Recombinant Lactobacillus plantarum tRNA (guanine-N (7)-)-methyltransferase (trmB)

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

Lactobacillus plantarum (recently reclassified as Lactiplantibacillus plantarum) is a gram-positive lactic acid bacterium that has gained significant attention as a versatile expression system for recombinant proteins. It is particularly valuable for delivering vaccines for mucosal immunity and has been shown to activate specific immune responses in numerous studies . L. plantarum offers several advantages as an expression system, including its Generally Recognized As Safe (GRAS) status, ability to survive gastrointestinal transit, and capacity to modulate gut microbiota composition and function.

How does recombinant Lactobacillus plantarum influence gut microbial diversity?

Recombinant L. plantarum has been shown to significantly alter gut microbiota composition and diversity. Research indicates that it can:

• Change the amount of operational taxonomic units (OTUs) in the gut

• Dramatically boost species diversity of gut bacteria (as measured by the Shannon-Wiener index)

• Alter the beta diversity, indicating changes in microbial community structure

What are tRNA-modifying enzymes and what functions do they serve in bacteria?

tRNA-modifying enzymes, including tRNA (guanine-N(7))-methyltransferase (trmB), are responsible for post-transcriptional modifications of transfer RNA molecules. These modifications are crucial for:

• Ensuring accurate codon recognition

• Maintaining tRNA structural stability

• Regulating translation efficiency and fidelity

• Adapting to environmental stresses

In bacterial systems like L. plantarum, these enzymes help fine-tune protein synthesis based on environmental conditions and metabolic requirements.

What experimental design considerations are critical when studying recombinant L. plantarum expressing tRNA-modifying enzymes?

When designing experiments involving recombinant L. plantarum expressing tRNA-modifying enzymes, researchers should consider the following methodological principles:

Additionally, researchers should establish clear distinctions between developmental stages (exploratory/probing experiments) and evaluation stages (validation of relationships, calibration of procedures) of their research .

How can one analyze the interaction between recombinant L. plantarum and gut microbiota?

To analyze interactions between recombinant L. plantarum and gut microbiota, researchers should employ a systematic approach:

• Characterize microbiota diversity changes using:

  • Alpha diversity metrics (Shannon-Wiener index)

  • Beta diversity analysis to assess structural changes in microbial communities

  • OTU (Operational Taxonomic Unit) quantification

• Analyze functional changes in gut bacteria related to:

  • Metabolism

  • Immune regulation

  • Host-microbe interactions

• Measure immunological parameters including:

  • Serum IgG and IgG1 levels

  • Fecal sIgA concentrations

  • CD4+ T cell and IgA+ B cell populations

This comprehensive approach enables researchers to establish causal relationships between recombinant L. plantarum administration and specific microbiota and immunological changes.

How does the structure of tRNA-related enzymes in L. plantarum influence their function?

The structure-function relationship of tRNA-related enzymes in L. plantarum is exemplified by studies of its glycyl-tRNA synthetase (GlyRS). L. plantarum GlyRS adopts an X-shaped heterotetrameric α₂β₂ structure, consisting of full-length α and β subunits . This structure influences tRNA recognition in several ways:

• The α and β subunits work together to recognize the L-shaped tRNA structure

• Both subunits interact with the 3′-end and acceptor region of tRNAGly

• The C-terminal domain of the β subunit specifically interacts with the anticodon region of tRNAGly

Biochemical analysis using tRNA variants has revealed specific determinants for efficient tRNA recognition, including:

• G1C72 and C2G71 base pairs

• C35, C36, and U73 positions in tRNAGly

• The combination of a purine base at position 4 and a pyrimidine base at position 69

This detailed understanding of structure-function relationships can inform strategies for engineering tRNA-modifying enzymes like trmB.

What expression systems are most effective for producing recombinant L. plantarum strains that express tRNA-modifying enzymes?

For expressing tRNA-modifying enzymes in L. plantarum, researchers should consider the following expression systems:

The choice of expression system should align with research objectives, considering factors such as expression level requirements, protein localization needs, and downstream applications.

What analytical methods are most appropriate for characterizing the activity of tRNA-modifying enzymes in recombinant L. plantarum?

To characterize the activity of tRNA-modifying enzymes such as trmB in recombinant L. plantarum, researchers should employ multiple complementary approaches:

• Biochemical Activity Assays:

  • Measure methyltransferase activity using radiolabeled substrates

  • Assess tRNA modification patterns using high-performance liquid chromatography (HPLC)

  • Quantify modified nucleosides using mass spectrometry

• Structural Characterization:

  • X-ray crystallography to determine three-dimensional structure, as successfully applied to L. plantarum GlyRS

  • Computational modeling to predict enzyme-substrate interactions

• Functional Assessment:

  • Analyze translation efficiency and accuracy

  • Measure bacterial fitness parameters under various conditions

  • Assess effects on protein expression profiles

These methods provide comprehensive insights into enzyme function, substrate specificity, and biological significance.

How can researchers address variability and ensure reproducibility in experiments with recombinant L. plantarum?

To address variability and ensure reproducibility in experiments with recombinant L. plantarum, researchers should implement rigorous experimental design principles:

• Develop a formal experimental design before initiating experiments, particularly for evaluation stages of research .

• Implement independent replication by repeating the complete set of procedures used to prepare samples and test specimens .

• Randomize the order of preparation and measurement of test specimens in each replicate set to avoid systematic biases .

• Identify and control for extraneous sources of variation (technician, day, batch) through appropriate blocking designs .

• Use coded labels when possible to ensure blindness, preventing technicians from identifying specific samples or replicates .

• Apply appropriate statistical methods for data analysis, including:

  • Analysis of variance (ANOVA)

  • Linear and non-linear regression

  • Response surface methods

• Document experimental procedures comprehensively to facilitate replication by other researchers.

How can recombinant L. plantarum expressing tRNA-modifying enzymes be applied in vaccine development?

Recombinant L. plantarum has demonstrated significant potential as a vaccine delivery system, with studies showing its ability to:

• Express and deliver antigens for mucosal immunity

• Activate specific immune responses

• Modulate gut microbiota to enhance immune function

For vaccine development utilizing tRNA-modifying enzymes:

• tRNA modifications could optimize codon usage for enhanced antigen expression

• Controlled modification of translation efficiency could fine-tune immune response magnitude

• Strategic engineering of tRNA modification patterns could potentially enhance bacterial survival in the gut, improving vaccine efficacy

Research has shown that recombinant NC8 L. plantarum expressing a fusion between the P14.5 protein of African swine fever virus and IL-33 enhanced gut bacteria functions in metabolism and immune regulation . Similar approaches could be applied to other antigens of interest.

What challenges exist in studying the effects of tRNA modifications on bacterial physiology?

Researchers face several methodological challenges when investigating the effects of tRNA modifications on bacterial physiology:

• Distinguishing direct effects of tRNA modifications from indirect effects on global translation

• Quantifying small but biologically significant changes in translation efficiency

• Accounting for complex interactions between multiple tRNA modifications

• Correlating in vitro enzymatic activities with in vivo physiological consequences

• Addressing potential pleiotropic effects of modifying tRNA processing enzymes

These challenges necessitate carefully designed experimental approaches that combine genetic, biochemical, and systems biology methods to establish causality and mechanism.

How might alterations in tRNA modification patterns affect the efficacy of recombinant L. plantarum as a probiotic?

Alterations in tRNA modification patterns could significantly impact the efficacy of recombinant L. plantarum as a probiotic through several mechanisms:

• Translation efficiency changes affecting:

  • Growth rate and competitive fitness in the gut

  • Stress response capacity under gastrointestinal conditions

  • Production of beneficial metabolites

• Immunomodulatory effects:

  • Altered surface protein expression affecting immune recognition

  • Changed production of immunomodulatory factors

  • Modified interaction with gut epithelium and immune cells

Research has shown that recombinant L. plantarum can increase levels of IgG and IgG1 in serum and sIgA in feces, as well as enrich CD4+ T cells and IgA+ B cells, indicating that the gut microbiota exerts an immunomodulatory role when mediated by recombinant L. plantarum . Manipulating tRNA modifications could potentially enhance these beneficial effects.

What controls should be included when studying recombinant L. plantarum expressing tRNA-modifying enzymes?

When studying recombinant L. plantarum expressing tRNA-modifying enzymes, researchers should include several critical controls:

Additionally, when analyzing experimental data, researchers should implement appropriate randomization and blinding procedures to minimize bias, as outlined in experimental design guidelines .

How can researchers verify the expression and activity of tRNA-modifying enzymes in recombinant L. plantarum?

Verification of expression and activity of tRNA-modifying enzymes in recombinant L. plantarum should follow a multi-step approach:

• Expression Verification:

  • Western blot analysis using specific antibodies

  • RT-qPCR to quantify mRNA expression levels

  • Epitope tagging for detection and purification

• Localization Assessment:

  • Subcellular fractionation followed by Western blotting

  • Immunofluorescence microscopy with labeled antibodies

  • Fusion to reporter proteins (e.g., GFP) for visualization

• Activity Confirmation:

  • In vitro enzymatic assays using purified protein

  • Analysis of tRNA modification profiles

  • Functional complementation of deficient strains

This comprehensive verification approach ensures that the recombinant enzyme is not only expressed but also correctly folded, properly localized, and functionally active.