Recombinant Mycoplasma mycoides subsp. mycoides SC tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG (mnmG1), partial

What is the molecular function of MnmG in bacterial tRNA modification?

MnmG functions as part of the MnmE-MnmG complex that catalyzes the GTP- and flavin adenine dinucleotide (FAD)-dependent incorporation of the carboxymethylaminomethyl (cmnm) group at position 5 of the wobble uridine in several tRNAs. This complex is essential for the precise modification of tRNAs that decode specific codons. The biochemical reaction involves the transfer of the cmnm group (CH2-NH-CH2-COOH) to position 5 of uridine, although the exact mechanistic steps remain under investigation . The modification enhances proper anticodon-codon interactions during translation, contributing to translational efficiency and accuracy.

How conserved is MnmG across bacterial species?

MnmG is a highly conserved FAD-binding protein across bacterial species. For instance, Escherichia coli MnmG shows approximately 49% sequence identity to human MTO1, its mitochondrial homolog . Within Mycoplasma species, there exists noteworthy conservation, though with some functional divergence. For example, Mycoplasma capricolum possesses two trmFO homologs (Mcap0476 and Mcap0613), with distinct functions - one evolved to methylate rRNA rather than tRNA . This conservation suggests the fundamental importance of tRNA modification systems across diverse bacterial lineages, while variations may reflect adaptations to specific ecological niches or metabolic requirements.

What is the significance of tRNA modifications for bacterial physiology?

tRNA modifications, including those mediated by MnmG, significantly impact bacterial physiology through multiple mechanisms:

• Translation efficiency: Modified tRNAs facilitate more efficient and accurate decoding of mRNAs during protein synthesis

• Codon-specific translation: Modifications can affect specific codons preferentially, influencing the expression of particular proteins

• Protein folding dynamics: The rate of translation affected by tRNA modifications can influence co-translational protein folding

• Cellular fitness: As demonstrated with related modification enzymes like TrmA and TruB, tRNA modifications can enhance cellular fitness under various growth conditions

Recent studies have revealed that tRNA modifications globally determine tRNA folding, aminoacylation, and translation in bacteria, with specific effects on codon-specific translation and the synthesis of large proteins .

How does the FAD cofactor binding influence MnmG conformation and activity?

Limited trypsinolysis experiments with E. coli MnmG suggest significant conformational changes upon FAD binding . The FAD binding appears to induce structural rearrangements that are crucial for the enzyme's catalytic function. Researchers investigating MnmG from Mycoplasma mycoides should consider implementing:

• Differential scanning fluorimetry to assess thermal stability changes with/without FAD

• Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon cofactor binding

• Site-directed mutagenesis of putative FAD-binding residues followed by activity assays

• Isothermal titration calorimetry to determine the thermodynamic parameters of FAD binding

These approaches would elucidate how FAD binding influences protein dynamics and catalytic efficiency specific to Mycoplasma mycoides MnmG, potentially revealing species-specific features.

What experimental approaches best assess the complex formation between MnmG and MnmE?

To characterize the MnmG-MnmE complex formation in Mycoplasma mycoides, researchers should consider a multi-method approach:

Studies with E. coli have demonstrated that MnmG and MnmE form a heterotetrameric α2β2 complex in vitro . Researchers working with Mycoplasma mycoides MnmG should validate if the same stoichiometry exists, as variations in complex assembly might explain functional differences between bacterial species.

How can researchers accurately assess the substrate specificity of MnmG?

Determining the tRNA substrate specificity of Mycoplasma mycoides MnmG requires systematic approaches:

• In vitro modification assays using purified recombinant MnmG and MnmE with:

  • Synthetic tRNA transcripts representing different isoacceptors

  • Native tRNAs isolated from wild-type and mnmG-deficient bacteria

  • Chimeric tRNAs with domain swaps to identify recognition elements

• Analysis of modified nucleosides using:

  • High-performance liquid chromatography (HPLC)

  • Mass spectrometry (MS) to detect cmnm5U formation

  • Two-dimensional thin-layer chromatography

• Binding affinity measurements:

  • Electrophoretic mobility shift assays with varying tRNA substrates

  • Filter binding assays to determine Kd values

  • Competition assays to establish relative binding preferences

Researchers should consider that in some Mycoplasma species, tRNAs have been reported to be devoid of certain modifications present in other bacteria , suggesting possible evolutionary divergence in substrate recognition patterns.

What is the optimal strategy for expressing and purifying recombinant Mycoplasma mycoides MnmG?

Based on successful approaches with other bacterial MnmG proteins, researchers should consider:

• Expression system optimization:

  • E. coli BL21(DE3) with a modified pET15b vector (or similar)

  • Low-temperature induction (16°C for ~16h) with moderate IPTG concentration (100 μM)

  • Codon-optimization for rare codons abundant in Mycoplasma

• Purification protocol:

  • Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography

  • Buffer conditions: 20 mM Tris-HCl (pH 8.0), 0.8 M NaCl, 5% glycerol, 5 mM DTT

  • Additional purification through ion exchange and size exclusion chromatography

• Protein stabilization considerations:

  • Addition of FAD to purification buffers (typically 1-10 μM)

  • Glycerol concentration optimization (5-10%)

  • Reducing agent presence throughout purification

This approach has yielded well-behaving protein for crystallization and biochemical assays in E. coli MnmG studies , and should be adaptable to the Mycoplasma homolog with appropriate modifications based on protein properties.

What crystallization conditions should be explored for structural studies of Mycoplasma mycoides MnmG?

Previous successful crystallization of E. coli MnmG suggests starting conditions:

• Protein concentration: ~8 mg/ml

• Crystallization method: Hanging-drop vapor diffusion

• Reservoir solution: 100 mM Tris-HCl (pH 7.5), 100 mM sodium formate, 6.5% polyethylene glycol 8000, 6% ethylene glycol

• Crystal optimization parameters:

  • pH range exploration (7.0-8.0)

  • PEG molecular weight variants (4000-10000)

  • Additive screening (particularly divalent cations)

  • Seeding techniques for crystal quality improvement

For phase determination, selenomethionine-labeled protein expression in a methionine auxotroph strain (e.g., DL41(DE3)) is recommended . Researchers should also consider co-crystallization with FAD and potential substrate analogs to capture different functional states.

How can genetic complementation studies validate MnmG function in vivo?

A comprehensive genetic approach to validate Mycoplasma mycoides MnmG function involves:

This genetic approach would definitively establish the in vivo function of Mycoplasma mycoides MnmG and identify any species-specific aspects of its activity.

How can researchers accurately measure the impact of MnmG on translation fidelity?

To assess MnmG's impact on translation fidelity in Mycoplasma mycoides, researchers should employ:

• Ribosome profiling analysis:

  • Preparation of ribosome-protected fragments

  • Deep sequencing to determine ribosome occupancy

  • Computational analysis of codon-specific translation effects

  • Comparison between wild-type and ΔmnmG strains

• Reporter-based assays:

  • Dual-luciferase reporters with programmed frameshifting sites

  • Stop codon readthrough reporters

  • Misincorporation reporters with strategic codon placements

• Mass spectrometry-based proteomics:

  • Quantitative proteomic comparison of wild-type and ΔmnmG strains

  • Analysis of protein synthesis rates using pulse-labeling

  • Error quantification using targeted mass spectrometry

Studies with related tRNA modification enzymes have revealed codon-specific translation defects in knockout strains , suggesting MnmG likely influences translation in a similarly specific manner.

What approaches can integrate experimental and computational methods to characterize MnmG activity?

An integrated approach combining experimental data with computational methods would provide comprehensive insights:

• Molecular dynamics simulations:

  • Structural modeling of Mycoplasma mycoides MnmG

  • Simulation of enzyme-substrate interactions

  • Analysis of conformational changes upon binding

• Augmented Markov Models (AMMs):

  • Integration of experimental observables with simulation data

  • Enhanced sampling of conformational states

  • Improved kinetic and thermodynamic predictions

• Experimental validation:

  • Site-directed mutagenesis guided by computational predictions

  • Spectroscopic measurements (RDCs, NMR spin-relaxation data)

  • Activity assays correlating structure with function

This approach has been successful in reconciling simulation and experimental data for molecular processes and would be valuable for understanding the complex dynamics of MnmG activity.

How does the absence of certain tRNA modifications in Mycoplasma species affect experimental design?

The reported absence of certain tRNA modifications in Mycoplasma species, such as the lack of m5U54 in Mycoplasma capricolum tRNAs , has significant implications for experimental design:

• Comparative analysis considerations:

  • Include controls from multiple bacterial species with known modification patterns

  • Employ complementation experiments across species boundaries

  • Analyze the co-evolution of modification enzymes and tRNA sequences

• Detection method adjustments:

  • Implement multiple orthogonal techniques to confirm modification status

  • Include internal standards for quantitative analyses

  • Develop Mycoplasma-specific analytical protocols

• Functional compensation investigation:

  • Examine alternative modification pathways

  • Analyze tRNA structural stability through thermal denaturation

  • Investigate specialized translation mechanisms

Researchers should note that Mycoplasma translation apparatus may have undergone compensatory changes to cope with modification deficiencies , necessitating broader experimental approaches when studying translation fidelity and efficiency.

How does MnmG activity relate to bacterial pathogenesis and antimicrobial resistance?

While primarily focusing on fundamental tRNA biology, MnmG research has potential implications for pathogenesis:

• Translation stress response:

  • MnmG-deficient bacteria may display altered stress responses

  • Modified translation profiles could affect virulence factor expression

  • Growth defects might impact host colonization efficiency

• Antimicrobial susceptibility:

  • Antibiotics targeting translation might show altered efficacy in MnmG mutants

  • Translation errors could affect membrane protein folding and permeability

  • Stress response alterations might impact adaptive resistance mechanisms

• Experimental approaches:

  • Animal infection models comparing wild-type and MnmG-deficient strains

  • Minimum inhibitory concentration determinations across antibiotic classes

  • Transcriptomic analysis under infection-relevant conditions

Given that Mycoplasma mycoides is a significant pathogen causing contagious bovine pleuropneumonia, these studies could provide valuable insights into its pathogenicity mechanisms .

What evolutionary insights can be gained from comparing MnmG across Mycoplasma species?

Comparative analysis of MnmG across Mycoplasma species offers evolutionary insights:

• Genomic context analysis:

  • Synteny conservation around the mnmG locus

  • Co-evolution with tRNA genes and other modification enzymes

  • Horizontal gene transfer evidence

• Functional divergence evaluation:

  • Comparison of substrate specificity across species

  • Analysis of modification patterns in different Mycoplasma lineages

  • Correlation with genome reduction events

• Research approaches:

  • Phylogenetic analysis of MnmG protein sequences

  • Ancestral sequence reconstruction and functional testing

  • Experimental complementation across species boundaries

Studies of Mycoplasma capricolum revealed that one trmFO homolog (Mcap0476) evolved to methylate rRNA rather than tRNA, demonstrating functional divergence within modification enzymes . Similar evolutionary shifts might exist for MnmG in Mycoplasma mycoides.