Recombinant Salmonella newport tRNA pseudouridine synthase A (truA)

What is the function of tRNA pseudouridine synthase A (truA) in Salmonella Newport?

tRNA pseudouridine synthase A (truA) in Salmonella Newport catalyzes the site-specific isomerization of uridine to pseudouridine (Ψ) at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. This enzymatic conversion breaks the N-glycosidic bond of the target uridine, rotates the uracil base, and forms a carbon-carbon glycosidic bond between C5 of the pyrimidine and C1' of the ribose sugar. This post-transcriptional modification is crucial for maintaining proper tRNA structure and function, thereby ensuring accurate and efficient protein synthesis. The modification affects the structural rigidity and base-stacking properties of the anticodon loop, which directly impacts codon-anticodon interactions during translation .

How does truA differ across Salmonella Newport lineages?

truA sequences exhibit conservation patterns that align with the distinct phylogenetic lineages of Salmonella Newport. Comparative genomic analysis of 28 S. Newport strains identified multiple sublineages with differences in genetic content. Specific variations in truA and surrounding genetic regions can be observed between Lineage II and Lineage III of S. Newport, which have different evolutionary histories and appear to have evolved largely independently. These variations in truA sequences can serve as molecular markers for differentiating between these lineages in epidemiological investigations and evolutionary studies .

What is the catalytic mechanism of truA in pseudouridine formation?

The catalytic mechanism of truA involves a conserved aspartate residue that acts as a nucleophilic catalyst. The enzyme adds to the 6-position of the pyrimidine ring of the target uridine to form a covalent intermediate. This addition leads to the breaking of the N-glycosidic bond, after which the uracil base is rotated and reconnected to the ribose via a carbon-carbon bond at the C5 position. The formation of a 5,6-dihydro-6-hydroxy intermediate has been observed when using 5-fluorouracil (FUra) in place of uracil, supporting this mechanism. The reaction completes with the elimination of the enzyme through hydrolytic cleavage, resulting in the formation of pseudouridine in the tRNA .

What protein domains and motifs are critical for truA function in Salmonella Newport?

The functional activity of truA in Salmonella Newport depends on several conserved domains and motifs. The catalytic domain contains a highly conserved aspartate residue that serves as the nucleophilic catalyst in the isomerization reaction. Additionally, the enzyme contains RNA-binding motifs that facilitate interaction with the tRNA substrate, particularly around the anticodon loop region. Structural studies have identified specific amino acid residues involved in substrate recognition and positioning of the target uridine in the catalytic pocket. Mutational analysis of these residues has demonstrated their importance for enzymatic activity, with substitutions often resulting in significant reduction or complete loss of pseudouridylation capacity .

How can recombinant truA from Salmonella Newport be expressed and purified for functional studies?

For expression and purification of recombinant truA from Salmonella Newport, the following methodology is recommended:

• Gene Cloning:

  • Amplify the truA gene from Salmonella Newport genomic DNA using PCR with gene-specific primers containing appropriate restriction sites

  • Clone the amplified gene into an expression vector (e.g., pET series) with a fusion tag (His-tag or GST-tag) for purification

• Expression System:

  • Transform the recombinant plasmid into E. coli expression strain (BL21(DE3) or Rosetta)

  • Induce protein expression with IPTG (typically 0.5-1 mM) at optimal temperature (often 16-25°C to enhance solubility)

• Purification Protocol:

  • Harvest cells and lyse using sonication or French press in appropriate buffer

  • Purify using affinity chromatography (Ni-NTA for His-tagged protein)

  • Further purify using ion-exchange and/or size exclusion chromatography

  • Confirm purity using SDS-PAGE and Western blot analysis

• Activity Preservation:

  • Store purified enzyme with glycerol (10-20%) at -80°C

  • Include reducing agents (DTT or β-mercaptoethanol) in storage buffer to maintain enzymatic activity

What methods are used to assess the enzymatic activity of recombinant truA?

Several complementary methods can be employed to assess the enzymatic activity of recombinant truA:

• Radioisotope-Based Assays:

  • Incorporate [14C]- or [3H]-labeled uridine into substrate tRNAs

  • Measure conversion to pseudouridine using thin-layer chromatography after enzymatic digestion

  • Quantify radioactivity in pseudouridine spots versus uridine spots

• HPLC Analysis:

  • Digest tRNA substrates after enzymatic reaction to nucleosides

  • Separate and quantify pseudouridine versus uridine by HPLC

  • Calculate modification efficiency based on peak areas

• Mass Spectrometry:

  • Analyze modified tRNAs using LC-MS/MS to detect pseudouridine formation

  • Identify specific modification sites within the tRNA sequence

  • Provide quantitative assessment of modification levels

• CMC-Primer Extension Assay:

  • Treat tRNAs with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC)

  • CMC specifically reacts with pseudouridine

  • Perform reverse transcription, which stops at CMC-modified bases

  • Analyze cDNA products by gel electrophoresis to identify modification sites

How is the truA gene organized in the genome of different Salmonella Newport lineages?

The truA gene in Salmonella Newport exhibits distinctive genomic organization patterns that vary between lineages. In Lineage II and III, the genomic region around truA shows evidence of genetic flow and homologous recombination events. Analysis of the loci around the mutS gene, which is proximal to truA, reveals significant differences between lineages. This region includes sequences at the 3' end of Salmonella Pathogenicity Island 1 (SPI-1), specifically between invH and mutS genes, the ste fimbrial operon, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated-proteins (cas). These genomic differences suggest that selective pressures have acted on this region differently across lineages, possibly reflecting adaptation to different environmental niches or hosts .

What evidence exists for horizontal gene transfer affecting truA in Salmonella Newport?

Horizontal gene transfer appears to have played a significant role in shaping the truA genomic region in Salmonella Newport. Comparative genomic analysis using progressive MAUVE alignment and ClonalFrame analysis identified locally collinear blocks (LCBs) that suggest recombination events around the truA-containing region. These analyses revealed that S. Newport Lineages II and III have divergent evolutionary histories in this region, with evidence of genetic exchange between Salmonella serovars. The presence of mobile genetic elements and phage-associated sequences near truA further supports the occurrence of horizontal gene transfer events. The genomic architecture surrounding truA exhibits mosaic patterns that likely resulted from multiple recombination events during the evolution of different Salmonella Newport lineages .

How can phylogenetic analysis of truA contribute to understanding Salmonella Newport evolution?

Phylogenetic analysis of truA sequences provides valuable insights into Salmonella Newport evolution through several approaches:

• Multi-locus Sequence Analysis (MLSA):

  • Include truA alongside other housekeeping genes

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Identify evolutionary relationships between Salmonella Newport lineages

• Whole Genome Comparison:

  • Analyze truA in context with complete genome sequences

  • Identify >140,000 informative SNPs for robust phylogenetic analysis

  • Establish clearer geographic structure and lineage differentiation

• Molecular Clock Analysis:

  • Estimate divergence times between lineages based on truA sequence differences

  • Correlate with known historical events or ecological transitions

  • Understand the temporal dynamics of Salmonella Newport evolution

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify selection pressures on truA

  • Determine if truA is under purifying, neutral, or positive selection

  • Correlate selection patterns with functional importance and adaptation

How can CRISPR-Cas9 be utilized to study truA function in Salmonella Newport?

CRISPR-Cas9 offers powerful approaches for studying truA function in Salmonella Newport:

• Gene Knockout Strategy:

  • Design sgRNAs targeting specific regions of the truA gene

  • Introduce Cas9 and sgRNA via plasmid transformation

  • Select for successful knockouts using appropriate markers

  • Verify deletion using PCR and sequencing

  • Assess phenotypic changes in growth, stress response, and virulence

• Base Editing for Point Mutations:

  • Use CRISPR-Cas9 base editors to introduce specific mutations

  • Target catalytic residues (e.g., the conserved aspartate)

  • Create variants with altered activity levels

  • Analyze the impact on pseudouridylation efficiency

• CRISPRi for Expression Modulation:

  • Deploy catalytically inactive Cas9 (dCas9) fused to repressors

  • Design sgRNAs targeting the truA promoter region

  • Create conditional knockdowns to study dosage effects

  • Monitor changes in tRNA modification levels and translation efficiency

• CRISPR Activation for Overexpression:

  • Utilize dCas9 fused to transcriptional activators

  • Target the truA promoter to enhance expression

  • Assess the consequences of truA overproduction

  • Determine if excess truA affects pathogenicity or stress tolerance

What cell-based assays can evaluate the impact of truA mutations on Salmonella Newport phenotypes?

Several cell-based assays can be employed to evaluate how truA mutations affect Salmonella Newport phenotypes:

• Growth Curve Analysis:

  • Compare growth rates of wild-type and truA mutant strains

  • Evaluate growth under various conditions (temperature, pH, salt stress)

  • Assess recovery after exposure to antibiotics or oxidative stress

  • Quantify differences in lag phase, doubling time, and final cell density

• Virulence Assessment in Cell Culture:

  • Measure invasion efficiency in epithelial cell lines (e.g., Caco-2, HT-29)

  • Quantify intracellular survival in macrophage models (e.g., RAW264.7)

  • Assess cytokine induction and inflammatory responses

  • Compare wild-type and mutant strains for virulence-associated phenotypes

• Biofilm Formation Assays:

  • Quantify biofilm formation using crystal violet staining

  • Analyze biofilm architecture using confocal microscopy

  • Measure extracellular matrix production

  • Evaluate antibiotic tolerance in biofilm versus planktonic states

• Stress Response Assessment:

  • Test survival under acid stress (mimicking gastric environment)

  • Evaluate tolerance to bile salts and digestive enzymes

  • Measure resistance to reactive oxygen species

  • Assess temperature stress tolerance (heat shock/cold shock)

How can structural biology approaches contribute to understanding truA function in Salmonella Newport?

Structural biology approaches provide critical insights into truA function through:

• X-ray Crystallography:

  • Crystallize purified recombinant truA alone and in complex with substrate tRNA

  • Determine three-dimensional structure at high resolution

  • Identify active site architecture and substrate binding pockets

  • Visualize conformational changes during catalysis

• Cryo-Electron Microscopy (Cryo-EM):

  • Analyze larger complexes involving truA and tRNA

  • Capture different conformational states during the reaction cycle

  • Visualize dynamic aspects of enzyme-substrate interactions

  • Obtain structural information under near-native conditions

• NMR Spectroscopy:

  • Study protein dynamics and conformational changes

  • Analyze chemical shift perturbations upon substrate binding

  • Investigate hydrogen/deuterium exchange patterns

  • Monitor structural changes during catalysis in solution

• Molecular Dynamics Simulations:

  • Model enzyme-substrate interactions based on experimental structures

  • Simulate reaction mechanisms and energy landscapes

  • Predict effects of mutations on structure and function

  • Identify potential allosteric sites and conformational transitions

How does truA activity influence virulence in Salmonella Newport infections?

The activity of truA influences Salmonella Newport virulence through several interconnected mechanisms:

What is the relationship between truA sequence variation and antimicrobial resistance in Salmonella Newport?

The relationship between truA sequence variation and antimicrobial resistance in Salmonella Newport involves both direct and indirect mechanisms:

• Co-localization with Resistance Determinants:

  • Analysis of genomic contexts shows that truA variants may be linked to mobile genetic elements carrying resistance genes

  • Certain truA alleles show stronger association with multidrug resistant (MDR) strains

  • Phylogenetic analysis indicates co-evolution of truA variants with plasmid-borne resistance genes

  • Recombination events around truA may facilitate acquisition of resistance determinants

• Impact on Expression of Resistance Genes:

  • truA-mediated tRNA modifications affect translation efficiency of resistance proteins

  • Proper expression of efflux pumps and drug-modifying enzymes depends on translational accuracy

  • Variations in truA may subtly modulate resistance gene expression levels

  • Stress response coordination influenced by truA affects antibiotic tolerance

• Epidemiological Patterns:

  • MDR Salmonella Newport strains carrying the blaCMY-2 gene show specific truA sequence patterns

  • Certain truA variants are more prevalent in cephalosporin-resistant MDR lineages

  • Pansusceptible isolates tend to have different truA alleles than resistant strains

  • These patterns suggest co-selection or genetic linkage between truA variants and resistance determinants

What computational approaches help predict substrate specificity of truA across different Salmonella strains?

Advanced computational approaches for predicting truA substrate specificity include:

• Homology Modeling and Molecular Docking:

  • Generate structural models of truA variants from different Salmonella strains

  • Perform docking simulations with various tRNA substrates

  • Calculate binding energies and interaction patterns

  • Rank potential substrates based on predicted affinity

• Sequence-Structure-Function Analysis:

  • Align truA sequences across Salmonella lineages

  • Identify conservation patterns in substrate recognition regions

  • Map sequence variations onto structural models

  • Correlate structural features with experimental substrate preferences

• Machine Learning Approaches:

  • Train neural networks on known enzyme-substrate interaction data

  • Incorporate features from sequence, structure, and experimental validation

  • Develop predictive models for substrate preferences

  • Validate predictions with biochemical assays

• Molecular Dynamics and Free Energy Calculations:

  • Simulate enzyme-substrate complexes to predict binding stability

  • Calculate free energy of binding for different tRNA substrates

  • Analyze conformational changes during substrate recognition

  • Predict energetic barriers for catalysis with different substrates

What challenges exist in developing selective inhibitors of bacterial truA for antimicrobial development?

The development of selective inhibitors targeting bacterial truA faces several significant challenges:

• Selectivity Challenges:

  • Maintaining specificity for bacterial truA over human pseudouridine synthases

  • Designing compounds that distinguish between bacterial species

  • Targeting conserved active sites while achieving selectivity

  • Avoiding off-target effects on other tRNA-modifying enzymes

• Structural Complexity:

  • Designing inhibitors for the large, complex truA-tRNA interface

  • Accounting for conformational changes during catalysis

  • Addressing the nucleic acid-protein dual recognition requirement

  • Developing compounds with appropriate pharmacokinetic properties

• Resistance Development:

  • Predicting potential resistance mechanisms against truA inhibitors

  • Designing inhibitors less prone to resistance development

  • Identifying collateral sensitivity patterns to counter resistance

  • Developing combination approaches to prevent resistance emergence

• Delivery and Bioavailability:

  • Ensuring inhibitors penetrate the bacterial cell envelope

  • Minimizing susceptibility to efflux pumps

  • Achieving sufficient intracellular concentrations

  • Maintaining stability under physiological conditions

How can systems biology approaches integrate truA function into broader Salmonella Newport metabolic and virulence networks?

Systems biology approaches can integrate truA function into broader Salmonella networks through:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Compare profiles between wild-type and truA mutant strains

  • Identify downstream effects of altered tRNA modification

  • Map perturbations across metabolic and virulence pathways

• Network Analysis:

  • Construct gene regulatory networks centered on truA

  • Identify hub genes affected by truA activity

  • Perform pathway enrichment analysis to identify affected systems

  • Model information flow through networks with and without functional truA

• Flux Balance Analysis:

  • Develop genome-scale metabolic models incorporating translation efficiency

  • Predict metabolic flux distributions with varying truA activity

  • Identify critical nodes connecting translation to metabolism

  • Simulate growth and virulence under different conditions

• Host-Pathogen Interaction Modeling:

  • Integrate bacterial and host cell responses

  • Model dynamic changes during infection processes

  • Predict infection outcomes based on truA functionality

  • Identify potential intervention points in the infection cycle

How does Salmonella Newport truA compare to homologs in other bacterial pathogens?

Comparative analysis of Salmonella Newport truA with homologs in other bacterial pathogens reveals:

The comparison indicates that while the core catalytic mechanism is conserved, subtle variations exist in substrate recognition and specificity that may contribute to species-specific adaptation strategies and virulence mechanisms .

What experimental approaches can determine if truA function influences host specificity in different Salmonella serovars?

To determine if truA function influences host specificity across Salmonella serovars, several experimental approaches can be employed:

• Comparative Infection Models:

  • Create isogenic truA mutants in multiple Salmonella serovars

  • Test colonization efficiency in different animal models

  • Compare tissue tropism between wild-type and mutant strains

  • Measure competitive indices in mixed infections

  • Assess bacterial loads in target organs across host species

• Cross-complementation Studies:

  • Swap truA alleles between host-restricted and broad-host serovars

  • Express truA from S. Newport in other serovars and vice versa

  • Evaluate changes in host range or preference

  • Measure virulence factor expression under different truA variants

  • Assess impact on host-specific stress response patterns

• Host Cell Interaction Analysis:

  • Compare invasion and intracellular survival in cell lines from different hosts

  • Evaluate cytokine responses triggered by different truA variants

  • Assess transcriptional response of host cells to bacterial strains

  • Measure impact on host-specific defense mechanism evasion

  • Analyze effects on host immune recognition patterns

How can evolutionary analysis of truA inform our understanding of Salmonella adaptation to different environmental niches?

Evolutionary analysis of truA provides valuable insights into Salmonella adaptation through:

• Selection Pressure Analysis:

  • Calculate dN/dS ratios across truA sequences from different environments

  • Identify sites under positive or purifying selection

  • Correlate selection patterns with environmental factors

  • Map selection hotspots onto protein structure

  • Compare selection patterns between clinical and environmental isolates

• Ancestral Sequence Reconstruction:

  • Infer ancestral truA sequences at key evolutionary nodes

  • Recreate and characterize ancestral enzymes

  • Compare catalytic properties with contemporary variants

  • Identify evolutionary trajectories during niche adaptation

  • Test functional divergence hypotheses experimentally

• Ecological Correlation Studies:

  • Associate truA sequence variants with isolation sources

  • Identify environment-specific patterns in truA sequences

  • Correlate genetic changes with ecological transitions

  • Analyze co-evolution with other genes in adaptation pathways

  • Map geographical and ecological distribution of truA variants

• Experimental Evolution:

  • Subject Salmonella strains to controlled environmental shifts

  • Monitor truA sequence changes over generations

  • Identify convergent evolutionary patterns

  • Correlate genetic changes with phenotypic adaptations

  • Validate the adaptive significance of observed mutations