

  • Determine Km and Vmax for SAM and RNA substrates

  • Assess product inhibition by SAH

  • Measure pH and temperature optima and stability

  • Evaluate metal ion dependencies and specificities

• Competition assays:

  • Test activity in the presence of competing methyl acceptors

  • Use structural analogs to probe binding site specificity

• Structural probing:

  • RNA structure mapping before and after methylation

  • Identify conformational changes upon methylation

  • Use chemical and enzymatic probes to access target site availability

• Comparative analysis:

  • Compare activity against rRNA from different bacterial species

  • Evaluate cross-specificity with other methyltransferases

Table 1: Key enzymatic parameters for characterizing rlmH activity and substrate specificity

What in vivo approaches can assess the physiological role of rlmH in M. succiniciproducens?

To investigate the physiological significance of rlmH in M. succiniciproducens, researchers can employ these in vivo approaches:

• Gene knockout construction:

  • Create rlmH deletion strains using methods similar to those reported for other M. succiniciproducens genes (e.g., MBEL55EΔ0784, MBEL55EΔ0909)

  • Complement with wild-type or mutant versions to confirm phenotypes

• Growth phenotype characterization:

  • Compare growth rates and yields in various media compositions

  • Test different carbon sources (glucose, fructose, sucrose at 10 g/L)

  • Evaluate stress resistance (temperature, pH, antibiotics)

  • Assess growth under capnophilic conditions (varying CO2 concentrations)

• Ribosome functional analysis:

  • Polysome profiling to examine translation efficiency

  • In vivo translation rate measurements using pulse-labeling

  • Mistranslation frequency assessment using reporter systems

  • Ribosome assembly kinetics during different growth phases

• Metabolic analysis:

  • Measure changes in succinic acid production capacity

  • Monitor carbon flux distribution using 13C-labeled substrates

  • Integrate with existing metabolic models of M. succiniciproducens

• Antibiotic sensitivity profiling:

  • Test sensitivity to ribosome-targeting antibiotics

  • Determine minimum inhibitory concentrations (MICs)

  • Assess adaptation to sublethal antibiotic exposure

These approaches should be performed under standardized conditions comparable to those used in previous M. succiniciproducens studies, such as growth in MH5S medium supplemented with appropriate carbon sources .

How can rlmH be integrated into metabolic engineering strategies for M. succiniciproducens?

Integrating rlmH into metabolic engineering approaches for M. succiniciproducens involves several strategic considerations:

• Translation efficiency optimization:

  • Modulate rlmH expression to potentially enhance global translation efficiency

  • Coordinate with existing metabolic engineering targets like zwf gene overexpression

  • Optimize ribosome function for improved expression of key pathway enzymes

• Strain robustness enhancement:

  • If rlmH contributes to ribosome stability under stress, its optimization could improve fermentation robustness

  • Co-engineer with stress response pathways for synergistic effects

  • Enhance tolerance to acidic conditions during succinic acid production

• Growth-production balancing:

  • Fine-tune translation efficiency to balance growth requirements with metabolic flux toward succinic acid

  • Consider dynamic regulation of rlmH expression during different production phases

• Synthetic biology approaches:

  • Design synthetic riboswitches incorporating rlmH-dependent rRNA structures

  • Develop orthogonal translation systems for targeted pathway expression

  • Create rlmH variants with altered specificity for specialized functions

The metabolic engineering strategies should build upon established approaches in M. succiniciproducens, such as those used in developing strains like LPK7 that showed improved succinic acid production through zwf gene overexpression .

What comparative analyses can reveal the evolutionary significance of rlmH in M. succiniciproducens?

To understand the evolutionary significance of rlmH in M. succiniciproducens, researchers should conduct these comparative analyses:

• Phylogenetic analysis:

  • Construct phylogenetic trees of rlmH across bacterial species

  • Compare evolutionary rates with other rRNA methyltransferases

  • Identify lineage-specific adaptations in the rumen microbiome

• Structural conservation assessment:

  • Analyze conservation of catalytic residues and binding motifs

  • Identify M. succiniciproducens-specific sequence features

  • Compare with methyltransferases from related Pasteurellaceae family members

• Genomic context analysis:

  • Examine gene neighborhood conservation across species

  • Identify potential horizontal gene transfer events

  • Compare operon structures and regulatory elements

• Functional conservation testing:

  • Perform cross-species complementation experiments

  • Test substrate specificity across homologs

  • Evaluate kinetic parameters of homologs from different ecological niches

• Co-evolutionary analysis:

  • Identify co-evolving residues within rlmH

  • Assess co-evolution with rRNA target sequences

  • Examine potential co-evolution with ribosomal proteins

This evolutionary perspective can provide insights into the adaptation of translation machinery in the context of M. succiniciproducens' specialized metabolic capabilities and rumen environment.

How does rlmH activity potentially influence antibiotic resistance in M. succiniciproducens?

The potential influence of rlmH on antibiotic resistance in M. succiniciproducens can be investigated through these approaches:

• Antibiotic susceptibility testing:

  • Compare MICs of various ribosome-targeting antibiotics between wild-type and rlmH mutant strains

  • Focus on macrolides, lincosamides, streptogramins, and oxazolidinones

  • Determine resistance profiles under different growth conditions

• Target site analysis:

  • Map the exact position of methylation in 23S rRNA

  • Correlate with known antibiotic binding sites

  • Use molecular modeling to predict steric effects on antibiotic binding

• Resistance development monitoring:

  • Track evolution of resistance under antibiotic selection pressure

  • Compare adaptation rates between strains with different rlmH expression levels

  • Identify compensatory mutations that arise in rlmH mutants

• Combination effects:

  • Assess interactions with other resistance mechanisms

  • Test synergy or antagonism with efflux pump systems

  • Evaluate the impact of membrane permeability alterations

• Clinical isolate comparison:

  • Analyze rlmH sequences in clinical or environmental isolates with varying resistance profiles

  • Correlate sequence variations with phenotypic differences

  • Identify naturally occurring mutations with functional consequences

This research has implications for both fundamental understanding of resistance mechanisms and practical applications in bioprocessing, where contamination control is essential for industrial fermentations.

What computational approaches can predict structure-function relationships of rlmH?

For predicting structure-function relationships of rlmH from M. succiniciproducens, researchers should employ these computational approaches:

• Homology modeling and structural analysis:

  • Generate 3D models based on crystal structures of related methyltransferases

  • Refine models using molecular dynamics simulations (100-500 ns)

  • Identify catalytic pocket and substrate binding regions

  • Analyze conservation mapping onto structural models

• Molecular docking studies:

  • Dock SAM and rRNA substrate fragments to the predicted binding sites

  • Calculate binding energies and identify key interaction residues

  • Perform virtual mutations to predict functional effects

• Reaction mechanism simulation:

  • Use QM/MM methods to model the methyl transfer reaction

  • Calculate activation energy barriers for catalysis

  • Identify transition states and rate-limiting steps

• Molecular dynamics simulations:

  • Analyze protein flexibility and conformational changes upon substrate binding

  • Identify allosteric sites and communication pathways

  • Simulate the effect of pH and temperature on protein dynamics

• Machine learning approaches:

  • Train models on existing methyltransferase data to predict substrate specificity

  • Use neural networks to identify functional motifs in the sequence

  • Develop classifiers for activity prediction based on sequence features

These computational approaches should be validated with experimental data whenever possible to ensure biological relevance of the predictions.

How can researchers design site-directed mutagenesis experiments to probe rlmH catalytic mechanism?

To systematically investigate the catalytic mechanism of rlmH through site-directed mutagenesis, researchers should:

• Target residue selection:

  • Conserved residues in methyltransferase motifs (I-X)

  • Predicted SAM-binding residues (typically G-X-G motif and acidic residues)

  • Predicted RNA-binding residues (basic and aromatic amino acids)

  • Residues unique to M. succiniciproducens rlmH compared to homologs

• Mutation design strategy:

  • Conservative mutations to probe specific chemical properties (e.g., D→E, K→R)

  • Non-conservative mutations to abolish function (e.g., D→A, K→A)

  • Charge reversal mutations to test electrostatic interactions (e.g., D→K)

  • Cysteine substitutions for subsequent chemical modification studies

• Experimental validation:

  • Expression and purification of mutant proteins under identical conditions

  • Structural integrity verification via circular dichroism or thermal shift assays

  • Kinetic parameter determination (kcat, Km) for SAM and RNA substrates

  • Product analysis to confirm methylation position and efficiency

• Structure-function correlation:

  • Map mutations onto the structural model

  • Correlate activity changes with structural perturbations

  • Identify networks of functionally coupled residues

Table 2: Priority residues for site-directed mutagenesis in methyltransferase enzymes

What techniques can determine the exact position of rRNA methylation by rlmH?

To precisely determine the methylation position in rRNA catalyzed by rlmH from M. succiniciproducens, researchers should employ these complementary techniques:

• Mass spectrometry approaches:

  • Tandem mass spectrometry (MS/MS) of enzymatically digested rRNA

  • Top-down MS of intact rRNA fragments

  • Comparative analysis of methylated vs. unmethylated samples

  • Exact mass determination to differentiate methylation isomers

• Reverse transcription-based methods:

  • Primer extension analysis (methylation may cause RT stops or pauses)

  • MALDI-TOF analysis of cDNA fragments

  • Next-generation sequencing to quantify methylation at single-nucleotide resolution

• Chemical probing methods:

  • Selective chemical modification of unmethylated positions

  • Bisulfite sequencing adaptations for RNA

  • Differential reactivity mapping before and after methylation

• NMR spectroscopy:

  • 1H and 13C NMR of isolated nucleosides

  • 2D NMR techniques to confirm methyl group position

  • Isotope labeling to enhance detection sensitivity

• Crystallographic approaches:

  • X-ray crystallography of rlmH in complex with substrate analogs

  • Cryo-EM of ribosomes or ribosomal subunits with and without rlmH activity

  • Visualization of methylated vs. unmethylated structures

The combination of these techniques provides complementary data that together can unambiguously identify the exact nucleotide and position methylated by rlmH, which is crucial for understanding its functional role in ribosome biogenesis and function.