Recombinant Lactobacillus plantarum Probable tRNA threonylcarbamoyladenosine biosynthesis protein Gcp (gcp)

What is threonylcarbamoyladenosine (t6A) and why is it important in tRNA biology?

Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs across all three kingdoms of life. This modification is located in the anticodon stem-loop at position 37 adjacent to the anticodon of tRNAs and is found in nearly all tRNAs that decode ANN codons .

The significance of t6A lies in its role as a critical component of the translation apparatus. It maintains translation accuracy by ensuring proper codon recognition during protein synthesis. In bacteria specifically, t6A functions as a strong positive determinant for aminoacylation of tRNA by bacterial-type isoleucyl-tRNA synthetases and might also serve as a determinant for the essential enzyme tRNA Ile-lysidine synthetase .

How is the t6A modification pathway organized in bacteria compared to other domains of life?

The t6A biosynthesis pathway shows both universal components and kingdom-specific variations:

• Universal core enzymes: Two core enzyme families are required for t6A synthesis in all domains of life .

• Bacterial pathway: In bacteria, the t6A synthesis pathway involves the TsaB, TsaC, TsaD, and TsaE proteins (previously known as YeaZ, YrdC, YgjD, and YjeE respectively) .

• Eukaryotic/archaeal pathway: These organisms have their own set of t6A biosynthesis enzymes distinct from bacteria .

• Mitochondrial pathway: Specialized t6A biosynthesis machinery exists for mitochondrial tRNAs .

The bacterial pathway specifically involves the formation of threonylcarbamoyl-adenylate (TC-AMP) as an intermediate, activated for condensation with adenosine-37 of tRNA .

What are the expression and purification methods for recombinant L. plantarum Gcp protein?

Recombinant L. plantarum protein expression typically follows these methodological steps:

• Construction of expression vector: The gene encoding the target protein is cloned into an appropriate expression vector, often with a fusion tag for purification .

• Expression conditions: Recombinant L. plantarum is cultured and induced as described in previous protocols. Typically, 1 × 10^6 CFU of bacteria are washed, followed by the addition of 1 mL of 1% BSA in PBS and incubation for 1 hour .

• Protein verification: Expression can be verified through:

  • Immunoblotting using specific antibodies

  • Flow cytometry after appropriate antibody labeling

  • Indirect immunofluorescence analysis

• Purification: The recombinant protein is typically purified using affinity chromatography based on the fusion tag included in the construct, followed by size exclusion chromatography if needed.

• Quality control: Purity assessment is performed using SDS-PAGE (>85% purity is typically desired) .

What are the storage conditions and stability considerations for recombinant Gcp protein?

For optimal stability and activity retention of recombinant tRNA threonylcarbamoyladenosine biosynthesis protein:

• Temperature: Store at -20°C/-80°C

• Form considerations:

  • Liquid form: shelf life of approximately 6 months

  • Lyophilized form: shelf life of approximately 12 months

• Reconstitution: Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Glycerol addition: Add 5-50% of glycerol (final concentration) and aliquot for long-term storage

• Working aliquots: Store at 4°C for up to one week

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended

How does the essentiality of t6A biosynthesis proteins vary among bacterial species, and what mechanisms underlie these differences?

The essentiality of t6A biosynthesis shows interesting patterns across bacterial species:

• Essential in most prokaryotes: Genome-wide essentiality studies demonstrate that genes for t6A synthesis are critical for survival in most bacterial species .

• Species with non-essential t6A: Interestingly, t6A is dispensable in specific bacteria including:

  • Deinococcus radiodurans

  • Thermus thermophilus

  • Synechocystis PCC6803

  • Streptococcus mutans

• Molecular basis for essentiality: In organisms where t6A is essential, the molecular mechanism appears related to:

  • t6A functioning as a determinant for aminoacylation by bacterial-type isoleucyl-tRNA synthetases

  • Its potential role as a determinant for the essential enzyme tRNA Ile-lysidine synthetase

The proteomics analysis of t6A-deficient D. radiodurans strains revealed an induction of the proteotoxic stress response, suggesting that while some bacteria can survive without t6A, they experience significant translational stress that must be compensated through alternative mechanisms .

What experimental approaches can be used to analyze the impact of t6A modification on translation in L. plantarum?

Several sophisticated methodological approaches can assess the impact of t6A modification on translation:

• Ribosome profiling analysis: This technique can reveal codon-specific changes in ribosome occupancy resulting from t6A deficiency, particularly at elevated temperatures relevant to host environments (37°C) .

• Dual-reporter assay system: This approach can be used to examine the role of t6A modifications in specific codon decoding:

  • Two sets of consecutive codons (e.g., AUA or AUC) are inserted at the beginning of a reporter gene (GFP)

  • A second reporter (e.g., mCherry) serves as an internal control

  • The decoding efficiency is evaluated by measuring normalized GFP signals

  • This approach was successfully used to demonstrate the role of tRNA modifications in AUA decoding

• LC-MS analysis of tRNA modifications: Liquid chromatography-mass spectrometry can be used to detect and quantify t6A modifications in tRNA preparations from L. plantarum under different conditions. Co-injection analyses of nucleosides from different sources can confirm the identity of specific modifications .

• Translation efficiency assays: In vitro translation systems using purified components can directly assess the impact of t6A-modified versus unmodified tRNAs on translation rates and accuracy.

What is the biochemical mechanism of the t6A modification pathway in L. plantarum, and how does it differ from other bacteria?

The t6A modification pathway in L. plantarum and other bacteria follows these key biochemical steps:

• Formation of TC-AMP intermediate: The TsaC protein (YrdC ortholog) catalyzes the formation of threonylcarbamoyl-adenylate (TC-AMP) .

• ATP utilization pattern: This process involves:

  • AMP production from TsaC (in a threonine-dependent process)

  • ADP production from the TsaD/TsaB/TsaE complex

• Mechanistic details: The pathway proceeds through:

  • Direct carboxylation of threonine by CO2 or HCO3-

  • Formation of TC-AMP with release of PPi

  • Transfer of the threonylcarbamoyl moiety to adenosine-37 of the target tRNA

Notable distinctions in L. plantarum include:

• Unlike some bacteria, L. plantarum lacks the capacity to synthesize either flavins or quinones, relying on exogenous sources

• This metabolic dependency affects the organism's redox balance and potentially influences the activity of enzymes involved in tRNA modification

How can genetic manipulation of the gcp gene be used to study t6A's role in L. plantarum fitness and host interactions?

Genetic manipulation approaches to study t6A's role in L. plantarum include:

• Gene deletion/knockout strategies:

  • CRISPR-Cas9 system for precise gene editing

  • Homologous recombination-based approaches

  • Assessment of knockout viability - potential lethality would support essentiality

• Conditional expression systems:

  • Arabinose-inducible promotor systems for controlled expression

  • Allow titration of gene expression to identify minimum threshold levels

  • Can be used to study effects of gradual depletion on cellular processes

• Complementation experiments:

  • Expression of wild-type gcp from a plasmid in a mutant background

  • Cross-species complementation to identify species-specific functions

  • Domain mutation approaches to identify critical functional regions

• Phenotypic assessment methods:

  • Growth rate measurements under different temperatures and stress conditions

  • Host cell adhesion and invasion assays using epithelial cell models

  • Virulence assessment in infection models

  • Immune response evaluation through dendritic cell activation markers (CD80, CD86, MHC-II)

How does temperature affect t6A modification activity in L. plantarum, and what are the implications for host-associated environments?

Temperature significantly impacts t6A modification in bacteria with important implications for host-pathogen interactions:

• Temperature-dependent ribosome occupancy: Ribosome profiling analyses reveal that t6A modification deficiency alters ribosome occupancy patterns particularly at 37°C—the body temperature of human hosts .

• Growth temperature effects:

  • Bacteria with t6A deficiencies often show temperature-sensitive growth phenotypes

  • This suggests the modification may be particularly important for translation accuracy at elevated temperatures

• Adaptation to host environments:

  • The 37°C temperature of the human host represents a significant stress for many microbes

  • t6A modifications may help maintain translational fidelity under these conditions

  • This could explain why t6A modifications are linked to virulence in some pathogenic species

• Experimental approaches to study temperature effects:

  • Comparative growth assays at different temperatures (25°C, 30°C, 37°C, 42°C)

  • Analysis of t6A levels at different temperatures using LC-MS

  • Measurement of translation error rates at different temperatures using reporter systems

What is the role of t6A modification in L. plantarum immune interactions and potential probiotics applications?

The t6A modification in L. plantarum may significantly impact immune interactions based on related research:

• Dendritic cell activation: Recombinant L. plantarum expressing modified proteins can activate dendritic cells in Peyer's patches (PPs) of mice, as evidenced by increased expression of activation markers:

  • CD80 (P < 0.001 compared to control groups)

  • CD86 (P < 0.01 compared to vaccine group)

  • MHC-II (P < 0.05 compared to PBS group)

• T-cell responses: Recombinant L. plantarum can induce:

  • CD4+IFN-γ+ T cells in mesenteric lymph nodes (MLNs)

  • CD8+IFN-γ+ T cells in both MLNs and spleen

  • T cell proliferation in response to specific antigens

• B-cell activation and antibody production:

  • Increased percentage of B220+IgA+ cells in PPs

  • Elevated levels of specific antibodies:

    • IgG in serum (significantly elevated at weeks 2, 4, and 10)

    • IgG1 and IgG2a in serum

    • IgA in feces

  • Enhanced IgA expression in lungs, duodenum, jejunum, and ileum

• Application considerations:

  • Probiotics delivery of recombinant proteins may depend on proper tRNA modification for accurate translation

  • t6A modification could affect the stability and immunogenicity of recombinant proteins

  • Understanding the role of t6A could lead to improved vaccine delivery systems using L. plantarum as a vector

What analytical techniques are most effective for verifying the function and activity of recombinant L. plantarum Gcp protein?

Multiple complementary techniques can verify the function and activity of recombinant Gcp protein:

• In vitro t6A formation assay:

  • Incubate purified recombinant Gcp with other t6A synthesis proteins (TsaB, TsaC, TsaE)

  • Add substrate tRNA lacking t6A modification

  • Detect t6A formation using LC-MS analysis of nucleosides

• Complementation assays:

  • Express recombinant Gcp in gcp-deficient bacterial strains

  • Evaluate restoration of growth and t6A modification levels

  • Compare complementation efficiency with Gcp proteins from different species

• ATP hydrolysis assay:

  • Monitor ATP consumption during t6A synthesis reaction

  • Measure AMP and ADP production as indicators of enzymatic activity

  • Use coupled enzyme assays or radioactive ATP to track conversion

• Protein-protein interaction studies:

  • Co-immunoprecipitation to detect interactions with other t6A pathway proteins

  • Surface plasmon resonance to measure binding affinities

  • Yeast two-hybrid or bacterial two-hybrid screening

• Structural validation:

  • Circular dichroism to confirm proper protein folding

  • Limited proteolysis to assess structural integrity

  • Thermal shift assays to evaluate protein stability

How can researchers efficiently generate and validate L. plantarum strains with modified gcp expression for experimental studies?

Efficient strategies for generating and validating modified L. plantarum strains include:

• Strain generation methods:

  • Homologous recombination using temperature-sensitive plasmids

  • CRISPR-Cas9 genome editing for precise modifications

  • Controlled expression systems using inducible promoters

  • Complementation with ectopic expression constructs

• Verification of genetic modifications:

  • PCR-based genotyping with primers flanking the modified region

  • Whole-genome sequencing to confirm modifications and rule out off-target effects

  • Reverse transcription quantitative PCR (RT-qPCR) to verify changes in gene expression

  • Western blotting to confirm protein expression levels

• Functional validation:

  • LC-MS analysis of t6A levels in tRNA as a direct measure of Gcp activity

  • Growth rate measurements under various conditions

  • Ribosome profiling to assess impacts on translation

  • Proteomics analysis to identify compensatory responses

• Controls and standards:

  • Include wild-type strains in all experiments

  • Use complemented mutants to confirm phenotype specificity

  • Include bacterial strains with known t6A modification defects as references

  • Implement appropriate statistical analyses for reproducibility assessment

What are the key considerations for designing experiments to investigate the role of t6A modification in codon-specific translation accuracy?

Designing robust experiments to investigate t6A's role in translation accuracy requires:

• Reporter system design:

  • Construct dual-reporter systems with GFP and mCherry

  • Insert specific codons (like AUA) at the beginning of the GFP gene

  • Use the ratio of GFP to mCherry signals to evaluate decoding efficiency

  • Include control constructs with synonymous codons (e.g., AUC)

• Codon bias considerations:

  • Analyze L. plantarum genome for codon usage patterns

  • Focus on ANN codons, particularly those that might be affected by t6A

  • Construct reporter systems with varying frequencies of target codons

• Quantification methods:

  • Flow cytometry for single-cell analysis of reporter expression

  • Plate reader assays for high-throughput screening

  • Western blotting for protein level confirmation

  • RT-qPCR for mRNA level normalization

• Experimental controls:

  • Wild-type versus t6A-deficient strains

  • Titrated depletion of t6A using conditional expression systems

  • Complementation with wild-type versus mutant Gcp

  • Temperature variation to assess condition-dependent effects

• Advanced analytical approaches:

  • Ribosome profiling to identify codon-specific translation pauses

  • Mass spectrometry to identify mistranslation events

  • In vitro translation systems with purified components

What interdisciplinary approaches would be most valuable for understanding the broader biological significance of t6A modification in L. plantarum?

Comprehensive understanding of t6A modification in L. plantarum requires interdisciplinary approaches:

• Structural biology:

  • X-ray crystallography or cryo-EM of Gcp protein structures

  • Structure-function relationships through targeted mutagenesis

  • Molecular dynamics simulations of tRNA-enzyme interactions

• Systems biology:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis of genes affected by t6A deficiency

  • Mathematical modeling of translation dynamics

• Evolutionary biology:

  • Comparative genomics across Lactobacillus species

  • Phylogenetic analysis of t6A pathway components

  • Investigation of selective pressures on tRNA modification systems

• Microbial ecology:

  • Co-culture experiments to study community interactions

  • Analysis of t6A modification during host colonization

  • Examination of environmental stress responses

• Host-microbe interactions:

  • Impact on adherence to and invasion of epithelial cells

  • Effects on immune system modulation

  • Potential role in probiotic and therapeutic applications

• Synthetic biology:

  • Design of engineered strains with optimized t6A modification systems

  • Creation of synthetic tRNA modification pathways

  • Development of biosensors for monitoring tRNA modification states

How can researchers differentiate between direct effects of t6A deficiency and secondary metabolic adaptations when studying L. plantarum gcp mutants?

Distinguishing direct from secondary effects in t6A research requires sophisticated experimental design:

• Temporal analysis approaches:

  • Time-course experiments following t6A depletion

  • Early timepoints capture direct effects before compensatory responses

  • Integration of multi-omics data at different timepoints

• Conditional expression systems:

  • Use of titratable promoters to create varying levels of t6A deficiency

  • Dose-response relationships can help identify primary targets

  • Pulse-chase experiments to track immediate consequences

• Specific codon reporter systems:

  • Construct reporters enriched in codons directly affected by t6A

  • Compare with control reporters using synonymous codons

  • Measure translation rates of specific reporter constructs

• Combined genomic/biochemical approaches:

  • Ribosome profiling to identify primary translation defects

  • Proteomics to identify stress response activation

  • Metabolomics to track metabolic pathway adjustments

• Genetic suppressor screens:

  • Identify mutations that alleviate t6A deficiency phenotypes

  • Suppressors often point to primary defects or compensation mechanisms

  • Complementation with related tRNA modification systems

• In vitro reconstitution:

  • Purified translation systems with defined components

  • Direct assessment of t6A effects on decoding without cellular adaptations

  • Comparison of in vitro and in vivo results to identify secondary effects