Recombinant Bacillus cereus 50S ribosomal protein L33 3 (rpmG3)

What is the genomic context of the rpmG3 gene in Bacillus cereus and how does it differ from other ribosomal protein genes?

The rpmG3 gene in Bacillus cereus encodes the 50S ribosomal protein L33 3, which is part of the large ribosomal subunit. Unlike many ribosomal protein genes that are organized in operons, genomic analysis reveals that rpmG3 has a distinctive context.

In B. cereus, the gene organization shows that rpmG3 is positioned near genes such as secE, nusG, and rplK genes. This organization pattern is conserved across several bacterial phyla . The rpmG3 gene is particularly interesting because B. cereus carries multiple paralogous copies of L33 ribosomal proteins (rpmG genes), which may serve different functional roles.

To study the genomic context:

• Use whole genome sequencing with long-read technologies such as PacBio or Oxford Nanopore to ensure accurate assembly around repeat regions

• Employ comparative genomics approaches to analyze synteny across the B. cereus group

• Perform transcriptomic analysis to identify co-transcribed genes and potential operons

How can recombinant rpmG3 protein be efficiently expressed and purified for structural studies?

For high-yield expression and purification of recombinant rpmG3:

Expression systems comparison:

Recommended protocol:

• Clone the rpmG3 gene into pET3a vector system with a His-tag for purification

• Transform E. coli BL21(DE3) with the resulting recombinant plasmid

• Grow cells in Luria-Bertani medium at 37°C to an OD600 of 0.5

• Induce with 1 mM IPTG for 6 hours at 30°C (lowered temperature improves solubility)

• Harvest cells and resuspend in binding buffer (20 mM Tris-HCl [pH 7.9], 0.5 M NaCl, 5 mM imidazole)

• Purify using nickel-charged Chelex-100 column

• Verify purity by SDS-PAGE (>85% purity is achievable)

For structural studies, add 5-50% glycerol as a final concentration for storage at -20°C/-80°C to maintain protein stability . Avoid repeated freeze-thaw cycles and store working aliquots at 4°C for up to one week.

What are the key differences between rpmG3 proteins in Bacillus cereus and Bacillus thuringiensis that enable species differentiation?

The rpmG3 protein serves as a valuable biomarker for differentiating between closely related Bacillus species. MALDI-TOF MS analysis reveals species-specific peaks that can be attributed to ribosomal proteins, including L33 proteins.

Species-specific mass differences:

These differences occur due to non-synonymous mutations resulting in amino acid substitutions in the corresponding proteins . For accurate species identification:

• Extract ribosomal proteins using trichloroacetic acid precipitation

• Analyze by MALDI-TOF MS focusing on the spectral range of 2,000-12,000 m/z

• Look for species-specific peaks at m/z 9160/9188 and 9214/9229

• Confirm by sequence analysis of the corresponding genes

This method achieves 97.22-100% accuracy in distinguishing B. cereus from B. thuringiensis, which is crucial for bacterial taxonomy .

How is rpmG3 expression regulated in response to zinc availability in Bacillus cereus?

Ribosomal protein expression, including rpmG3, can be regulated by zinc availability through zinc-responsive regulatory systems. Research indicates that the Zur (zinc uptake regulator) protein controls zinc homeostasis and affects ribosomal protein expression.

Regulation mechanism:

• Under zinc-replete conditions, Zur binds zinc and acts as a repressor

• During zinc limitation, Zur releases from DNA, allowing transcription

• The rpmG3 gene may be partially induced by zinc depletion (e.g., EDTA treatment)

Experimental approach to study zinc-dependent regulation:

• Grow B. cereus strains in YEME or chelated minimal medium to an OD600 of 0.3-0.4

• Extract RNA and perform Northern blot analysis using PCR-generated probes

• Design primers for the rpmG3 gene with appropriate controls

• Label probe DNA fragments with [γ-32P]

• Compare expression levels between wild-type and zur mutant strains under varying zinc conditions

The relationship between zinc and rpmG3 may be particularly important as zinc ion binding is critical for protein stability in some ribosomal proteins, as demonstrated in studies of metallo-beta-lactamases from Bacillus cereus .

What role does rpmG3 play in bacterial pathogenicity within the Bacillus cereus group?

The connection between ribosomal proteins like rpmG3 and pathogenicity in the B. cereus group is complex. While rpmG3 itself is primarily a structural component of the ribosome, several lines of evidence suggest potential roles in virulence:

• Phylogenetic association: rpmG3 sequence variations correlate with pathogenic potential within the B. cereus group. Strains that cause similar diseases tend to have similar rpmG3 sequences .

• Co-regulation with virulence factors: Genomic analysis shows that genes encoding ribosomal proteins may be co-regulated with virulence factors. In B. cereus, PlcR (a pleiotropic regulator of extracellular virulence factors) regulates genes encoding degradative enzymes and enterotoxins .

• Role in stress response: Ribosomal proteins may contribute to stress adaptation mechanisms that are crucial for pathogen survival in host environments.

To investigate rpmG3's potential role in pathogenicity:

• Generate knockout mutants using CRISPR-Cas9 or homologous recombination

• Perform comparative proteomics between wild-type and mutant strains

• Evaluate virulence in infection models (cell culture or animal models)

• Assess expression during different infection stages using RT-qPCR

Understanding this relationship may provide insights into B. cereus pathogenicity, which causes a spectrum of diseases from food poisoning to systemic infections .

How can multi-locus sequence typing (MLST) incorporating rpmG3 improve phylogenetic analysis of the Bacillus cereus group?

MLST is a powerful technique for phylogenetic analysis of closely related bacterial species. Incorporating rpmG3 sequences into MLST schemes can enhance resolution when differentiating members of the B. cereus group.

Existing MLST schemes vs. proposed rpmG3-inclusive scheme:

Methodological approach:

• Amplify the rpmG3 gene using universal primers that target conserved flanking regions

• Sequence the amplicons using high-fidelity Sanger sequencing

• Concatenate sequences with other MLST loci

• Construct phylogenetic trees using Maximum Likelihood or Bayesian methods

• Test for congruence between individual gene trees using the incongruence length difference test

Including rpmG3 could help resolve ambiguities between B. cereus and B. thuringiensis, which show "more congruence than expected by chance, indicating a generally clonal structure to the population" but with significant exceptions suggesting horizontal gene transfer events.

What is the relationship between rpmG3 and the NLRP3 inflammasome during Bacillus cereus infection?

The interaction between B. cereus components and the NLRP3 inflammasome represents a critical aspect of host-pathogen interaction. While direct evidence for rpmG3's role in inflammasome activation is limited, several mechanisms may connect ribosomal proteins to this immune response:

• Potential PAMPs: Bacterial ribosomal proteins can act as pathogen-associated molecular patterns (PAMPs) recognized by host pattern recognition receptors

• Inflammasome context: B. cereus enterotoxins (Nhe and Hbl) trigger NLRP3 activation and subsequent pyroptosis

• Upstream signals: Inflammasome activation involves K+ efflux, Ca2+ flux, and organelle disruption , which could be influenced by bacterial ribosomal proteins

Experimental approach to investigate this relationship:

• Generate recombinant rpmG3 protein with high purity (>90%)

• Treat macrophage cell lines (THP-1, RAW264.7) with purified rpmG3

• Measure inflammasome activation markers:

  • IL-1β and IL-18 secretion by ELISA

  • Caspase-1 activation by Western blot

  • GSDMD cleavage by Western blot

  • Cell death by LDH release assay

• Use NLRP3 inhibitors (MCC950) as controls to confirm specificity

• Compare responses to whole bacteria and other virulence factors

Understanding this interaction could provide insights into B. cereus pathogenesis and potential therapeutic targets, as "NLRP3 inflammasome activation often leads to GSDMD-mediated pyroptosis" .

How can natural competence induction in Bacillus cereus be optimized for rpmG3 genetic manipulation?

Genetic manipulation of rpmG3 requires efficient transformation methods. Recent research has demonstrated that natural competence can be induced in B. cereus, providing an alternative to electroporation.

Optimized protocol for natural competence induction:

• Clone ComK from B. subtilis (ComKBsu) behind an inducible promoter (Phyperspank) in a suitable vector (e.g., pNW33N)

• Transform B. cereus by electroporation with this construct

• Grow transformed cells in competence-stimulating medium to an OD600 of 0.7

• Induce with 1 mM IPTG to express ComKBsu

• Add target DNA (for rpmG3 modification) and incubate for 30 minutes at 30°C with shaking

• Add recovery medium and continue incubation for 1 hour

• Plate on selective media to identify transformants

Key optimization factors:

• Only a small percentage of cells (typically <5%) will express competence genes and become transformable

• Flow cytometry can be used to monitor competence development using a PcomGA-gfp reporter

• Transformation efficiency will be relatively low compared to standard model organisms

This approach enables sophisticated genetic manipulations such as:

• Precise point mutations in rpmG3 to study structure-function relationships

• Reporter gene fusions to study expression patterns

• Deletion mutants to assess phenotypic effects

What techniques are most effective for studying ribosomal protein-protein interactions involving rpmG3?

Understanding rpmG3's interactions with other ribosomal and non-ribosomal proteins is crucial for elucidating its functions. Several complementary techniques can be employed:

In vitro interaction analysis:

For analytical ultracentrifugation specifically:

• Purify recombinant rpmG3 to >90% purity

• Perform experiments at various protein concentrations

• Collect data at 280 nm during centrifugation

• Analyze using the equation: C(r) = Cb exp[A*M(r²-rb²)] + ε
  Where:

  • C(r) is the concentration at radial position r

  • Cb is the concentration at cell bottom

  • A is (1-υρ)ω²/2RT

  • M is molecular mass

  • ε is baseline error

In vivo interaction analysis:

• Bacterial two-hybrid system

• In vivo cross-linking followed by mass spectrometry

• Fluorescence resonance energy transfer (FRET)

• Proximity-dependent biotin identification (BioID)

These approaches can reveal both structural interactions within the ribosome and potential moonlighting functions of rpmG3 outside ribosomal context.

How does recombinant rpmG3 compare structurally and functionally to native protein, and what are the implications for research applications?

When using recombinant rpmG3 for research, it's essential to understand how closely it mimics the native protein in structure and function:

Structural considerations:

• Folding accuracy: Recombinant expression in heterologous systems (particularly E. coli) may yield proteins with subtly different folding compared to native B. cereus ribosomes

• Post-translational modifications: Native rpmG3 may undergo modifications not replicated in recombinant systems

• Metal coordination: Ribosomal proteins like L33 can bind zinc via zinc-finger motifs, which must be preserved in recombinant proteins

Functional assessment methods:

• Circular dichroism spectroscopy to compare secondary structure elements

• Thermal shift assays to evaluate stability differences

• Ribosome assembly assays using in vitro reconstitution systems

• Translation efficiency measurements using cell-free translation systems

Research implications and recommendations:

• Use baculovirus expression systems for structural studies requiring native-like protein

• Include appropriate metal ions (particularly zinc) in purification buffers

• Verify activity through functional assays specific to the research question

• Consider tag position and removal strategies to minimize structural interference

• For antimicrobial development studies, validate findings with native protein contexts

The presence of zinc in particular may be critical, as zinc depletion can stimulate regulatory systems and affect protein structure, similarly to what has been observed with metallo-beta-lactamases from B. cereus .