Recombinant Bradyrhizobium japonicum 50S ribosomal protein L14 (rplN)

What Is the Functional Role of rplN in Bradyrhizobium japonicum Ribosomal Assembly and Translation Fidelity?

The 50S ribosomal protein L14 (rplN) is integral to ribosome structure and function. In B. japonicum, rplN stabilizes the 23S rRNA tertiary structure by forming cross-subunit bridges with the 30S subunit, ensuring proper alignment during translation initiation . Methodological approaches to confirm its role include:

• Cryo-EM Structural Mapping: Resolve ribosome structures from wild-type and ΔrplN mutants to identify conformational disruptions (e.g., altered inter-subunit spacing or rRNA folding) .

• Genetic Complementation: Introduce plasmid-borne rplN into deletion strains and quantify translational fidelity via β-galactosidase reporter assays under varying stress conditions (e.g., high salinity) .

• Cross-Linking Mass Spectrometry: Identify rplN interaction partners using formaldehyde fixation followed by tryptic digestion and LC-MS/MS .

Table 1: Phenotypic Comparison of Wild-Type vs. ΔrplN Strains

How Should Researchers Optimize Heterologous Expression of Recombinant rplN for Structural Studies?

Recombinant rplN production requires addressing B. japonicum’s codon bias and solubility challenges:

• Codon Optimization: Use algorithms like GeneArt to match E. coli or Pichia pastoris codon usage while retaining native protein folding signals .

• Fusion Tags: Employ N-terminal His-tags with TEV cleavage sites for IMAC purification, followed by size-exclusion chromatography (SEC) to remove aggregates .

• Solubility Screening: Test expression in E. coli BL21(DE3) pLysS at 18°C with 0.5 mM IPTG and evaluate lysate solubility via SDS-PAGE and Coomassie staining .

Critical Note: B. japonicum’s GC-rich genome (64%) necessitates PCR amplification with high-fidelity polymerases (e.g., Q5) to avoid nonspecific mutations during cloning .

How Can Discrepancies in rplN Structural Data Be Resolved?

Conflicting crystallographic and NMR models often arise from dynamic regions or crystallization artifacts. Mitigation strategies include:

• Multi-Method Validation: Compare cryo-EM densities (3.5–4.0 Å resolution) with X-ray crystallography (1.8 Å) to identify flexible loops or disordered domains .

• Molecular Dynamics Simulations: Run 100-ns simulations in GROMACS to assess conformational stability of disputed regions (e.g., C-terminal α-helix) .

• Mutagenesis Studies: Introduce point mutations (e.g., K78A, R92A) to test whether disputed residues contribute to rRNA binding via electrophoretic mobility shift assays (EMSAs) .

What Regulatory Elements Control rplN Expression Under Microaerobic Conditions?

The rplN promoter in B. japonicum is regulated by oxygen-sensitive transcription factors:

• FixLJ-FixK2 Cascade: Under low O₂, FixL autophosphorylates and activates FixJ, which induces FixK2 to bind the rplN promoter .

• qRT-PCR Validation: Compare rplN mRNA levels in wild-type vs. ΔfixJ mutants grown in 0.5% O₂ using SYBR Green assays (primers: 5’-ATGGCGCTCAAC-3’ and 5’-TCAGGCGATAGT-3’) .

• Promoter Deletion Analysis: Clone serial truncations of the rplN 5’ UTR into a lacZ reporter vector and measure β-galactosidase activity in microaerobic chemostats .

Table 2: rplN Expression Under Varying O₂ Levels

What Advanced Techniques Are Recommended for Studying rplN-rRNA Interactions?

To map rRNA binding sites with nucleotide precision:

• Hydroxyl Radical Footprinting: Treat ribosomes with Fe(II)-EDTA and H₂O₂ to generate cleavage patterns protected by rplN binding. Resolve fragments via PAGE and autoradiography .

• Single-Molecule FRET: Label rRNA helices 18 and 34 with Cy3/Cy5 to monitor real-time conformational changes upon rplN binding using a Total Internal Reflection Fluorescence (TIRF) microscope .

• Cross-Linking Immunoprecipitation (CLIP-seq): UV-irradiate B. japonicum cultures, immunoprecipitate rplN-RNA complexes, and sequence bound rRNA regions .

How Do Post-Translational Modifications Affect rplN Function in Symbiotic Nitrogen Fixation?

B. japonicum rplN undergoes phosphorylation (Ser15) and acetylation (Lys29) during symbiosis:

• Phosphoproteomics: Enrich phosphorylated peptides from bacteroid extracts using TiO₂ columns and identify sites via LC-MS/MS on a Q Exactive HF-X .

• Site-Directed Mutagenesis: Replace Ser15 with alanine (S15A) and assess nodulation efficiency on soybean roots (cv. Williams 82) in hydroponic assays .

Key Finding: S15A mutants reduce nitrogen fixation rates by 40% compared to wild-type, implicating phosphorylation in regulating ribosome activity under symbiotic conditions .

How Can Researchers Address Contamination in rplN Preparations During Purification?

Common contaminants include chaperones (GroEL) and nucleic acids:

• Ion-Exchange Chromatography: Use a HiTrap Q HP column with a 0.1–1.0 M NaCl gradient to separate rplN (pI 9.8) from acidic contaminants .

• Benzonase Treatment: Incubate lysates with 50 U/mL Benzonase for 1 hr at 4°C to digest co-purifying rRNA fragments .

• SEC-MALS: Confirm monodispersity using a Superdex 200 Increase column coupled with multi-angle light scattering (MALS) to detect aggregates or degraded fragments .

What Genetic Tools Are Available for rplN Manipulation in Bradyrhizobium japonicum?

The USDA110 strain’s sequenced genome (9.1 Mbp) enables precise editing:

• CRISPR-Cas9 Knockouts: Design sgRNAs targeting rplN (locus tag: blr0318) and transform via conjugation with pK18mobsacB .

• Inducible Overexpression: Clone rplN into pBBR1-MCS2 under a xylose-inducible promoter and verify plasmid stability in B. japonicum USDA110 .

• Transcriptional Fusion Reporters: Insert gfp downstream of rplN in a neutral site (e.g., intergenic region) to monitor real-time expression in root nodules .

How Does rplN Contribute to Antibiotic Tolerance in Bradyrhizobium japonicum?

rplN mutations alter ribosome-targeting antibiotic susceptibility:

• MIC Assays: Test tetracycline (30S inhibitor) and erythromycin (50S inhibitor) sensitivity in rplN point mutants (e.g., G71V) using broth microdilution .

• Ribosome Profiling: Treat cultures with sublethal antibiotic doses, isolate ribosomes, and quantify stalled ribosome footprints via deep sequencing .

Table 3: Antibiotic MICs for rplN Mutants

What Are the Implications of rplN Sequence Divergence Across Bradyrhizobium Species?

Phylogenetic analysis reveals rplN as a molecular marker for speciation:

• Comparative Genomics: Align rplN sequences from 12 Bradyrhizobium species using MUSCLE and construct a maximum-likelihood tree in RAxML .

• Positive Selection Analysis: Apply PAML’s site models to identify codons under diversifying selection (e.g., ω = dN/dS > 1) .

Key Insight: Residues 45–58 in rplN exhibit ω = 2.3, suggesting adaptive evolution linked to host-specific symbiosis .