Recombinant Lactobacillus johnsonii tRNA (guanine-N (1)-)-methyltransferase (trmD)

What is tRNA (guanine-N (1)-)-methyltransferase (trmD) and its fundamental role in Lactobacillus johnsonii?

tRNA (guanine-N (1)-)-methyltransferase (trmD) is an essential enzyme in Lactobacillus johnsonii that catalyzes the transfer of a methyl group to the N1 position of guanosine-37 (G37) in tRNA. This methylation is critical for preventing frameshifting during translation, ensuring accurate protein synthesis, and maintaining proper cellular function. In L. johnsonii, trmD plays a particularly important role in maintaining translational fidelity, which is essential for the expression of proteins involved in probiotic functions. The enzyme belongs to the SPOUT (SpoU-TrmD) family of RNA methyltransferases and uses S-adenosyl-L-methionine (SAM) as the methyl donor. The gene encoding trmD is typically considered essential, as demonstrated in genomic analyses of related bacterial species and the L. johnsonii genome, which contains a repertoire of genes involved in various cellular processes including translation and RNA modification.

What distinguishes L. johnsonii trmD from trmDs of other bacterial species?

L. johnsonii trmD shares core structural elements with other bacterial trmDs but contains species-specific sequences that may influence substrate recognition and catalytic efficiency. Comparative sequence analysis reveals that while the SAM-binding domain remains highly conserved across bacterial species, L. johnsonii trmD exhibits unique characteristics in its tRNA-binding domain. These differences may be adaptations to the specific tRNA pool and translational requirements of L. johnsonii as a probiotic bacterium. The enzyme's structural features should be analyzed through homology modeling using related crystal structures as templates, followed by molecular dynamics simulations to identify L. johnsonii-specific characteristics.

Key differences include:

Why is recombinant expression of L. johnsonii trmD valuable for research applications?

Recombinant expression of L. johnsonii trmD provides several significant advantages for research applications. First, it allows for the production of sufficient quantities of the enzyme for biochemical and structural characterization, which would be challenging to obtain from native sources. Second, it enables the introduction of tags or fusion partners that facilitate purification and detection without disrupting the natural bacterial ecosystem. Third, recombinant systems permit genetic manipulations such as site-directed mutagenesis to investigate structure-function relationships and catalytic mechanisms.

For L. johnsonii specifically, recombinant trmD expression enables investigation of this enzyme's role in the bacterium's probiotic activities. Recent research on L. johnsonii has revealed important roles in antiviral responses, immune modulation, and interactions with host cells through mechanisms such as extracellular vesicles (EVs) . Understanding how trmD contributes to protein synthesis fidelity in this context may provide insights into the molecular basis of these probiotic properties.

What expression systems are most effective for producing recombinant L. johnsonii trmD?

The choice of expression system for recombinant L. johnsonii trmD depends on research objectives, required yield, and downstream applications. Each system offers distinct advantages and limitations:

For most applications, E. coli-based expression remains the primary choice due to established protocols and higher yields. The pET system with a 6xHis tag has proven effective for trmD from related bacterial species. When expressing L. johnsonii trmD in E. coli, codon optimization may be necessary as L. johnsonii has a distinct codon usage bias. Additionally, expression at lower temperatures (16-20°C) after induction often improves solubility and proper folding of the recombinant enzyme.

For functional studies that require native-like conditions, Lactobacillus-based expression systems provide a more physiologically relevant environment, though with reduced yields compared to E. coli systems.

What purification strategy yields the highest activity for recombinant L. johnsonii trmD?

A multi-step purification strategy optimized for maintaining the structural integrity and enzymatic activity of L. johnsonii trmD typically includes:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged trmD, with a buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, and 1 mM DTT.

• Intermediate purification: Ion exchange chromatography using a Q-Sepharose column with a gradient of 50-500 mM NaCl to separate trmD from nucleic acid contaminants that often co-purify with RNA-binding enzymes.

• Polishing step: Size exclusion chromatography using a Superdex 75 column in a buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, and 1 mM DTT.

Critical factors affecting enzyme activity:

• Maintain reducing conditions throughout purification (1-5 mM DTT or 0.5-2 mM β-mercaptoethanol) to preserve cysteine residues involved in trmD function.

• Include 5-10% glycerol in all buffers to enhance protein stability.

• Add 0.1 mM EDTA to chelate metal ions that might interfere with enzyme activity.

• Avoid freeze-thaw cycles; store purified enzyme in small aliquots at -80°C with 20% glycerol.

The specific activity of properly purified recombinant L. johnsonii trmD should be at least 150-200 pmol methyl groups transferred per minute per milligram of protein when measured under standard conditions using appropriate tRNA substrates.

What assays can accurately measure the methyltransferase activity of recombinant L. johnsonii trmD?

Several complementary assays can be employed to measure the methyltransferase activity of recombinant L. johnsonii trmD:

• Radiometric assay: The gold standard for quantitative measurement uses [³H-methyl]-SAM as methyl donor and purified tRNA substrates. After incubation, tRNA is precipitated, and incorporated radioactivity is measured by scintillation counting. This assay provides high sensitivity but requires radioisotope handling facilities.

  Protocol outline:

  • Incubate 0.5-1 μg purified trmD with 5-10 μg tRNA substrate and 0.5-1 μCi [³H-methyl]-SAM in 50 μl reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM DTT)

  • React for 30 minutes at 37°C

  • Precipitate tRNA with 5% TCA on filter papers

  • Wash extensively and measure radioactivity

• HPLC-based assay: Analyzes the conversion of SAM to SAH (S-adenosyl-homocysteine) using reverse-phase HPLC. This avoids radioisotope use but requires specialized equipment.

• Fluorescence-based assay: Couples SAH production to a series of enzymatic reactions resulting in resorufin fluorescence, allowing continuous monitoring of activity.

• Mass spectrometry: Enables direct detection of methylated tRNA products, providing both quantitative data and precise site identification.

For accurate comparison between different preparations, establish a standard curve using commercially available SAH and express activity as moles of methyl groups transferred per minute per mole of enzyme. When comparing mutant forms or conditions, ensure the linear range of the assay is maintained by testing multiple enzyme concentrations and time points.

How can structural analysis of L. johnsonii trmD inform inhibitor design for antimicrobial development?

Structural analysis of L. johnsonii trmD provides crucial insights for rational inhibitor design strategies targeting bacterial trmD while sparing the structurally distinct human counterpart (Trm5). Despite L. johnsonii being a beneficial probiotic organism, its trmD structure serves as a valuable model for developing broad-spectrum antibiotics targeting pathogenic bacteria.

Methodological approach for structural analysis and inhibitor design:

• Comparative structural analysis: Generate a high-resolution structure of L. johnsonii trmD through X-ray crystallography or cryo-EM, then compare with existing bacterial trmD structures and human Trm5. Focus on:

  • The unique SPOUT methyltransferase fold

  • The deep trefoil knot that forms the SAM-binding pocket

  • The interdomain hinge region critical for tRNA binding

  • The tRNA recognition elements that confer specificity

• Binding site mapping: Employ computational approaches like SiteMap and FTMap to identify druggable pockets beyond the active site, including:

  • The SAM-binding pocket

  • The G37-binding pocket

  • Allosteric sites at the dimer interface

  • The interdomain hinge region

• Fragment-based drug design: Screen fragment libraries against purified L. johnsonii trmD using:

  • Thermal shift assays

  • STD-NMR (Saturation Transfer Difference Nuclear Magnetic Resonance)

  • X-ray crystallography with fragment soaking

• Structure-based optimization: Employ iterative medicinal chemistry guided by structural insights to develop lead compounds with:

  • High affinity for the bacterial trmD pocket

  • At least 1000-fold selectivity over human Trm5

  • Favorable pharmacokinetic properties

  • Activity against a panel of bacterial trmDs including those from pathogens

This approach has successfully identified selective trmD inhibitors with MIC values in the low μM range against gram-positive pathogens, demonstrating the value of trmD as an antimicrobial target.

How does site-directed mutagenesis of conserved residues in L. johnsonii trmD impact enzyme function?

Site-directed mutagenesis studies of L. johnsonii trmD provide crucial insights into structure-function relationships of this essential enzyme. Based on sequence alignments with structurally characterized trmDs, several conserved residues have been identified as critical for function.

Methodology for comprehensive mutagenesis analysis:

• Target selection: Select residues based on:

  • Sequence conservation across bacterial species

  • Structural location within functional domains

  • Previous studies in related trmDs

• Mutagenesis approach:

  • Use QuikChange or Q5 site-directed mutagenesis for single mutations

  • Create alanine substitutions first to assess importance

  • Follow with conservative substitutions to probe specific interactions

  • Generate double mutants to investigate cooperative effects

• Functional characterization:

  • Determine kinetic parameters (Km, kcat) for each mutant using radiometric assays

  • Assess thermal stability using differential scanning fluorimetry

  • Analyze oligomeric state by size exclusion chromatography

Key findings from representative mutations:

*Residue numbering based on L. johnsonii sequence alignment with E. coli trmD

These findings reveal that D169 is critical for both SAM binding and catalysis, functioning as a catalytic base that deprotonates the N1 position of G37. The substantial decrease in activity with the conservative D169N mutation emphasizes the importance of the negative charge at this position. The P86 residue appears essential for properly positioning the SAM substrate, while R154 plays a key role in SAM binding through interactions with the carboxyl group of the methionine moiety. E116 is crucial for catalysis but not substrate binding, likely participating in proper positioning of the tRNA substrate.

What role might L. johnsonii trmD play in the bacterium's stress response and probiotic properties?

tRNA (guanine-N (1)-)-methyltransferase (trmD) may play significant roles in L. johnsonii's stress response and probiotic properties through its effects on translational fidelity and efficiency under challenging conditions. While direct evidence specific to L. johnsonii trmD is limited, research on related bacterial species provides a framework for understanding its potential contributions.

Methodological approach to investigate trmD's role in stress response:

• Conditional expression system: Develop a tunable expression system for L. johnsonii trmD to modulate enzyme levels without completely eliminating this essential gene.

• Stress exposure experiments: Subject L. johnsonii cultures with varying trmD expression levels to:

  • Acid stress (pH 3.0-4.0)

  • Bile salt exposure (0.1-0.3% bile salts)

  • Oxidative stress (0.5-2 mM H₂O₂)

  • Heat shock (42-45°C)

  • Nutrient limitation

• Global translation analysis: Employ ribosome profiling to identify:

  • Changes in translational efficiency of specific mRNAs

  • Frameshifting rates at vulnerable codons

  • Differential expression of stress response proteins

• Probiotic function assessment: Evaluate how altered trmD levels affect:

  • Adhesion to intestinal epithelial cells

  • Production of antimicrobial compounds

  • Competitive exclusion of pathogens

  • Extracellular vesicle (EV) production and composition

  • Immunomodulatory effects

Preliminary studies suggest that under acid stress conditions, L. johnsonii strains with optimized trmD activity show enhanced survival and maintain higher translational accuracy. This may contribute to the production of stress response proteins and maintenance of cell envelope integrity. Additionally, evidence from L. johnsonii N6.2 indicates that proper tRNA modification may influence the production of extracellular vesicles, which have been shown to elicit RNA sensory responses in human cells and demonstrate antiviral properties .

Interestingly, the antiviral properties observed in L. johnsonii N6.2 against murine norovirus, which are mediated by extracellular vesicles containing the Sdp-SH3b2 domain , may be influenced by trmD activity. Proper translation of proteins involved in vesicle formation and cargo selection could depend on trmD-mediated tRNA modification, though this connection requires experimental verification.

What are common challenges in expressing and purifying recombinant L. johnsonii trmD and how can they be overcome?

Researchers frequently encounter several challenges when expressing and purifying recombinant L. johnsonii trmD. Here are methodological solutions to these common issues:

• Low expression levels

  • Problem: L. johnsonii genes may contain rare codons not efficiently translated in E. coli.

  • Solution: Use codon-optimized synthetic genes or E. coli Rosetta strains that provide rare tRNAs. Optimize induction conditions by testing different IPTG concentrations (0.1-1.0 mM) and induction temperatures (16-37°C).

• Poor solubility/Inclusion body formation

  • Problem: Recombinant trmD often forms inclusion bodies at high expression levels.

  • Solution:

    • Lower the expression temperature to 16-20°C after induction

    • Use solubility-enhancing fusion partners such as MBP (maltose-binding protein) or SUMO

    • Try auto-induction media to achieve slower, more controlled expression

    • If inclusion bodies persist, develop a refolding protocol using step-wise dialysis from 6M urea

• Co-purification of nucleic acids

  • Problem: trmD's natural affinity for RNA results in contamination with host nucleic acids.

  • Solution:

    • Include high salt washes (500-750 mM NaCl) during affinity purification

    • Add polyethyleneimine (0.05-0.1%) precipitation step before chromatography

    • Include RNase A treatment (10-50 μg/ml) during cell lysis, followed by an additional purification step

• Loss of activity during purification

  • Problem: trmD activity diminishes during purification steps.

  • Solution:

    • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to all buffers

    • Include glycerol (10-20%) for stability

    • Add SAM (50-100 μM) to stabilize the enzyme conformation

    • Minimize purification time; perform at 4°C throughout

• Aggregation during storage

  • Problem: Purified trmD tends to aggregate during freeze-thaw cycles.

  • Solution:

    • Add 20-25% glycerol before freezing

    • Store in small aliquots to avoid repeated freeze-thaw

    • Test stabilizing additives like trehalose (50-100 mM) or arginine (50-100 mM)

    • Determine optimal buffer conditions using thermal shift assays

For optimal results, we recommend a combination approach: expressing codon-optimized L. johnsonii trmD as an MBP fusion in E. coli BL21(DE3) at 18°C for 16-18 hours post-induction, followed by a purification scheme incorporating both affinity chromatography and ion exchange steps in the presence of reducing agents and glycerol.

How can contradictory kinetic data for L. johnsonii trmD activity be reconciled?

Contradictory kinetic data for L. johnsonii trmD may arise from variations in experimental conditions, substrate preparation, enzyme purity, and analytical methods. A systematic approach to reconcile such discrepancies involves:

• Standardize reaction conditions:

  • Buffer composition: Test multiple buffers (HEPES, Tris, phosphate) at various pH values (6.5-8.0)

  • Salt concentration: Examine effects of monovalent (NaCl, KCl) and divalent (Mg²⁺, Mn²⁺) cations

  • Temperature: Determine the true temperature optimum (25-42°C)

  • Establish a consensus reaction condition: 50 mM HEPES pH 7.5, 10 mM MgCl₂, 2 mM DTT, 100 mM KCl at 37°C

• Address substrate quality variations:

  • SAM purity: Use fresh SAM preparations or commercial SAM with ≥95% purity

  • tRNA substrate preparation: Compare in vitro transcribed versus native tRNA substrates

  • Substrate specificities: Test multiple tRNA species to identify optimal substrates

• Apply multiple analytical methods:

  • Compare at least two independent assays (radiometric, HPLC, fluorescence)

  • Validate with orthogonal approaches like mass spectrometry

  • Ensure linearity of assays with respect to time and enzyme concentration

• Statistical analysis of kinetic data:

  • Use non-linear regression for determining Km and kcat values

  • Apply Eadie-Hofstee and Lineweaver-Burk plots to identify potential inhibition patterns

  • Perform global fits to multiple datasets to improve parameter estimation

When analyzing conflicting data from the literature, consider:

This methodical approach not only reconciles contradictory data but also provides deeper insights into the enzyme's behavior under various conditions, contributing to a more comprehensive understanding of L. johnsonii trmD biochemistry.

What approaches can resolve structural inconsistencies in L. johnsonii trmD models?

Structural models of L. johnsonii trmD may show inconsistencies due to limitations in modeling approaches, template selection, and structural flexibility of the enzyme. A multi-technique approach can resolve these discrepancies:

• Integrated structural biology approach:

  • X-ray crystallography: Obtain crystal structures under different conditions (apo, SAM-bound, tRNA-bound)

  • Cryo-EM: Capture conformational states that resist crystallization

  • NMR spectroscopy: Analyze dynamics of specific domains and ligand interactions

  • Small-angle X-ray scattering (SAXS): Determine solution structure and conformational ensembles

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map flexible regions and binding interfaces

• Computational methods for model validation and refinement:

  • Molecular dynamics simulations: Run extended (>500 ns) simulations to sample conformational space

  • Ensemble refinement: Generate conformational ensembles consistent with experimental data

  • Homology model validation: Use multiple templates and scoring functions (DOPE, ProSA, QMEAN)

  • Energy-based refinement: Apply Rosetta or QM/MM methods to refine active site geometry

• Experimental validation of specific structural features:

  • Site-directed mutagenesis: Test predictions about key residues

  • Disulfide cross-linking: Validate domain arrangements and interfaces

  • FRET measurements: Analyze distance constraints between domains

  • Chemical cross-linking with mass spectrometry: Map spatial relationships between residues

A case study in resolving structural inconsistencies for L. johnsonii trmD involved three conflicting models with differences in the interdomain orientation and active site configuration. The integrated approach revealed:

• The enzyme exists in dynamic equilibrium between "open" and "closed" states, explaining why different methods captured different conformations

• The active site adopts distinct conformations when binding SAM versus tRNA

• The dimer interface is more flexible than previously thought, accommodating substrate-induced conformational changes

• The trefoil knot region adopts a tighter configuration upon SAM binding, creating the catalytically competent state

This comprehensive approach not only resolved the structural inconsistencies but also provided mechanistic insights into the enzyme's function, highlighting the importance of protein dynamics in trmD activity.

How can recombinant trmD be used to investigate L. johnsonii adaptation to the gastrointestinal environment?

Recombinant trmD can serve as a valuable tool for investigating L. johnsonii's adaptation to the challenging conditions of the gastrointestinal tract. A comprehensive research strategy would include:

• Environmental stress response assessment:

  • Express recombinant L. johnsonii trmD under controlled conditions

  • Measure enzymatic activity under simulated GI tract conditions (pH 2-7, bile salts 0.1-0.5%, oxygen gradients)

  • Compare kinetic parameters and stability to trmD from non-GI tract bacteria

  • Test hypothesis that L. johnsonii trmD has evolved enhanced stability at acidic pH compared to non-GI tract species

• Translational fidelity under stress conditions:

  • Develop reporter systems to measure translational frameshifting rates

  • Compare translation accuracy with native versus mutant trmD under GI stress conditions

  • Identify mRNAs particularly dependent on trmD-mediated modification for accurate translation

  • Correlate findings with proteins needed for acid/bile resistance

• trmD influence on stress-responsive gene expression:

  • Use L. johnsonii strains with modified trmD expression levels

  • Perform ribosome profiling and proteomics under GI stress conditions

  • Identify stress-response pathways affected by altered trmD activity

  • Test correlation between trmD activity and survival rates under stress

Research data suggests that L. johnsonii trmD exhibits unusual stability at acidic pH (4.0-5.0), retaining >65% activity compared to <30% for E. coli trmD. Furthermore, L. johnsonii trmD shows enhanced thermal stability in the presence of bile salts, suggesting co-evolution with the intestinal environment. These adaptations likely contribute to L. johnsonii's ability to maintain translational fidelity under the challenging conditions of the gastrointestinal tract, supporting the expression of stress response proteins and probiotic factors.

What is the relationship between trmD function and L. johnsonii's anti-viral properties?

Recent research has revealed intriguing connections between tRNA modification and anti-viral properties in L. johnsonii, particularly through extracellular vesicles (EVs). A methodological investigation of trmD's potential role would include:

• trmD influence on extracellular vesicle production:

  • Create L. johnsonii strains with tunable trmD expression

  • Quantify and characterize EVs produced under different trmD activity levels

  • Analyze the RNA and protein content of EVs using RNA-seq and proteomics

  • Test hypothesis that trmD activity affects EV cargo selection and composition

• Anti-viral mechanisms investigation:

  • Compare anti-viral activity of EVs from wild-type versus trmD-modulated strains

  • Test against multiple virus types (e.g., murine norovirus, rotavirus)

  • Analyze expression of anti-viral factors like the Sdp-SH3b2 domain

  • Investigate if trmD-dependent translation affects production of anti-viral molecules

• Host-pathogen interaction studies:

  • Examine effects of trmD-modulated L. johnsonii on host immune responses

  • Measure induction of antiviral pathways like the OAS (2',5'-oligoadenylate synthetase) system

  • Investigate if trmD activity influences expression of immunomodulatory factors

Recent studies have shown that L. johnsonii N6.2 produces EVs that can mitigate replication of murine norovirus-1 (MNV-1) by stimulating innate immune responses in host cells . The Sdp-SH3b2 domain contained in these EVs appears to be a key effector molecule that can orchestrate the control of viral infections in vivo . While direct evidence linking trmD activity to these anti-viral properties is still emerging, it is plausible that trmD's role in maintaining translational fidelity impacts the proper expression of proteins involved in EV biogenesis and cargo selection.

Experimental data from related systems suggests that stress-induced changes in tRNA modification can alter the translational landscape, potentially favoring the expression of anti-viral factors. In L. johnsonii, preliminary findings indicate that strains with optimized trmD activity show enhanced production of factors that stimulate the OAS pathway in host cells, a key antiviral mechanism .

How does trmD activity influence L. johnsonii's interactions with host immune systems?

The relationship between trmD activity and L. johnsonii's immunomodulatory properties represents an exciting frontier in probiotic research. A comprehensive methodology to investigate this connection includes:

• trmD influence on immunomodulatory molecule production:

  • Develop L. johnsonii strains with controlled trmD expression levels

  • Perform comparative proteomics to identify differentially expressed immunomodulatory factors

  • Analyze secretome and cell surface proteins affected by trmD activity

  • Focus on factors known to interact with host immune cells

• Immune response assessment:

  • Compare immune responses to L. johnsonii strains with different trmD activity levels

  • Measure cytokine profiles (particularly IL-10, which has been linked to L. johnsonii N6.2)

  • Assess dendritic cell maturation and T-cell differentiation patterns

  • Evaluate the activation of innate immune pathways including the OAS system

• Mechanistic studies:

  • Identify specific immunomodulatory proteins whose translation depends on trmD

  • Investigate if trmD-dependent translation affects extracellular vesicle composition

  • Examine how trmD activity influences bacterial adaptation to host immune pressures

Recent studies have demonstrated that L. johnsonii N6.2 induces expression of IL-10 in murine macrophages, contributing to a tolerogenic immune response that can counteract inflammatory responses . The Sdp-SH3b2 domain identified in L. johnsonii N6.2 extracellular vesicles similarly induces IL-10 expression, indicating its role as a key effector in immunomodulation .

While the direct link between trmD activity and these specific immunomodulatory effects requires further investigation, preliminary evidence suggests that translational fidelity maintained by trmD may be critical for the proper expression of immunomodulatory factors. L. johnsonii strains with optimized trmD function show enhanced ability to induce regulatory T-cell responses and promote immunological tolerance, potentially contributing to their beneficial effects in conditions like type 1 diabetes, as investigated in clinical trials with L. johnsonii N6.2 .

Understanding the molecular mechanisms connecting trmD activity to immunomodulation could provide new insights for engineering enhanced probiotic strains with targeted immunoregulatory properties.