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

What is Lactobacillus plantarum and why is it significant for recombinant protein studies?

Lactobacillus plantarum (recently reclassified as Lactiplantibacillus plantarum) is a versatile probiotic bacterium naturally found in the human mouth, gut, and various fermented foods. This gram-positive bacterium has gained significant attention as a potential starter and health-promoting probiotic with diverse applications in biotechnology. L. plantarum serves as an excellent host for recombinant protein expression due to its GRAS (Generally Recognized As Safe) status, ability to survive gastrointestinal transit, and capacity to deliver heterologous proteins to mucosal surfaces. These characteristics make it particularly valuable for expressing proteins like tRNA (guanine-N(1))-methyltransferase that require proper folding and function in a bacterial system .

How can I express recombinant trmD in Lactobacillus plantarum?

Expression of recombinant trmD in L. plantarum typically involves a multi-step process that begins with gene isolation and vector construction. The trmD gene can be amplified from L. plantarum genomic DNA using PCR with specific primers designed to include appropriate restriction sites. After cloning the gene into a suitable expression vector containing regulatory elements functional in L. plantarum, the construct can be transformed into competent L. plantarum cells. Current genetic tools for L. plantarum include CRISPR/Cas9-assisted recombineering methods that enable precise genetic modifications, including gene knockouts, insertions, and point mutations . Expression can be optimized through various approaches, including codon optimization, use of strong promoters, and cultivation under optimal growth conditions, which for L. plantarum is typically near 37°C (where it demonstrates a specific growth rate of 0.7683 h⁻¹ and doubling time of 54 minutes) .

What strategies can optimize the expression and purification of functional recombinant trmD from L. plantarum?

Optimizing recombinant trmD expression in L. plantarum requires a multifaceted approach addressing both genetic and environmental factors. At the genetic level, several strategies have proven effective:

• Promoter selection and engineering: Using strong, inducible promoters to control expression timing and level

• Codon optimization: Adapting the trmD coding sequence to match L. plantarum's codon usage bias

• Signal peptide incorporation: Adding appropriate secretion signals if extracellular expression is desired

• Fusion tags: Including affinity tags (His, GST, etc.) for simplified purification while ensuring minimal impact on enzyme activity

For purification, a multi-stage approach is typically required:

• Initial clarification of cell lysate through centrifugation and filtration

• Capture phase using affinity chromatography (leveraging fusion tags)

• Intermediate purification via ion exchange chromatography

• Polishing step using size exclusion chromatography

• Optional tag removal through specific proteases if the tag affects enzyme function

CRISPR/Cas9-assisted genome editing techniques allow for chromosome integration of the trmD gene with precise control over expression levels. Phosphorothioate modification can improve double-stranded DNA insertion efficiency, while adenine-specific methyltransferase overexpression enhances single-stranded DNA recombination efficiency, as demonstrated with other recombinant proteins in L. plantarum .

How do post-translational modifications affect trmD functionality in recombinant expression systems?

The functionality of recombinant trmD in L. plantarum is significantly influenced by post-translational modifications (PTMs) that occur after protein synthesis. These modifications can include:

• Phosphorylation: Potential regulatory mechanism affecting enzyme activity

• Acetylation: May influence protein stability and interaction with tRNA substrates

• Proteolytic processing: Can impact enzyme maturation and activity

• Disulfide bond formation: Critical for proper tertiary structure

Research indicates that the bacterial host environment significantly impacts these modifications. L. plantarum, as a gram-positive bacterium, may introduce different PTMs compared to commonly used E. coli expression systems. Comparative studies between native and recombinant trmD often reveal activity differences attributable to PTMs.

To assess the impact of PTMs on enzyme functionality, researchers typically employ:

• Mass spectrometry to identify specific modifications

• Site-directed mutagenesis to evaluate the importance of modified residues

• Activity assays comparing native and recombinant enzymes

• Structural studies correlating modifications with protein conformation

Optimizing expression conditions, including growth temperature, media composition, and induction parameters, can help ensure proper PTMs and maximize functional enzyme yield .

What are the implications of trmD mutations on L. plantarum fitness and metabolic capabilities?

Mutations in trmD can have profound effects on L. plantarum fitness due to the enzyme's essential role in translation fidelity. Comprehensive mutational analysis reveals several categories of effects:

• Lethal mutations: Complete loss of trmD function is typically lethal, underscoring its essential nature

• Growth defects: Partial function mutations often result in slower growth kinetics

• Stress sensitivity: Many trmD mutants show increased sensitivity to environmental stressors

• Metabolic alterations: Changes in translation fidelity can cascade to affect numerous metabolic pathways

These fitness effects manifest through translational frameshifting, increased error rates, and protein misfolding. The methyl modification catalyzed by trmD prevents base pairing between G37 and the adjacent C36, maintaining the correct reading frame during translation.

Table 1: Correlation between trmD mutation types and L. plantarum phenotypes

Exploiting trmD mutations through genetic engineering approaches presents opportunities for modulating L. plantarum metabolism for specific biotechnological applications, similar to how metabolic engineering has been used to enhance N-acetylglucosamine production in this organism .

What are the most effective protocols for measuring trmD enzymatic activity in recombinant L. plantarum systems?

Accurately measuring trmD enzymatic activity in recombinant L. plantarum requires robust, sensitive assays that can distinguish between native and recombinant enzyme contributions. The following methodological approaches are recommended:

• Radiometric methylation assay:

  • Incubate purified trmD with substrate tRNA and [³H]-S-adenosylmethionine (SAM)

  • Measure incorporation of radioactive methyl groups into tRNA substrate

  • Quantify using scintillation counting after precipitation and washing

• Mass spectrometry-based approaches:

  • Digest tRNA with RNase after methylation reaction

  • Analyze modified nucleosides by LC-MS/MS

  • Quantify G37 methylation levels versus unmodified controls

• Fluorescence-based assays:

  • Use fluorescently labeled tRNA substrates

  • Monitor changes in fluorescence properties upon methylation

  • Enables real-time kinetic measurements

For in vivo assessment of trmD function in recombinant systems:

• Frameshifting reporter assays using dual luciferase constructs

• Monitoring growth rates under various stress conditions

• Ribosome profiling to assess translational fidelity

Optimal reaction conditions for L. plantarum trmD typically include pH 7.0-8.0, temperature of 30-37°C (aligning with the organism's optimal growth temperature), and appropriate concentrations of divalent metal ions (Mg²⁺ typically at 5-10 mM) .

How can CRISPR/Cas9 technology be applied to study trmD function in L. plantarum?

CRISPR/Cas9 technology offers powerful approaches for investigating trmD function in L. plantarum through precise genome editing. Effective implementation requires careful consideration of several methodological aspects:

• CRISPR/Cas9 system design for L. plantarum:

  • Selection of appropriate Cas9 variant with optimal activity in L. plantarum

  • Design of sgRNA targeting specific regions of the trmD gene

  • Development of efficient delivery methods (typically electroporation for L. plantarum)

• Editing strategies for functional analysis:

  • Knockout studies through frameshift mutations or complete gene deletion

  • Point mutations targeting catalytic residues for structure-function analysis

  • Promoter modifications to control expression levels

  • Domain swapping with homologs from other species

• Technical optimizations for L. plantarum:

  • Phosphorothioate modification of double-stranded DNA to improve insertion efficiency

  • Adenine-specific methyltransferase overexpression to enhance recombination efficiency

  • Temperature optimization (near 37°C) for optimal cellular machinery function

• Validation approaches:

  • PCR and sequencing to confirm genetic modifications

  • RT-qPCR to assess transcriptional changes

  • Western blotting to evaluate protein expression levels

  • Activity assays to confirm functional consequences

Recent advances in CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering have enabled seamless genome editing in L. plantarum, facilitating precise genetic modifications without introducing exogenous genes or plasmids. This approach has been successfully applied to metabolic engineering in L. plantarum WCFS1, suggesting its potential applicability to trmD studies .

What immunological techniques can assess the impact of recombinant trmD expression on L. plantarum host responses?

Evaluating the immunological impact of recombinant trmD expression in L. plantarum requires sophisticated techniques that assess both bacterial physiology and host immune responses. The following methodological approaches have proven effective:

• Flow cytometry-based assays:

  • Quantification of surface marker expression on L. plantarum

  • Assessment of bacterial cell viability and stress responses

  • Analysis of host immune cell activation (dendritic cells, T cells, B cells)

• T cell response evaluation:

  • Measurement of CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cell populations

  • T cell proliferation assays using CFSE staining

  • Cytokine profiling to assess polarization patterns

• Dendritic cell activation assessment:

  • Evaluation of dendritic cell maturation markers

  • Antigen presentation capacity testing

  • Migration assays to assess chemotactic responses

• B cell and antibody responses:

  • Quantification of B220⁺IgA⁺ cells in Peyer's patches

  • Measurement of specific antibody production (IgG, IgA)

  • Assessment of antibody functionality through neutralization assays

These techniques have been successfully applied to other recombinant L. plantarum strains, such as those expressing influenza virus antigens. Similar approaches can be adapted to study trmD-expressing strains, allowing researchers to evaluate whether trmD expression modifies the inherent immunomodulatory properties of L. plantarum .

How do I interpret discrepancies between in vitro and in vivo trmD activity data?

Discrepancies between in vitro and in vivo trmD activity measurements are common and can provide valuable insights when properly interpreted. The following systematic approach can help researchers resolve such inconsistencies:

• Identify potential sources of variation:

  • Substrate availability differences (purified tRNA vs. cellular pool)

  • Cofactor concentrations (SAM levels, magnesium availability)

  • Presence of regulatory proteins or small molecules

  • Post-translational modifications present in vivo but absent in purified systems

• Design validation experiments:

  • Cell extract supplementation studies to identify missing cofactors

  • Protein complex isolation to identify interaction partners

  • Metabolomic analysis to identify potential allosteric regulators

  • Targeted mutagenesis to evaluate regulatory modifications

• Quantitative framework for comparison:

  • Calculate apparent kinetic parameters in both systems

  • Develop mathematical models accounting for cellular context

  • Establish correction factors for consistent interpretation

• Growth condition considerations:

  • Evaluate trmD activity across different growth phases

  • Test activity under various stress conditions

  • Consider temperature effects (optimal growth at 37°C with specific growth rate of 0.7683 h⁻¹)

Understanding that L. plantarum has complex regulatory networks that may influence trmD activity in vivo is essential. For example, growth temperature significantly affects L. plantarum metabolism, potentially modifying enzyme activity in ways not replicable in vitro .

What strategies can address low expression or inactivity of recombinant trmD in L. plantarum?

When faced with challenges in expressing active recombinant trmD in L. plantarum, researchers can implement a systematic troubleshooting approach:

• Expression-level optimization:

  • Promoter strength adjustment through promoter library screening

  • Ribosome binding site optimization for efficient translation initiation

  • Codon optimization based on L. plantarum-specific usage patterns

  • Induction timing and conditions refinement (temperature, inducer concentration)

• Protein folding and stability enhancement:

  • Co-expression with molecular chaperones (GroEL/ES, DnaK system)

  • Addition of stability-enhancing fusion partners

  • Inclusion of disulfide bond formation facilitators if appropriate

  • Cultivation at lower temperatures (20-30°C) to slow folding

• Enzyme activity restoration:

  • Supplementation with potential cofactors or metal ions

  • Expression of accessory proteins that may form functional complexes

  • Directed evolution to select for variants with improved activity

  • Rational design based on structural information

Table 2: Troubleshooting strategies for recombinant trmD expression in L. plantarum

The CRISPR/Cas9-assisted genome editing techniques described for L. plantarum can be particularly valuable for addressing expression issues through precise manipulation of the genomic context, potentially integrating trmD into highly expressed genomic loci or modifying regulatory elements .

What emerging technologies might advance our understanding of trmD function in L. plantarum?

Several cutting-edge technologies are poised to revolutionize research on trmD function in L. plantarum:

• Single-cell technologies:

  • Single-cell RNA sequencing to capture cell-to-cell variability in trmD expression

  • Single-molecule fluorescence for tracking trmD dynamics in living cells

  • Microfluidic approaches for correlating trmD activity with cellular phenotypes

• Advanced genome editing platforms:

  • Base editing technologies for precise nucleotide substitutions

  • Prime editing for targeted insertions and deletions

  • Multiplex genome engineering for systematically modifying trmD and related pathways

• Structural biology innovations:

  • Cryo-EM for visualizing trmD-tRNA complexes at near-atomic resolution

  • Hydrogen-deuterium exchange mass spectrometry for mapping dynamic regions

  • AlphaFold2 and related AI approaches for predicting variant impacts

• Systems biology integration:

  • Multi-omics approaches linking trmD activity to global cellular physiology

  • Metabolic flux analysis for assessing translation-metabolism connections

  • Network modeling to predict impacts of trmD modulation

The recently developed CRISPR/Cas9-assisted recombineering methods in L. plantarum provide a foundation for these advanced approaches, enabling precise genetic manipulations that were previously challenging in this organism. These technologies will likely accelerate our understanding of how trmD contributes to L. plantarum physiology and its potential biotechnological applications .

How might trmD engineering contribute to developing L. plantarum as a biotherapeutic platform?

Engineering trmD in L. plantarum presents exciting opportunities for advancing this organism as a biotherapeutic platform:

• Translation efficiency optimization:

  • Fine-tuning trmD activity to enhance recombinant protein production

  • Modulating translational fidelity for expressing challenging therapeutic proteins

  • Creating synthetic trmD variants with expanded substrate specificity

• Metabolic engineering applications:

  • Leveraging translation-metabolism connections to enhance beneficial metabolite production

  • Developing stress-resistant strains through targeted trmD modifications

  • Optimizing growth characteristics for industrial production

• Immunomodulatory potential:

  • Investigating whether trmD-modified L. plantarum elicits distinct immune responses

  • Developing strains with calibrated immunostimulatory or immunoregulatory properties

  • Creating recombinant L. plantarum expressing both trmD variants and therapeutic antigens

• Delivery system refinement:

  • Engineering trmD to improve L. plantarum survival in gastrointestinal conditions

  • Developing controlled lysis systems for targeted release of therapeutic molecules

  • Creating biosensor-actuator systems with trmD-mediated translational control

Previous research demonstrating L. plantarum's ability to activate dendritic cells in Peyer's patches and induce robust T and B cell responses suggests that trmD-engineered strains could potentially serve as effective mucosal delivery vehicles for vaccines or therapeutic proteins .