Recombinant Bacillus cereus Ribonuclease 3 (rnc)

What is the functional role of RNase III (rnc) in Bacillus cereus, and how does it differ from homologs in other Bacillus species?

RNase III (rnc) is a ribonuclease critical for processing precursor rRNA (pre-rRNA) into mature 16S, 23S, and 5S rRNAs. In B. subtilis, RNase III cleaves double-stranded regions in pre-rRNA, while B. subtilis Mini-III, an RNase III-like enzyme, matures 23S rRNA by trimming extremities . For B. cereus, homologs may share core catalytic functions but exhibit divergence in substrate specificity or auxiliary domains due to genetic drift or horizontal gene transfer .

How should experimental designs be optimized for studying B. cereus RNase III recombinant expression and purification?

Key considerations include:

• Host Selection: E. coli (BL21(DE3)) for high-yield expression, or B. subtilis (for native post-translational modifications).

• Tagging: C-terminal His-tag for affinity chromatography, avoiding interference with the catalytic domain.

• Solubility: Use chaperones (e.g., GroEL) or slow induction at 16–18°C to prevent aggregation.

Example Protocol:

What biochemical assays are most effective for characterizing B. cereus RNase III activity?

• In Vitro Cleavage Assays: Use synthetic double-stranded RNA (dsRNA) substrates mimicking rRNA processing sites. Monitor cleavage via native PAGE or Northern blot .

• rRNA Processing in Nucleic Extracts: Mix recombinant RNase III with B. cereus pre-rRNA and analyze mature rRNA products via agarose gel electrophoresis.

• Structural Studies: X-ray crystallography or cryo-EM to resolve interactions between RNase III and rRNA substrates.

Data Contradiction Handling: Discrepancies between in vitro and in vivo activity may arise from subunit assembly dependencies. For example, B. subtilis Mini-III acts efficiently only on assembled 50S subunits , suggesting B. cereus RNase III may require ribosomal context for optimal activity.

How does CRISPR/Cas9 enable functional studies of B. cereus rnc?

CRISPR/Cas9 allows precise gene editing in B. cereus:

• Knockout Generation: Design sgRNAs targeting rnc exons to create null mutants. Verify disruption via PCR and sequencing.

• Gene Replacement: Introduce point mutations (e.g., catalytic site residues) to study substrate specificity.

• Phenotypic Screening: Assess rRNA maturation defects in mutants via RNA-seq or ribosomal profiling .

Example Workflow:

• sgRNA Design: Target conserved rnc regions (e.g., catalytic motifs).

• Transformation: Electroporate B. cereus with Cas9 and sgRNA plasmids.

• Screening: Select for antibiotic-resistant colonies and validate editing via Sanger sequencing.

What are the challenges in analyzing B. cereus rnc diversity across population genetics?

The Bacillus cereus group exhibits high genetic diversity, with horizontal gene transfer (HGT) influencing rnc evolution . Challenges include:

• Phylogenetic Incongruence: rnc gene trees may conflict with housekeeping gene trees due to HGT, complicating strain classification.

• Functional Redundancy: Paralogs (e.g., Mini-III in B. subtilis) may compensate for rnc loss, masking phenotypic effects.

Solution: Integrate multi-locus sequence typing (MLST) with functional assays to link rnc variants to rRNA processing efficiency.

How to resolve conflicting data on RNase III substrate specificity between Bacillus species?

Conflicts may arise from differences in:

• rRNA Secondary Structures: B. cereus pre-rRNA may form distinct helices compared to B. subtilis.

• Auxiliary Proteins: Chaperones or RNA-binding factors in B. cereus may modulate RNase III activity.

Resolution Strategy:

• Comparative Bioinformatics: Align rnc sequences from B. cereus and B. subtilis to identify conserved catalytic residues.

• Cross-Species Complementation: Test B. cereus rnc in B. subtilis Δrnc mutants to assess functional conservation.

What advanced techniques are recommended for studying RNase III interactions with ribosomal precursors?

• Proteomics: Co-immunoprecipitation (Co-IP) to identify RNase III-binding partners in B. cereus.

• RNA Interomics: CLIP-seq (crosslinking and immunoprecipitation) to map RNase III binding sites on pre-rRNA.

• Single-Molecule Fluorescence: Monitor real-time cleavage kinetics of fluorescently labeled pre-rRNA.

Example Application:

How does plcR regulatory gene variation impact B. cereus rnc studies?

The plcR gene regulates virulence factors in B. cereus and may indirectly influence rnc expression. Strain-specific plcR polymorphisms could alter:

• rnc Expression Levels: plcR mutations may dysregulate stress-responsive pathways affecting rnc transcription.

• Experimental Reproducibility: Ensure plcR status is consistent across experimental strains to avoid confounding results.

Recommendation: Screen strains for plcR variants (e.g., via PCR) before using them in rnc functional studies .

What computational tools are essential for predicting B. cereus RNase III targets?

• RNA Secondary Structure Prediction: Tools like mfold or RNAfold to model pre-rRNA helices.

• Homology Modeling: Use B. subtilis RNase III crystal structures (e.g., PDB: 1I1B) as templates for B. cereus rnc.

• CRISPR Design: CRISPRscan or Benchling for sgRNA design targeting rnc.

Workflow Example:

• Target Identification: Align B. cereus rnc to B. subtilis RNase III active sites.

• Structure Prediction: Model B. cereus rRNA helices for cleavage site mapping.

How to address solubility issues in recombinant B. cereus RNase III production?

• Tag Optimization: Test N-terminal MBP or GST tags to enhance solubility.

• Co-Expression: Include molecular chaperones (e.g., DnaK, GroEL) during expression.

• Denaturation/Refolding: Use GuHCl/urea denaturation followed by gradual refolding in dialysis.

Troubleshooting Table: