Recombinant Lactobacillus johnsonii Valine--tRNA ligase (valS), partial

What is the Valine--tRNA ligase (valS) gene in Lactobacillus johnsonii and what is its function?

The valS gene in L. johnsonii encodes Valine--tRNA ligase (also known as valyl-tRNA synthetase), an essential enzyme that catalyzes the attachment of valine to its cognate tRNA during protein synthesis. This aminoacylation reaction is critical for accurate translation of the genetic code. The enzyme belongs to the class I aminoacyl-tRNA synthetase family and plays a fundamental role in protein biosynthesis by ensuring valine is correctly incorporated into nascent polypeptide chains according to mRNA sequences .

The valS gene typically encodes a protein of approximately 900 amino acids, similar to what has been observed in related Lactobacillus species like L. casei . The enzyme contains conserved HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases, which are involved in ATP binding and catalysis.

How conserved is the valS gene across Lactobacillus species and strains?

The valS gene is highly conserved across Lactobacillus species due to its essential function in protein synthesis. Comparative genomic analyses show significant sequence similarity between valS genes from different Lactobacillus species. This high conservation reflects the evolutionary pressure to maintain the critical function of accurate aminoacylation.

Studies examining genetic variation within L. johnsonii strains isolated from various hosts have shown that essential housekeeping genes like valS typically exhibit less variation compared to adaptive or strain-specific genes . The conservation pattern often follows phylogenetic separation observed in L. johnsonii strains isolated from different animal hosts, suggesting some degree of host-specific adaptation while maintaining core functionality .

What is known about the genomic context and expression patterns of valS in L. johnsonii?

In L. johnsonii, valS appears as a single-copy gene in the core genome. Based on genomic analyses of various L. johnsonii strains, including FI9785, valS gene expression may be regulated as part of the translation machinery . Quantitative proteomics studies have detected valS protein in L. johnsonii FI9785, with relative abundance measurements showing modest fluctuations (-1.37 log2 fold change) under certain experimental conditions .

The genomic neighborhood of valS in L. johnsonii may include genes involved in ribosomal structure and protein synthesis, which reflects its functional role in translation. Like many bacterial genes involved in protein synthesis, valS expression is likely modulated based on growth rates and nutrient availability, though specific regulatory elements controlling its expression in L. johnsonii have not been fully characterized.

What are the most effective strategies for cloning the valS gene from L. johnsonii?

For successful cloning of the L. johnsonii valS gene, researchers should consider:

• Genomic DNA extraction: Use specialized protocols for Gram-positive bacteria, such as those involving enhanced cell wall lysis steps with lysozyme treatment.

• PCR amplification: Design primers based on published L. johnsonii genome sequences with appropriate restriction sites. For a gene of this size (~2.7 kb), high-fidelity DNA polymerases like Phusion or Q5 are essential to minimize errors.

• Vector selection: For functional complementation studies, shuttle vectors that can replicate in both E. coli and Lactobacillus are preferable. Vectors like pG+host9 have been successfully used for L. johnsonii gene manipulation .

• Cloning strategy: Consider using methods that have been successful for other L. johnsonii genes:

  • For homologous recombination approaches, splice overlap extension PCR (SOE-PCR) has been effective for creating constructs with L. johnsonii sequences .

  • For heterologous expression, vectors with inducible promoters like pNZ8148 (NICE system) or pPG612 (xylose-inducible) have been used successfully with L. johnsonii genes .

What expression systems are optimal for producing recombinant L. johnsonii valS?

Based on successful expression of other L. johnsonii recombinant proteins, several expression systems should be considered:

• E. coli-based systems:

  • The pET system with BL21(DE3) host strains has been widely used for tRNA synthetases.

  • The T7 Express or Rosetta strains can help with codon usage differences between L. johnsonii and E. coli.

• Lactobacillus-based systems:

  • The pNZ8148 vector with the NICE (Nisin-Controlled gene Expression) system has been used for L. johnsonii recombinant protein expression .

  • The pPG612 vector with xylose-inducible promoter has been successfully used for L. johnsonii gene expression .

• Expression conditions optimization:

  • Lower induction temperatures (16-25°C) often improve solubility of large enzymes like tRNA synthetases.

  • Addition of glycine (0.3-1%) to growth media can enhance protein expression in Lactobacillus systems .

  • For L. johnsonii-based expression, media containing 0.3M sucrose has shown improved transformation and expression efficiency .

What purification strategies work best for recombinant valS from L. johnsonii?

Effective purification strategies should include:

• Affinity tags approach:

  • His-tag purification has been successfully applied to recombinant L. johnsonii proteins .

  • Consider tag position carefully; C-terminal tags may be preferable to avoid interfering with enzyme activity.

• Chromatographic methods:

  • Ion exchange chromatography as a polishing step (typically cation exchange as tRNA synthetases often have high pI).

  • Size exclusion chromatography for final purification and buffer exchange.

• Buffer optimization:

  • Include 10-25% glycerol to enhance stability.

  • Consider adding ATP (1-5 mM) and valine (1-2 mM) in purification buffers to stabilize the enzyme's active conformation.

  • Dithiothreitol (DTT) or β-mercaptoethanol (2-5 mM) may help maintain cysteine residues in reduced form.

• Activity preservation:

  • Minimize freeze-thaw cycles by storing as single-use aliquots.

  • Test storage in 50% glycerol at -20°C versus flash-freezing in liquid nitrogen for long-term stability.

How can the aminoacylation activity of recombinant L. johnsonii valS be assessed?

Several methodologies can be employed to assess valS enzymatic activity:

• Aminoacylation assays:

  • ATP-pyrophosphate exchange assay to measure the first step of the aminoacylation reaction.

  • Direct measurement of Val-tRNAVal formation using either:

    • Radioactive assays with [³H]-valine or [¹⁴C]-valine

    • Acid gel electrophoresis to separate charged and uncharged tRNAs

    • HPLC-based methods for detection of aminoacylated tRNA

• Kinetic parameters determination:

  • Measure Km and kcat for valine, ATP, and tRNAVal substrates.

  • Determine metal ion dependencies (typically Mg²⁺).

  • Assess pH and temperature optima, particularly relevant for comparing valS from different host-adapted L. johnsonii strains.

• In vivo complementation:

  • Test functional complementation of E. coli valS temperature-sensitive mutants (similar to what has been done with L. casei valS) .

  • Measure growth rates and protein synthesis capacity in complemented strains.

What applications could recombinant L. johnsonii valS have in research and biotechnology?

Recombinant L. johnsonii valS has several potential applications:

• Fundamental research:

  • Structural and biochemical studies of host-specific adaptations in aminoacyl-tRNA synthetases.

  • Investigation of species-specific differences in substrate recognition and catalytic efficiency.

  • Understanding the molecular basis of translation fidelity in probiotic bacteria.

• Biotechnology applications:

  • Development of in vitro translation systems optimized for Lactobacillus gene expression.

  • Engineering L. johnsonii strains with enhanced translational efficiency for increased production of therapeutic proteins or metabolites.

  • Creating chimeric aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids into recombinant proteins.

• Therapeutic development:

  • Structure-based design of antibiotics targeting bacterial valS while sparing human orthologs.

  • Exploring the immunogenic properties of bacterial tRNA synthetases for potential vaccine development.

How does recombinant valS contribute to understanding L. johnsonii adaptation in different hosts?

Recombinant valS can provide insights into bacterial adaptation through:

• Comparative biochemistry of host-adapted strains:

  • Analysis of L. johnsonii valS from different hosts (human, chicken, rodent, swine) may reveal host-specific adaptations in catalytic efficiency or substrate specificity .

  • Different L. johnsonii strains show host-specific genetic clustering, which may extend to functional adaptations in essential genes like valS .

• Translation optimization:

  • Comparing valS properties with valine codon usage patterns in highly expressed genes across L. johnsonii strains from different hosts.

  • Understanding if translational machinery adaptations correlate with host colonization efficiency.

• Co-evolution analysis:

  • Investigating possible co-evolution between valS and tRNAVal genes in host-adapted strains.

  • Exploring if valS adaptation correlates with other host-specific genomic features in L. johnsonii.

What are common challenges in expressing and purifying recombinant valS from L. johnsonii?

Researchers commonly encounter several challenges:

• Solubility issues:

  • tRNA synthetases are large enzymes prone to aggregation when overexpressed.

  • Solution: Expression at lower temperatures (16-20°C), co-expression with chaperones, or fusion with solubility-enhancing tags like MBP.

• Codon usage bias:

  • L. johnsonii has different codon preferences than common expression hosts like E. coli.

  • Solution: Use Rosetta strains supplying rare tRNAs or perform codon optimization of the valS sequence.

• Protein stability issues:

  • ValS may exhibit limited stability during purification.

  • Solution: Include stabilizing additives in purification buffers (glycerol, ATP, valine) and optimize buffer conditions (pH 7.5-8.0, 100-300 mM NaCl).

• Heterogeneous product:

  • Partial proteolytic degradation during expression or purification.

  • Solution: Add protease inhibitors during purification and use protease-deficient expression strains.

• Activity loss during purification:

  • Recombinant valS may lose activity during purification steps.

  • Solution: Monitor activity throughout purification process and optimize conditions to maintain enzyme functionality.

How can codon optimization improve expression of L. johnsonii valS?

Codon optimization can significantly improve expression by:

• Addressing codon bias differences:

  • L. johnsonii has a different GC content (~34-35%) compared to E. coli (~50%), affecting codon usage patterns.

  • Replacing rare codons in the expression host with synonymous commonly used codons can prevent translational pausing and improve yields.

• Systematic approach to optimization:

  • Analyze the valS sequence for rare codons in the expression host.

  • Adjust GC content to match host preferences.

  • Eliminate potential RNA secondary structures in the transcript.

  • Remove internal Shine-Dalgarno-like sequences that might cause translational issues.

• Optimization strategies comparison:

  Optimization Approach	Advantages	Disadvantages
  Whole gene optimization	Maximizes expression potential	More expensive; may affect folding dynamics
  Targeted optimization of rare codon clusters	Cost-effective; maintains natural folding dynamics	May not address all expression limitations
  Harmonization (matching host codon usage frequency)	May preserve co-translational folding	More complex to design

• Validation strategy:

  • Compare expression levels between native and optimized sequences.

  • Test protein solubility and activity to ensure functional expression.

What strategies can overcome issues with enzymatic activity of recombinant L. johnsonii valS?

To address activity issues with recombinant valS:

• Buffer optimization:

  • Test various buffer compositions (HEPES, Tris, phosphate) at pH 7.0-8.5.

  • Optimize divalent cation concentration (Mg²⁺, Mn²⁺) which is critical for aminoacylation.

  • Add stabilizing components (glycerol, BSA, reducing agents) to maintain enzyme conformation.

• Substrate quality control:

  • Ensure high-quality ATP preparation without significant ADP contamination.

  • Use freshly prepared tRNA substrates to avoid degradation issues.

  • Verify valine purity and prepare fresh stock solutions.

• Expression system selection:

  • Compare activity of valS expressed in different systems (E. coli vs. Lactobacillus).

  • Test different affinity tags and their positions (N-terminal vs. C-terminal).

  • Consider tag removal if it interferes with enzymatic function.

• Storage conditions:

  • Determine optimal storage conditions to maintain long-term activity.

  • Test stabilizing additives (glycerol, DTT, substrates) for storage.

  • Evaluate the impact of freeze-thaw cycles on enzyme activity.

How might CRISPR-Cas9 techniques be applied to study valS function in L. johnsonii?

CRISPR-Cas9 techniques offer powerful approaches for studying valS:

• Gene editing strategies:

  • Create precise point mutations in catalytic residues or binding sites to study structure-function relationships.

  • Generate conditional knockdowns using inducible promoters to study the essentiality of valS under various conditions.

  • Introduce specific mutations observed in different host-adapted strains to test their functional significance.

• Implementation considerations for L. johnsonii:

  • Adaptation of CRISPR-Cas9 systems for efficient editing in Lactobacillus requires optimized transformation protocols.

  • Electroporation conditions similar to those used for other genetic manipulations in L. johnsonii (2.1 kV, 3 ms) would likely be effective .

  • Media supplements like 0.3M sucrose have improved transformation efficiency in L. johnsonii genetic studies .

• Potential applications:

  • Generate L. johnsonii strains with modified valS to study effects on translation fidelity and efficiency.

  • Create reporter fusions to study valS expression patterns under different conditions.

  • Perform domain swapping between valS genes from different hosts to identify regions responsible for host-specific adaptations.

What can structural biology approaches reveal about L. johnsonii valS?

Structural biology approaches can provide critical insights:

• Structural determination methods:

  • X-ray crystallography of purified recombinant valS (alone and in complex with substrates).

  • Cryo-EM studies to visualize valS interactions with tRNA and ribosomal components.

  • NMR studies of specific domains to understand dynamic interactions.

• Key structural questions to address:

  • Conformational changes during catalysis and substrate binding.

  • Structural basis for species-specific differences in substrate recognition.

  • Identification of potential allosteric sites for regulation.

  • Interaction surfaces with other components of the translation machinery.

• Computational structural biology:

  • Homology modeling based on related valS structures.

  • Molecular dynamics simulations to study enzyme flexibility and substrate interactions.

  • In silico docking studies to identify potential inhibitor binding sites.

How can multi-omics approaches advance understanding of valS regulation in L. johnsonii?

Integrative multi-omics approaches can reveal:

• Transcriptomic insights:

  • RNA-seq under various conditions to identify factors affecting valS expression.

  • Identification of potential non-coding RNAs regulating valS expression.

  • Ribosome profiling to assess translation efficiency of valS mRNA.

• Proteomic analyses:

  • Quantitative proteomics to measure valS protein levels under different conditions.

  • Post-translational modifications affecting valS activity.

  • Protein-protein interaction networks involving valS.

• Integration with metabolomics:

  • Correlations between valine metabolism and valS expression/activity.

  • Effects of nutritional status on aminoacylation efficiency.

  • Host-derived metabolites that might affect valS function.

• Comparative multi-omics:

  Approach	Application to valS Research	Key Insights
  Transcriptomics	Expression patterns in different conditions	Regulatory mechanisms
  Proteomics	Protein levels, modifications, interactions	Post-transcriptional regulation
  Metabolomics	Amino acid pools, ATP availability	Substrate availability effects
  Genomics	Strain variations in valS sequence/context	Evolutionary adaptations

What are the critical parameters for designing a valS gene knockout or complementation study in L. johnsonii?

For successful genetic manipulation of valS in L. johnsonii:

• Knockout strategy considerations:

  • Since valS is likely essential, conditional knockouts or partial deletions would be required.

  • Temperature-sensitive plasmid systems (like pG+host9) have been successful for gene replacements in L. johnsonii .

  • Recovery of valS mutations may require specific conditions, similar to what has been observed with other essential gene manipulations in L. johnsonii .

• Complementation design:

  • Expression vectors like pFI2560 have been successfully used for complementation in L. johnsonii .

  • Inducible promoters allow controlled expression levels to prevent toxicity.

  • Include the native promoter region if studying regulatory aspects.

• Growth conditions:

  • For manipulations of translation-related genes, slower growth at lower temperatures (30°C) may be necessary .

  • Media supplementation with specific components might be required to compensate for growth defects.

How can researchers analyze the impact of valS mutations on L. johnsonii physiology?

Comprehensive analysis should include:

• Growth and viability assessments:

  • Growth curve analysis under various conditions (temperature, pH, stress).

  • Colony morphology examination on solid media.

  • Microscopic examination of cell morphology and division patterns.

• Translation efficiency measurements:

  • Pulse-chase labeling with radioactive amino acids to measure protein synthesis rates.

  • Polysome profiling to assess translation initiation and elongation efficiency.

  • Reporter gene assays (GFP, luciferase) to quantify translation of specific mRNAs.

• Stress response analysis:

  • Susceptibility to antibiotics targeting translation.

  • Resistance to environmental stresses (oxidative, acid, bile).

  • Expression of stress-response genes using qRT-PCR or RNA-seq.

• Host interaction studies:

  • Adhesion to epithelial cell lines.

  • Survival in simulated gastrointestinal conditions.

  • Immunomodulatory effects using cell culture models.

What are the recommended controls for studying recombinant L. johnsonii valS?

Rigorous controls should include:

• Enzymatic activity controls:

  • Inactive enzyme variants (site-directed mutants of catalytic residues).

  • Heat-inactivated enzyme preparations.

  • Reactions without essential components (ATP, tRNA, valine).

  • Commercial valyl-tRNA synthetase from related species as positive control.

• Expression and purification controls:

  • Empty vector controls for expression studies.

  • Non-transformed host cells processed in parallel.

  • Other recombinant proteins purified using the same protocol.

• Functional complementation controls:

  • Complementation with native L. johnsonii valS.

  • Complementation with valS from closely related species.

  • Empty vector negative control.

• Strain authentication:

  • PCR verification of genetic modifications.

  • Whole genome sequencing to confirm genetic changes and detect potential compensatory mutations.

  • Phenotypic verification of expected traits.

How might valS research contribute to developing improved recombinant L. johnsonii for therapeutic applications?

Fundamental valS research could enable:

• Enhanced protein production systems:

  • Optimizing translation efficiency for therapeutic protein expression in L. johnsonii through valS engineering.

  • Developing strains with improved growth and stress resistance through targeted modification of translation machinery.

  • Creating expression systems with controlled rates of recombinant protein synthesis.

• Host adaptation improvements:

  • Engineering L. johnsonii strains with valS variants optimized for specific host environments.

  • Enhancing survival and colonization in targeted body sites through translation machinery adaptations.

  • Fine-tuning protein expression profiles for specific therapeutic applications.

• Novel applications:

  • Development of L. johnsonii as delivery vehicles for therapeutic proteins using knowledge gained from valS studies .

  • Designing strains with unique amino acid incorporation abilities for specialized protein production.

  • Creating reporters and biosensors based on translation efficiency in different environments.

What emerging technologies could advance L. johnsonii valS research?

Several cutting-edge technologies hold promise:

• Single-cell analyses:

  • Single-cell RNA-seq to examine valS expression heterogeneity within populations.

  • Microfluidics approaches to study translation dynamics at the single-cell level.

  • Real-time imaging of protein synthesis using fluorescent reporters.

• High-throughput screening:

  • Deep mutational scanning of valS to comprehensively map functional residues.

  • Synthetic genetic arrays to identify genetic interactions with valS.

  • CRISPR interference libraries to identify pathways interacting with valS function.

• Advanced structural techniques:

  • Time-resolved structural studies using X-ray free electron lasers.

  • Hydrogen/deuterium exchange mass spectrometry for dynamics analysis.

  • Single-particle tracking to visualize valS movement and interactions in living cells.

• Synthetic biology approaches:

  • Minimal genome approaches to define essential translation components.

  • Orthogonal translation systems engineered into L. johnsonii.

  • Cell-free expression systems optimized with recombinant L. johnsonii components.

How does comparative analysis of valS across L. johnsonii strains inform bacterial adaptation mechanisms?

Comparative analyses can reveal:

• Evolutionary patterns:

  • Selection pressures on valS in different host environments.

  • Co-evolution with other components of the translation machinery.

  • Horizontal gene transfer events involving translation-related genes.

• Host-specific adaptations:

  • Correlation between valS sequence variations and host range.

  • Functional consequences of strain-specific polymorphisms.

  • Connection between translation machinery and niche adaptation.

• Research implications:

  • Identification of strain-specific valS properties may explain host specificity patterns observed in L. johnsonii populations .

  • Understanding if valS contributes to the co-evolutionary relationship between L. johnsonii and its hosts could reveal fundamental mechanisms of bacterial adaptation .

  • Insights into translation machinery adaptation could inform broader understanding of how probiotics evolve for specific environments.