Recombinant Lactobacillus plantarum tRNA pseudouridine synthase B (truB)

What is the role of tRNA pseudouridine synthase B (truB) in Lactobacillus plantarum?

TruB in L. plantarum catalyzes the isomerization of uridine to pseudouridine at position 55 in the TΨC loop of tRNA molecules. This post-transcriptional modification is critical for maintaining proper tRNA tertiary structure and stability. Methodologically, researchers can investigate this function by:

• Conducting comparative genomic analysis between L. plantarum strains

• Performing gene knockout experiments and observing phenotypic changes

• Using RNA-seq to analyze changes in tRNA populations in truB-deficient strains

• Employing radioactive labeling of tRNAs followed by thin-layer chromatography to detect pseudouridine formation

The enzyme's function can be further understood within the context of L. plantarum's adaptation mechanisms, as this bacterium contains multiple nucleoside hydrolysis-related enzymes that play roles in nucleoside metabolism .

How does truB expression differ among various Lactobacillus plantarum strains?

Expression patterns of truB can vary significantly among L. plantarum strains, particularly those isolated from different ecological niches. To investigate these differences methodologically:

• Use quantitative PCR to measure relative expression levels across strains

• Perform Western blot analysis using anti-truB antibodies to quantify protein levels

• Employ reporter gene fusions (e.g., truB promoter-GFP) to monitor expression in different conditions

• Analyze RNA-seq data across multiple strains to identify regulatory patterns

When investigating strain differences, consider that L. plantarum strains show considerable genomic diversity, as evidenced by studies that have isolated functional strains from diverse sources such as HUA geese .

What are the standard protocols for cloning and expressing the truB gene from Lactobacillus plantarum?

For successful cloning and expression of L. plantarum truB, researchers should follow these methodological steps:

• Design primers with appropriate restriction sites based on the published L. plantarum genome

• Amplify the truB gene using high-fidelity PCR

• Clone the gene into a suitable expression vector (e.g., pWCF vectors have been successfully used for L. plantarum recombinant proteins )

• Transform into an appropriate expression host (E. coli for initial cloning, followed by L. plantarum)

• Verify correct insertion by sequencing and restriction digestion

• Optimize expression conditions (temperature, inducer concentration, time)

For higher yields, researchers may employ the strategy demonstrated with other L. plantarum recombinant proteins, where sonication and freeze-thaw cycles have been effective for protein extraction .

How can I optimize the functional expression of recombinant truB in Lactobacillus plantarum?

To achieve optimal functional expression of recombinant truB, implement this methodological approach:

• Test multiple promoter systems (constitutive vs. inducible)

• Evaluate different signal peptides for protein targeting

• Optimize codon usage for L. plantarum

• Test various growth temperatures (25-37°C) and induction periods

• Evaluate media composition effects on expression levels

• Compare different cell lysis methods (sonication vs. enzymatic vs. mechanical disruption)

Based on successful expression of other recombinant proteins in L. plantarum, researchers should consider incorporating a verification method such as immunoblotting or flow cytometry to confirm expression, as demonstrated in the expression of influenza virus antigens in L. plantarum .

What methodologies can accurately assess the enzymatic activity of recombinant truB?

For precise measurement of recombinant truB enzymatic activity, employ these methodological approaches:

• In vitro pseudouridylation assay:

  Component	Concentration	Purpose
  Purified recombinant truB	0.1-1 μM	Enzyme
  Substrate tRNA	1-5 μM	Target molecule
  Buffer (Tris-HCl, pH 7.5)	50 mM	Maintain pH
  MgCl₂	5 mM	Cofactor
  DTT	2 mM	Maintain reducing environment
  Incubation temperature	37°C	Optimal enzyme activity
  Incubation time	30-60 min	Allow reaction completion

• Use HPLC analysis to quantify pseudouridine formation

• Employ tritium release assays with [³H]UTP-labeled tRNAs

• Utilize mass spectrometry to identify modified nucleosides

These approaches can be combined with the analytical methods used for characterizing other L. plantarum recombinant proteins, where immunoblotting and flow cytometry have successfully verified functional expression .

What strategies are effective for investigating truB-substrate interactions in Lactobacillus plantarum?

To investigate truB-substrate interactions in L. plantarum, implement these methodological approaches:

• Cross-linking studies: UV cross-linking of truB with radiolabeled tRNA substrates

• Electrophoretic mobility shift assays (EMSA): Titrate increasing amounts of purified truB with constant tRNA concentration

• Surface plasmon resonance (SPR): Measure real-time binding kinetics between truB and various tRNA substrates

• Structural studies: X-ray crystallography or cryo-EM of truB-tRNA complexes

• Mutational analysis: Site-directed mutagenesis of key residues in truB followed by binding assays

When analyzing the interactions, researchers can draw parallels with nucleoside hydrolase interactions in L. plantarum, where specific enzymes like IunH have been shown to participate in nucleoside metabolism pathways .

How does genetic background affect truB expression and function in recombinant systems?

The genetic background significantly impacts truB expression and function. To methodically investigate these effects:

• Compare expression in various L. plantarum strains with different genetic backgrounds

• Analyze expression in deletion strains lacking related RNA modification enzymes

• Evaluate expression in strains with different metabolic capacities

• Test complementation of truB-deficient strains with recombinant truB variants

Consider the approaches used in L. plantarum recombinant studies where strain NC8Δ has been successfully employed as an expression host for recombinant proteins .

What are the most effective vector systems for expressing truB in Lactobacillus plantarum?

For optimal expression of truB in L. plantarum, these vector systems and methodological considerations are recommended:

• pWCF vector system: Has demonstrated success with other recombinant proteins in L. plantarum

• pSIP expression vectors: Offer inducible expression with tight regulation

• pNZ8148 vector: Provides NICE system compatibility for controlled expression

Key methodological considerations include:

• Selection of appropriate antibiotic resistance markers

• Inclusion of species-specific origin of replication

• Choice of constitutive vs. inducible promoters

• Addition of secretion signals if extracellular expression is desired

• Incorporation of epitope tags for detection and purification

The pWCF vector system has been validated for successful expression of recombinant proteins in L. plantarum, as demonstrated in immunological studies .

How can CRISPR-Cas9 be utilized to study truB function in Lactobacillus plantarum?

For CRISPR-Cas9 based investigation of truB in L. plantarum, implement this methodological workflow:

• Design phase:

  • Identify target sequences within truB gene

  • Design sgRNAs with minimal off-target effects

  • Select appropriate Cas9 variant (SpCas9 or SaCas9)

  • Design repair templates for precise gene editing

• Implementation phase:

  • Clone sgRNAs into CRISPR vector

  • Transform constructs into L. plantarum

  • Screen transformants for successful editing

  • Verify edits by sequencing

• Functional analysis:

  • Create point mutations in catalytic residues

  • Generate domain deletions

  • Introduce reporter tags for localization studies

  • Create conditional knockdowns using inducible promoters

This approach can be integrated with the techniques used for genetic manipulation of other L. plantarum genes, where targeted modifications have successfully altered functional properties .

What approaches help resolve inconsistent truB activity in recombinant expression systems?

When encountering inconsistent truB activity, implement this systematic troubleshooting methodology:

• Protein expression verification:

  • Confirm expression by Western blot with anti-truB antibodies

  • Verify protein solubility in different fractions

  • Assess protein degradation patterns

• Activity optimization:

  • Test multiple buffer compositions for optimal activity

  • Evaluate cofactor requirements and concentrations

  • Analyze temperature and pH dependencies

  • Examine enzyme stability under various storage conditions

• Substrate considerations:

  • Ensure tRNA substrates are properly folded

  • Test multiple tRNA species as substrates

  • Examine potential inhibitors in the reaction mixture

Similar troubleshooting approaches have been successfully applied to other recombinant L. plantarum proteins, where flow cytometry and immunoblotting were used to verify proper expression .

How should researchers interpret contradictory results between in vitro and in vivo truB studies?

To methodically reconcile contradictory results between in vitro and in vivo truB studies:

• Systematic comparison:

  Parameter	In vitro system	In vivo system	Potential reconciliation approach
  Enzyme concentration	Typically higher	Physiological	Dilution series to match physiological levels
  Substrate accessibility	Unrestricted	Competed for	Add competitor molecules to in vitro reactions
  Cofactor availability	Controlled	Variable	Manipulate cofactor concentrations
  Post-translational modifications	Often absent	Present	Purify native enzyme for comparison
  Cellular compartmentalization	Absent	Present	Study in membrane mimetics or cell extracts

• Examine differences in tRNA folding states between the two systems

• Consider the impact of cellular factors absent in purified systems

• Analyze the role of potential regulatory proteins in vivo

When interpreting conflicting results, researchers can draw insights from studies on other L. plantarum enzymes, where both in vitro and in vivo approaches have revealed complementary aspects of enzyme function .

What computational tools are most effective for analyzing truB sequence conservation and predicting functional domains?

For comprehensive computational analysis of truB sequence and function, employ these methodological tools:

• Multiple sequence alignment tools:

  • MUSCLE or CLUSTAL for basic alignment

  • T-Coffee for improved accuracy with distant homologs

  • MAFFT for large-scale alignments

• Evolutionary analysis:

  • MEGA for phylogenetic tree construction

  • Consurf for mapping evolutionary conservation onto protein structure

  • PAML for detecting positive selection

• Structure prediction:

  • AlphaFold2 for accurate 3D structure prediction

  • SWISS-MODEL for homology modeling

  • I-TASSER for ab initio and template-based modeling

• Functional domain prediction:

  • InterProScan for integrated domain analysis

  • NCBI Conserved Domain Database

  • Pfam for protein family identification

These computational approaches can complement experimental methods used in L. plantarum studies, where genome-wide analyses have successfully identified functional domains in enzymes involved in nucleoside metabolism .

How can recombinant truB be utilized to study tRNA modifications' impact on Lactobacillus plantarum stress response?

To methodically investigate how truB-mediated tRNA modifications influence L. plantarum stress responses:

• Experimental design approach:

  • Generate truB overexpression and knockout strains

  • Expose strains to various stressors (acid, oxidative, temperature, osmotic)

  • Measure growth kinetics under stress conditions

  • Analyze tRNA modification profiles using mass spectrometry

  • Perform transcriptome and proteome analysis of stress response genes

• Phenotypic characterization:

  • Assess biofilm formation capabilities

  • Measure survival rates under extreme conditions

  • Evaluate metabolic shifts using metabolomics

  • Analyze membrane integrity and morphological changes

This research direction can build upon observations that L. plantarum strains modulate gene expression and enzyme activity in response to environmental conditions, as demonstrated in studies of its role in hyperuricemia .

What advances in structural biology techniques have improved our understanding of truB function?

Recent methodological advances in structural biology have enhanced our understanding of truB function through:

• Cryo-electron microscopy (cryo-EM):

  • Enables visualization of truB-tRNA complexes at near-atomic resolution

  • Allows analysis of conformational changes during catalysis

  • Reveals dynamic aspects of enzyme-substrate interactions

• X-ray crystallography enhancements:

  • Serial femtosecond crystallography captures transient states

  • Room-temperature crystallography provides physiologically relevant structures

  • Neutron crystallography offers insights into hydrogen bonding networks

• Nuclear magnetic resonance (NMR) applications:

  • Residue-specific dynamics of truB during catalysis

  • Identification of flexible regions involved in substrate recognition

  • Characterization of weak, transient interactions with tRNAs

• Integrative structural biology approaches:

  • Combining multiple techniques (cryo-EM, X-ray, NMR, mass spectrometry)

  • Computational modeling to integrate diverse experimental data

  • Molecular dynamics simulations to explore conformational landscapes

These structural biology techniques complement experimental approaches used to characterize other L. plantarum proteins, where protein structure has been linked to functional properties .

How does truB activity correlate with Lactobacillus plantarum adaptation to different environmental niches?

To methodically investigate the relationship between truB activity and L. plantarum environmental adaptation:

• Comparative analysis framework:

  • Isolate L. plantarum strains from diverse environments (dairy, fermented vegetables, GI tract)

  • Sequence and compare truB genes across isolates

  • Measure truB expression levels under conditions mimicking natural habitats

  • Correlate tRNA modification patterns with environmental parameters

• Adaptation markers:

  • Growth rates in environment-specific media

  • Metabolic profiling under different conditions

  • Competitive fitness in mixed cultures

  • Stress resistance correlations with truB activity levels

This research approach aligns with observations that L. plantarum strains show environment-specific adaptations, as seen in strains isolated from hyperuricemic geese that demonstrated specific metabolic capabilities .