Recombinant Bradyrhizobium japonicum Leucine--tRNA ligase (leuS), partial

What is the function of leucine-tRNA ligase in Bradyrhizobium japonicum?

Leucine-tRNA ligase (LeuRS), encoded by the leuS gene, catalyzes the attachment of leucine to its cognate tRNA molecules during protein synthesis. In Bradyrhizobium japonicum, this enzyme is essential for translation and protein production, particularly in the context of symbiotic nitrogen fixation. The enzyme functions through a two-step reaction: first activating leucine using ATP to form leucyl-adenylate, then transferring the leucine to the appropriate tRNA molecule. This aminoacylation process ensures accurate incorporation of leucine during protein synthesis, which is critical for the production of functional proteins involved in nodulation and nitrogen fixation processes. The activity of LeuRS is particularly important in Bradyrhizobium strains that form symbiotic relationships with leguminous plants, as these interactions require precise protein synthesis for successful nodule formation and function .

How can researchers isolate functional leuS from Bradyrhizobium japonicum?

Isolating functional leuS from B. japonicum requires a methodical approach combining genomic and proteomic techniques. An effective isolation protocol typically follows these steps:

• Genomic DNA extraction from B. japonicum cultures (preferably strain USDA110 or USDA6, which have well-characterized genomes)

• PCR amplification of the leuS gene using primers designed based on the annotated genome sequence

• Cloning of the amplified gene into an appropriate expression vector

• Expression in a suitable host system (typically E. coli BL21 or similar strains)

• Protein purification using affinity chromatography (His-tag methods are common)

For optimal expression and isolation, researchers should consider using the GenoSuite pipeline or similar proteogenomic tools to confirm the exact translation initiation site (TIS) of the leuS gene, as incorrect annotation of start codons can lead to non-functional recombinant proteins . Experimental validation of the purified enzyme's activity should be conducted through aminoacylation assays that measure the attachment of radiolabeled leucine to tRNA substrates.

What experimental techniques are used to study leuS expression patterns in Bradyrhizobium?

Several complementary techniques can be employed to study leuS expression patterns:

• RT-qPCR: Real-time quantitative PCR allows precise measurement of leuS transcript levels under various environmental conditions or developmental stages. This approach requires careful selection of reference genes stable under the conditions being tested.

• RNA-Seq: Transcriptome-wide analysis enables researchers to place leuS expression in the broader context of global gene expression patterns. This is particularly valuable when studying expression during symbiosis or under stress conditions .

• Proteomics: Mass spectrometry-based approaches can quantify LeuRS protein levels directly, providing insights into post-transcriptional regulation. The GenoSuite pipeline has been effectively used for proteogenomic analysis of B. japonicum, confirming protein expression and potentially identifying post-translational modifications .

• Reporter Gene Fusions: Constructing translational fusions between leuS promoter regions and reporter genes (such as GFP or β-glucuronidase) allows visualization of expression patterns in different tissues or cell types.

How does leuS sequence conservation compare across Bradyrhizobium species?

Analysis of sequence conservation requires:

• Multiple sequence alignment of leuS genes from different Bradyrhizobium strains (USDA110, USDA6, ORS3257, ORS86, etc.)

• Calculation of sequence identity percentages and identification of conserved domains

• Mapping of variable regions to the protein structure

• Phylogenetic analysis to correlate sequence variation with evolutionary relationships

Research indicates that while the core enzymatic functions are preserved across strains, variations often appear in regions that might influence regulatory interactions or enzyme kinetics. Particularly, strains with different symbiotic capabilities, such as those that nodulate different plant hosts or use different nodulation mechanisms (T3SS-dependent vs. Nod factor-dependent), may show specific adaptations in aminoacyl-tRNA synthetase genes that correlate with their symbiotic lifestyle .

How can statistical design of experiments (DOE) optimize recombinant leuS expression?

Optimizing recombinant leuS expression from B. japonicum requires a systematic approach to experimental design. Rather than traditional one-factor-at-a-time methods, applying DOE principles can significantly enhance efficiency and outcomes:

• Fractional Factorial Screening: Initially screen 4-6 factors (temperature, inducer concentration, media composition, host strain, growth phase at induction, and expression duration) to identify significant variables affecting leuS expression.

• Response Surface Methodology (RSM): Based on screening results, develop a mathematical model relating significant factors to expression levels and enzyme activity. This approach has been successfully used for optimization in protein expression systems and can be applied to leuS .

• Definitive Screening Design (DSD): This efficient design allows simultaneous screening and optimization with fewer experimental runs than traditional approaches, ideal for recombinant protein expression.

An example RSM design for optimizing leuS expression might include:

Statistical analysis of such data enables development of regression models to predict optimal conditions for soluble, active enzyme production. This approach has been shown to increase protein yield by 2-4 fold compared to non-optimized conditions in similar expression systems .

What are the implications of leuS mutations on Bradyrhizobium symbiotic capacity?

Mutations in the leuS gene may significantly impact B. japonicum's symbiotic capabilities through several mechanisms:

• Protein Synthesis Accuracy: Mutations affecting the catalytic site or tRNA recognition domains could lead to mischarging of tRNAs, resulting in amino acid misincorporation during protein synthesis. This would particularly affect the production of nodulation factors and nitrogen fixation enzymes.

• Symbiosis-Specific Adaptation: During symbiosis, Bradyrhizobium undergoes substantial metabolic and physiological changes. Research suggests that aminoacyl-tRNA synthetases may have specialized functions during these transitions, potentially interacting with symbiosis-specific regulatory proteins.

• Stress Response Integration: LeuRS may function as a regulatory node linking translational fidelity to stress responses encountered during nodule formation and nitrogen fixation.

Experimental approaches to investigate these effects include:

• Construction of point mutations in conserved domains using site-directed mutagenesis

• Complementation studies with mutated leuS variants in leuS-deficient strains

• Analysis of nodulation phenotypes on host plants such as soybean or Lotus species

• Transcriptomic analysis comparing wild-type and mutant strains during free-living growth versus symbiotic conditions

Research has shown that mutations in genes involved in protein synthesis machinery can affect the bacteroid differentiation process and nitrogen fixation efficiency. This is consistent with the observation that many symbiosis-related genes require precise regulation and expression for successful nodulation .

How does the proteogenomic approach improve annotation of leuS and related genes in Bradyrhizobium?

Proteogenomic analysis represents a powerful approach for improving gene annotation in Bradyrhizobium japonicum, particularly for leuS and other genes involved in translation. Traditional genome annotation often relies on computational prediction algorithms that may inaccurately identify gene boundaries, translation initiation sites, or miss genes entirely. The GenoSuite pipeline and similar proteogenomic approaches offer significant advantages:

• Translation Initiation Site (TIS) Refinement: In B. japonicum USDA110, proteogenomic analysis has refined 49 gene models for their translation initiation sites. Similar refinement of the leuS gene ensures that the correct protein sequence is used in functional studies .

• Novel Gene Discovery: The proteogenomic approach has identified 59 novel protein-coding regions in B. japonicum that were missed by traditional annotation methods. This comprehensive approach helps ensure that genes functionally related to leuS or that interact with LeuRS are properly annotated .

• Post-Translational Modification Identification: Analysis of N-terminally acetylated peptides and other modifications provides insight into the regulation of protein function, including potential regulatory modifications of LeuRS itself.

• Cross-Species Verification: Ortho-proteogenomic analysis enables verification of gene models across different Bradyrhizobium strains, such as USDA110 and USDA6T, ensuring consistent annotation of essential genes like leuS .

To implement proteogenomic analysis for leuS characterization, researchers should:

• Collect high-quality mass spectrometry data from diverse growth conditions

• Search spectra against a six-frame translation of the B. japonicum genome

• Apply rigorous statistical filtering (≤1% FDR) to peptide spectrum matches

• Map identified peptides back to genomic coordinates to verify or refine the leuS gene model

• Identify any novel open reading frames potentially related to tRNA charging or protein synthesis

This approach has expanded the proteomic landscape of B. japonicum and offers valuable insights for researchers focusing on translation-related genes .

What experimental design approaches can differentiate leuS function in symbiotic versus free-living Bradyrhizobium?

Differentiating leuS function between symbiotic and free-living states requires carefully designed comparative experiments:

• Dual RNA-Seq/Proteomics: Simultaneous analysis of transcriptomes and proteomes from bacteroids (symbiotic state) and cultured bacteria (free-living state) can reveal condition-specific expression patterns of leuS. This comparative approach should include multiple time points to capture the transition from free-living to symbiotic states .

• Conditional Mutagenesis: Developing inducible expression systems or temperature-sensitive alleles of leuS allows researchers to manipulate LeuRS activity at different stages of symbiosis, revealing stage-specific requirements.

• Protein-Protein Interaction Studies: Techniques such as bacterial two-hybrid screens or co-immunoprecipitation followed by mass spectrometry can identify different interaction partners of LeuRS between free-living and symbiotic states.

• Metabolic Labeling: Pulse-chase experiments using isotope-labeled leucine can measure differences in protein synthesis rates and accuracy between the two states.

A comprehensive experimental design might look like:

This approach has revealed that many genes involved in protein synthesis show differential expression between free-living and symbiotic states, suggesting specialized roles during symbiosis. Particularly, studies of Bradyrhizobium strains that nodulate through T3SS-dependent mechanisms versus Nod factor-dependent mechanisms may reveal different patterns of aminoacyl-tRNA synthetase regulation .

What purification strategies yield the highest activity of recombinant LeuRS?

Obtaining highly active recombinant LeuRS from Bradyrhizobium japonicum requires careful attention to purification strategies that preserve enzyme structure and function:

• Tag Selection and Positioning: While 6xHis tags are commonly used, their position (N- or C-terminal) can significantly affect LeuRS activity. C-terminal tags often preserve activity better for aminoacyl-tRNA synthetases due to the important role of the N-terminus in substrate binding.

• Expression Temperature: Lower expression temperatures (16-20°C) generally yield more soluble and active LeuRS compared to standard conditions (37°C), as they reduce inclusion body formation.

• Buffer Optimization: LeuRS activity is highly dependent on buffer composition:

  • HEPES or Tris buffer (pH 7.5-8.0)

  • Divalent cations (5-10 mM MgCl₂)

  • Monovalent salts (50-100 mM KCl)

  • Reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Glycerol (10-20%) for stability during storage

• Purification Protocol:

  • Initial capture via affinity chromatography (Ni-NTA for His-tagged proteins)

  • Intermediate purification using ion exchange chromatography

  • Polishing step via size exclusion chromatography to remove aggregates

  • Avoid freeze-thaw cycles; aliquot and flash-freeze in liquid nitrogen for storage

• Activity Preservation: Addition of stabilizing agents such as bovine serum albumin (0.1 mg/mL) and glycerol (20%) can maintain activity during storage.

Activity assays should be performed after each purification step to track enzyme functionality. The standard aminoacylation assay measures the attachment of [³H]-leucine to purified tRNA, but alternative non-radioactive assays such as the ATP-pyrophosphate exchange reaction can also be used to assess enzyme activity.

How can researchers analyze contradictory data in leuS expression studies?

When confronted with contradictory data regarding leuS expression or function, researchers should implement a systematic troubleshooting approach:

• Technical Variability Assessment:

  • Ensure biological and technical replicates are sufficient (minimum n=3 for each)

  • Verify primer specificity for RT-qPCR through melt curve analysis and sequencing

  • Check for batch effects in RNA-seq or proteomics datasets

  • Validate antibody specificity for Western blot or immunoprecipitation studies

• Biological Context Consideration:

  • Growth phase differences: leuS expression may vary significantly between lag, log, and stationary phases

  • Environmental conditions: pH, temperature, oxygen availability can affect expression

  • Strain variation: subtle genetic differences between laboratory strains can cause expression differences

• Integration of Multiple Methods:

  • Combine transcriptomics (RT-qPCR, RNA-seq) with proteomics approaches

  • Validate results using reporter gene constructs

  • Employ ribosome profiling to assess translation efficiency

• Statistical Re-evaluation:

  • Apply appropriate statistical tests based on data distribution

  • Consider using non-parametric tests when assumptions of parametric tests are violated

  • Implement multiple testing correction for genome-wide studies

• Experimental Design Revision:

  • Apply factorial design principles to systematically test interactions between variables

  • Use time-course experiments rather than endpoint analyses

  • Consider mixed-effects models to account for random variation

When analyzing contradictory results regarding leuS expression during symbiosis, a particular consideration is the heterogeneity of bacteroid populations within nodules. Single-cell approaches or careful fractionation of nodule tissues may reveal expression patterns missed by whole-nodule analyses .

What are the critical controls for studying leuS function in Bradyrhizobium mutants?

Rigorous experimental design for studying leuS function in Bradyrhizobium mutants requires several critical controls:

• Genetic Controls:

  • Wild-type strain grown under identical conditions

  • Complemented mutant (mutant strain containing a plasmid with functional leuS)

  • Empty vector control (mutant strain containing the plasmid backbone without leuS)

  • Unrelated gene mutant (mutation in a gene not expected to affect leuS function)

• Phenotypic Validation Controls:

  • Growth curves in minimal versus rich media to assess auxotrophy

  • Protein synthesis rates measured via pulse-labeling

  • Microscopic examination for morphological changes

  • Competitive index in mixed inoculation experiments

• Symbiosis-Specific Controls:

  • Non-inoculated plants as negative controls

  • Plants inoculated with wild-type strain as positive controls

  • Measurement of multiple symbiotic parameters (nodule number, size, leghemoglobin content, nitrogen fixation rates)

  • Bacteroid isolation and viability assessment

  • Gene expression analysis of both bacterial and plant symbiotic genes

• Biochemical Assay Controls:

  • Heat-inactivated enzyme preparations

  • Reactions without ATP or tRNA substrates

  • Inhibitor controls (specific aminoacyl-tRNA synthetase inhibitors)

  • Comparison with purified E. coli LeuRS as a reference standard

The systematic use of these controls helps differentiate direct effects of leuS mutation from indirect effects caused by general growth defects or stress responses. This comprehensive approach has been successfully used in studies of other symbiotic genes in Bradyrhizobium .

How can computational modeling enhance understanding of leuS structure-function relationships?

Computational modeling provides valuable insights into structure-function relationships of Bradyrhizobium japonicum LeuRS:

• Homology Modeling: Since the crystal structure of B. japonicum LeuRS has not been determined, homology modeling based on related bacterial LeuRS structures (such as those from E. coli or Thermus thermophilus) can predict the 3D structure. Key steps include:

  • Template selection based on sequence identity and coverage

  • Sequence alignment optimization focusing on conserved domains

  • Model building using software such as MODELLER or SWISS-MODEL

  • Refinement through energy minimization

  • Validation using Ramachandran plots and quality assessment tools

• Molecular Dynamics Simulations: These simulations reveal dynamic aspects of LeuRS function:

  • Conformational changes during substrate binding

  • Effects of temperature or pH on enzyme flexibility

  • Impact of specific mutations on protein stability

  • Identification of allosteric sites

• Docking Studies: In silico docking predicts interactions between LeuRS and its substrates:

  • Leucine binding mode in the active site

  • ATP positioning and interactions

  • tRNA recognition and binding determinants

  • Potential binding sites for inhibitors or regulatory molecules

• Evolutionary Analysis: Combining structural modeling with evolutionary analysis provides context:

  • Identification of evolutionarily conserved residues through multiple sequence alignment

  • Mapping conservation patterns onto the structural model

  • Correlation of sequence variation with symbiotic lifestyle differences

  • Detection of potential adaptation signatures specific to B. japonicum

When applied to aminoacyl-tRNA synthetases like LeuRS, these computational approaches have successfully predicted critical residues involved in substrate specificity and catalysis. For B. japonicum LeuRS, modeling may reveal unique features associated with its symbiotic lifestyle and adaptation to the nodule environment .

How might leuS be involved in regulating the transition between free-living and symbiotic states?

The transition from free-living to symbiotic states in Bradyrhizobium japonicum involves extensive reprogramming of gene expression and metabolism. Current evidence suggests LeuRS may play regulatory roles beyond its canonical function in protein synthesis:

• Potential Moonlighting Functions: Aminoacyl-tRNA synthetases in other organisms have been shown to function as regulatory proteins through interactions with transcription factors or signaling molecules. LeuRS might have similar moonlighting functions in Bradyrhizobium, particularly during symbiotic transitions.

• Stress Response Integration: The symbiosome environment presents numerous stresses (low oxygen, altered pH, nutritional shifts). LeuRS might serve as a sensor linking translational fidelity to stress adaptation, similar to the role of GCN2 kinase in eukaryotes.

• Involvement in T3SS Regulation: In strains with T3SS-dependent nodulation, LeuRS could potentially interact with regulatory components of this machinery. Research has shown that T3SS effectors like ErnA trigger specific plant signaling pathways, and proper translation of these effectors may be particularly dependent on LeuRS function .

Future research should employ:

• Interactome analysis comparing LeuRS binding partners between free-living and symbiotic states

• Targeted mutagenesis of non-catalytic domains to identify regulatory functions

• Ribosome profiling to identify mRNAs particularly dependent on LeuRS function during symbiosis

• Comparative analysis across different Bradyrhizobium strains with varying symbiotic capabilities

What novel experimental approaches could better characterize leuS regulation in Bradyrhizobium?

Emerging technologies offer new opportunities to characterize leuS regulation with unprecedented precision:

• CRISPR Interference (CRISPRi): Allows tunable repression of leuS expression without complete knockout, enabling the study of partial loss-of-function phenotypes that might be lethal in conventional deletion mutants.

• Single-Cell Transcriptomics: Reveals heterogeneity in leuS expression across bacterial populations, particularly important when studying bacteroids within nodules.

• Proximity Labeling Proteomics (BioID or APEX2): Identifies proteins physically near LeuRS in living cells, potentially uncovering novel interaction partners under different conditions.

• Ribosome Profiling (Ribo-seq): Provides genome-wide information on translation efficiency and could reveal condition-specific regulation of leuS translation.

• RNA Structurome Analysis: Characterizes changes in mRNA secondary structure under different conditions, potentially revealing regulatory RNA elements affecting leuS expression.

• Nanopore Direct RNA Sequencing: Detects RNA modifications without conversion to cDNA, potentially revealing epitranscriptomic regulation of leuS mRNA.

• Microfluidics-Based Single-Cell Phenotyping: Enables real-time monitoring of gene expression in individual bacteria during host infection or under changing environmental conditions.

• Mathematical Modeling: Integrates experimental data to predict system-level behavior:

These approaches, particularly when applied within the framework of statistical design of experiments (DOE), promise to revolutionize our understanding of leuS regulation in the context of Bradyrhizobium symbiosis .