Recombinant Lactobacillus plantarum Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) and what is its primary function?

Queuine tRNA-ribosyltransferase (tgt) is an enzyme (EC 2.4.2.29) responsible for the post-transcriptional modification of tRNA by catalyzing the insertion of queuine at the wobble anticodon position of specific tRNAs (tRNAAsn, tRNAAsp, tRNAHis, and tRNATyr). In Lactobacillus plantarum, the tgt gene encodes a 380-amino acid protein that functions as a guanine insertion enzyme or tRNA-guanine transglycosylase .

How does L. plantarum tgt compare with eukaryotic QTRT1?

While L. plantarum tgt and human QTRT1 catalyze similar reactions, they differ in several important aspects:

The human QTRT1 enzyme requires queuine as a substrate, which is exclusively produced by microorganisms and obtained through diet or gut microbiome. Research has demonstrated that human QTRT1 and tRNA Q-modification influence cell proliferation, cellular junctions, and microbiome composition in tumors and intestinal tissues, playing a significant role in breast cancer development .

What expression systems are commonly used for producing recombinant L. plantarum tgt?

Multiple expression systems have been successfully employed for the production of recombinant L. plantarum tgt, each offering specific advantages:

• E. coli expression system: Most commonly used due to high yield and ease of manipulation. Available with different tags (e.g., CSB-EP773445LMS) .

• Yeast expression system: Provides eukaryotic post-translational modifications while maintaining high yield (e.g., CSB-YP773445LMS) .

• Baculovirus expression system: Used for more complex protein folding requirements (e.g., CSB-BP773445LMS) .

• Mammalian cell expression system: Offers the most sophisticated post-translational modifications when necessary for specific functional studies (e.g., CSB-MP773445LMS) .

For specialized applications, biotinylated variants using Avi-tag technology (e.g., CSB-EP773445LMS-B) are available, where E. coli biotin ligase (BirA) catalyzes the covalent attachment of biotin to the AviTag peptide, enabling protein detection and immobilization strategies .

How can researchers leverage tgt activity to study microbiome-host interactions?

Researchers can utilize L. plantarum tgt to investigate microbiome-host interactions through several strategic approaches:

• Modification of tgt expression levels: Creating L. plantarum strains with upregulated or downregulated tgt expression allows researchers to assess how tRNA modification impacts colonization efficiency and persistence in the gut.

• Engineered tgt variants: Introducing specific mutations in the tgt gene can help determine how altered tRNA modification patterns influence bacterial adaptation to host environmental conditions.

• Co-culture systems: Establishing co-culture systems with host cells and L. plantarum strains varying in tgt expression provides insights into how tRNA modifications affect host-microbe communication.

• Fluorescent tagging: Implementing fluorescently tagged tgt proteins enables visualization of enzyme localization and activity dynamics during host-microbe interactions.

L. plantarum has demonstrated significant capabilities in modulating host immune responses through various mechanisms, including the engagement of TLR2/TLR6 heterodimers, which promote regulatory T cell responses and anti-inflammatory activities . The tgt enzyme, through its role in tRNA modification, may influence the expression of bacterial surface components that interact with these host immune receptors.

What role might tgt play in L. plantarum's probiotic properties?

The tgt enzyme potentially contributes to L. plantarum's probiotic properties through several mechanisms:

• Translational efficiency: By modifying specific tRNAs, tgt may enhance translational efficiency under the stress conditions encountered in the gastrointestinal tract.

• Protein expression regulation: tRNA modifications can influence the expression of proteins involved in probiotic functions, including those mediating adhesion to intestinal mucosa and immunomodulatory effects.

• Stress adaptation: tgt-mediated tRNA modifications may contribute to L. plantarum's remarkable ability to adapt to various ecological niches, including the human gastrointestinal tract .

• Colonization persistence: L. plantarum demonstrates superior colonization capabilities compared to other probiotics, with studies showing persistence for months after administration . tgt-related translational control might contribute to this trait.

Research has shown that L. plantarum can modulate NF-κB dependent pathways in the intestinal mucosa, inducing genes associated with anti-inflammatory activities such as BCL3, ADM, and IκB . This suggests a potential role for tgt in establishing an immunoregulatory environment in the host.

How does tgt function relate to the immunomodulatory properties of L. plantarum?

The relationship between tgt function and L. plantarum's immunomodulatory properties may involve:

• Regulation of immunogenic surface components: tgt-mediated tRNA modifications could influence the translation of proteins involved in synthesizing cell surface components that interact with host immune receptors.

• Adaptation to immune microenvironments: The tgt enzyme may enable L. plantarum to adjust its protein expression profile in response to host immune signals.

• Metabolite production: tRNA modifications could affect the production of bacterial metabolites that influence host immune responses.

L. plantarum has been shown to activate dendritic cells in Peyer's patches, increase CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes, and enhance B220+IgA+ cells in Peyer's patches . The exact contribution of tgt to these immunomodulatory effects remains to be fully elucidated but may involve translational regulation of key bacterial components.

What purification strategies yield high-activity recombinant L. plantarum tgt?

To obtain high-activity recombinant L. plantarum tgt, researchers should consider the following purification strategy:

• Expression system selection: For most research applications, E. coli-based expression (e.g., CSB-EP773445LMS) provides the best balance of yield and functionality .

• Affinity purification: Implementing His-tag or other affinity tag-based purification methods is recommended for initial capture.

• Ion exchange chromatography: Following affinity purification, ion exchange chromatography helps remove contaminants with different charge properties.

• Size exclusion chromatography: As a final polishing step, size exclusion chromatography separates aggregates and degradation products.

• Buffer optimization: Maintaining proper buffer conditions (pH 7.5-8.0, with reducing agents) throughout purification preserves enzyme activity.

This multi-step approach typically yields protein with >85% purity as assessed by SDS-PAGE , providing material suitable for enzymatic and structural studies.

What are effective methods for measuring tgt enzymatic activity?

Several complementary approaches can be employed to assess tgt enzymatic activity:

• Radioisotope incorporation assay: Measuring the incorporation of radiolabeled guanine or queuine substrates into tRNA provides a direct quantification of enzymatic activity.

• HPLC-based assay: High-performance liquid chromatography can detect modified nucleosides in tRNA samples before and after tgt treatment.

• Mass spectrometry: LC-MS/MS analysis offers precise identification and quantification of modified nucleosides in tRNA following tgt activity.

• Fluorescence-based assays: Using fluorescently labeled substrate analogs allows real-time monitoring of enzymatic activity.

• Coupled enzyme assays: Systems that couple tgt activity to production of a detectable product (e.g., fluorescent or chromogenic) enable high-throughput screening.

For comprehensive activity assessment, researchers should employ multiple methods and include appropriate controls such as heat-inactivated enzyme and substrate-free reactions.

What considerations are important when designing vectors for L. plantarum tgt expression?

When designing expression vectors for L. plantarum tgt, researchers should consider:

• Codon optimization: Adjusting codons to match the preference of the expression host increases translation efficiency.

• Promoter selection: Using strong, inducible promoters allows controlled expression and reduces potential toxicity.

• Tag placement: N-terminal tags generally have less impact on enzymatic activity than C-terminal tags for tgt.

• Fusion partners: Including solubility-enhancing fusion partners like SUMO or MBP can improve expression and folding.

• Purification strategy: Incorporating appropriate affinity tags (His, GST, etc.) facilitates downstream purification.

• Protease cleavage sites: Including specific protease recognition sequences enables tag removal with minimal residual amino acids.

• Secretion signals: For certain applications, incorporating secretion signals directs protein export from the cell.

The full-length L. plantarum tgt protein (380 amino acids) has been successfully expressed with various tags, with the tag type determined during the manufacturing process based on specific research requirements .

How can recombinant L. plantarum tgt be used to study tRNA modification in microbiome research?

Researchers can utilize recombinant L. plantarum tgt in microbiome research through several approaches:

• Comparative tRNA modification profiling: Analyzing differences in tRNA modification patterns between L. plantarum strains from various ecological niches provides insights into adaptation mechanisms.

• In vitro modification of microbiome-derived tRNAs: Using purified recombinant tgt to modify tRNAs extracted from different microbiome communities helps understand cross-species modification dynamics.

• Heterologous expression in other gut bacteria: Expressing L. plantarum tgt in other bacterial species allows assessment of its impact on their colonization and interaction with the host.

• Competitive colonization experiments: Comparing colonization efficiency between wild-type and tgt-modified L. plantarum strains can reveal the importance of tRNA modification in gut persistence.

L. plantarum has shown remarkable colonization capabilities in human studies, with one trial demonstrating that 95% of treated infants were colonized after 28 days of treatment, with some maintaining colonization for up to 6 months . This presents a valuable model for studying how tgt activity might contribute to microbiome establishment and persistence.

What techniques are effective for studying tgt's impact on bacterial adaptation?

To investigate how tgt influences bacterial adaptation, researchers can employ:

• Growth curve analysis under stress conditions: Comparing growth of wild-type and tgt-modified strains under various stressors (pH, bile salts, oxygen levels) reveals adaptation differences.

• Transcriptome analysis: RNA-seq comparing wild-type and tgt-modified strains identifies genes differentially expressed due to altered tRNA modification.

• Proteome analysis: Quantitative proteomics determines how tgt activity affects the bacterial protein expression profile during adaptation.

• Ribosome profiling: This technique reveals how tRNA modifications influence translation efficiency of specific mRNAs during adaptation.

• Metabolite profiling: Metabolomic analysis identifies differences in cellular metabolites between wild-type and tgt-modified strains.

L. plantarum's ability to inhabit diverse ecological niches is attributed to its unique evolutionary history and genetic flexibility . The tgt enzyme may contribute to this adaptability by fine-tuning translation through tRNA modifications, particularly under changing environmental conditions.

How does tgt activity in L. plantarum potentially influence host disease states?

Research into the connection between L. plantarum tgt activity and host disease states can explore:

• Cancer progression models: Studies similar to those on QTRT1 in cancer could investigate whether L. plantarum tgt activity in the gut microbiome influences tumor development.

• Inflammatory disease models: Examining how L. plantarum strains with varying tgt activity affect inflammatory conditions such as IBD or colitis.

• Gut barrier function: Assessing the impact of tgt-modified L. plantarum on intestinal permeability and tight junction proteins.

• Immune response modulation: Investigating how tgt activity influences L. plantarum's effects on regulatory T cells and cytokine production.

Studies have shown that human QTRT1 and tRNA Q-modification influence cell proliferation, junctions, and microbiome composition in tumors and intestinal tissues, playing a critical role in breast cancer development . The presence of Q-modifying bacteria in the gut microbiome, including L. plantarum with its tgt enzyme, may therefore have implications for host disease processes through similar mechanisms.

What emerging technologies might advance research on L. plantarum tgt?

Several cutting-edge technologies promise to enhance research on L. plantarum tgt:

• CRISPR-Cas9 genome editing: Precise genetic manipulation of tgt and related genes in L. plantarum without marker genes.

• Single-molecule enzymology: Real-time observation of individual tgt molecules interacting with tRNA substrates.

• Cryo-electron microscopy: High-resolution structural analysis of tgt in complex with its tRNA substrates.

• Nanopore direct RNA sequencing: Direct detection of tRNA modifications without the need for reverse transcription.

• Organoid co-culture systems: Advanced 3D intestinal organoid models co-cultured with tgt-modified L. plantarum strains.

These technologies will provide unprecedented insights into the structure-function relationships of tgt and its role in bacterial physiology and host-microbe interactions.

How might engineered variants of L. plantarum tgt contribute to probiotic development?

Engineered L. plantarum tgt variants could advance probiotic development through:

• Enhanced colonization ability: Modified tgt enzymes that optimize tRNA modification patterns for improved gut persistence.

• Targeted immunomodulation: Engineered tgt variants that influence the expression of bacterial components with specific immunomodulatory effects.

• Improved stress resistance: tgt modifications that enhance L. plantarum survival through gastrointestinal transit.

• Synbiotic optimization: Designing tgt variants that enhance utilization of specific prebiotics like fructooligosaccharides.

L. plantarum has already demonstrated significant potential as a probiotic in clinical trials, with one landmark study involving more than 4,500 newborns showing that a synbiotic containing L. plantarum and fructooligosaccharide effectively prevented infant sepsis . Engineering the tgt enzyme could further enhance such beneficial effects by optimizing L. plantarum's adaptive capabilities.

Research suggests that L. plantarum's "nomadic" lifestyle and genetic flexibility may make it particularly amenable to engineering, potentially facilitating the production of highly effective probiotic strains with enhanced beneficial properties .

What strategies can resolve poor expression or activity of recombinant L. plantarum tgt?

Researchers encountering issues with recombinant L. plantarum tgt can implement these solutions:

• Expression optimization:

  • Reduce induction temperature (16-20°C)

  • Decrease inducer concentration

  • Supplement media with rare amino acids

  • Co-express molecular chaperones

• Solubility enhancement:

  • Add solubility-enhancing tags (SUMO, MBP)

  • Include stabilizing agents in lysis buffer (10% glycerol, 1mM DTT)

  • Test different detergents for membrane association

• Activity restoration:

  • Ensure proper metal cofactor inclusion (often Zn2+)

  • Optimize buffer conditions (pH, ionic strength)

  • Add reducing agents to prevent oxidation of catalytic residues

  • Test protein refolding protocols if necessary

When working with lyophilized tgt protein, centrifugation prior to opening the vial is recommended to ensure all product is collected . Proper reconstitution and storage conditions are critical for maintaining enzymatic activity.

How can researchers overcome challenges in detecting tRNA modifications?

Detecting tRNA modifications presents several challenges that can be addressed through:

• Sample preparation optimization:

  • Develop gentle extraction methods to preserve labile modifications

  • Implement size-selection techniques to enrich for tRNA molecules

  • Use deacylation steps to improve resolution in subsequent analyses

• Detection method selection:

  • For global analysis: LC-MS/MS with multiple reaction monitoring

  • For site-specific detection: Reverse transcription stops or misincorporation

  • For visualization: Northern blotting with specific probes for modified versus unmodified tRNAs

• Data analysis approaches:

  • Implement machine learning algorithms for modification pattern recognition

  • Develop databases of modification-specific mass signatures

  • Use statistical methods to identify significant changes across conditions

These approaches collectively enhance the sensitivity and specificity of tRNA modification detection, enabling more accurate assessment of tgt activity in various experimental settings.

How does L. plantarum tgt research connect to translational medicine?

L. plantarum tgt research intersects with translational medicine through several avenues:

• Microbiome-based therapeutics: Understanding how tgt contributes to L. plantarum's beneficial properties informs the development of next-generation probiotics.

• Cancer research connections: The parallel between bacterial tgt and human QTRT1 provides insights into tRNA modification in cancer development, as QTRT1 has been shown to affect cell proliferation, junctions, and microbiome in tumors .

• Immune modulation applications: L. plantarum's demonstrated ability to activate dendritic cells, influence T cell populations, and affect B cell activation may be partially regulated through tgt-mediated translational control.

• Drug delivery platforms: Engineered L. plantarum with modified tgt activity could serve as vehicles for delivering therapeutic molecules to specific gut locations.

The demonstrated efficacy of L. plantarum in preventing infant sepsis in a large clinical trial involving over 4,500 newborns underscores its translational potential, with tgt potentially contributing to the strain's colonization efficiency and beneficial effects.

What interdisciplinary approaches would advance L. plantarum tgt research?

Advancing L. plantarum tgt research would benefit from interdisciplinary collaboration between:

• Structural biologists and biochemists: To determine high-resolution structures of L. plantarum tgt and characterize its enzymatic mechanisms.

• Microbiologists and immunologists: To investigate how tgt-mediated tRNA modifications influence bacterial-host immune interactions, particularly the engagement of TLR2/TLR6 heterodimers that promote regulatory T cell responses .

• Bioinformaticians and systems biologists: To model how tRNA modifications affect the bacterial proteome and metabolic pathways.

• Clinicians and microbiome experts: To translate findings into therapeutic applications, building on evidence that L. plantarum can modulate gene expression in intestinal mucosa, inducing genes associated with anti-inflammatory activities .

• Synthetic biologists and bioengineers: To design and construct optimized L. plantarum strains with modified tgt for specific applications.