Recombinant Bacillus cereus UPF0180 protein BCE33L1278 (BCE33L1278)

What is the complete biochemical characterization of BCE33L1278?

BCE33L1278 is an 82 amino acid protein with the sequence MRIMARIGVENSLTDVQQALKQQGHEVVTLNSEQDAQGCDCCVVTGQDSNMMGIADASIKGSVITAHGLTTDEVCQQVESRT and a molecular mass of 8.8 kDa . This protein belongs to the UPF0180 (Uncharacterized Protein Family 0180) found across multiple Bacillus species. The protein contains four cysteine residues (positions 39, 40, 41, and 77), which may form disulfide bonds critical for structural integrity or function. Analysis using hydrophobicity plots would likely reveal both hydrophobic and hydrophilic regions consistent with globular proteins rather than membrane-spanning domains.

For comprehensive biochemical characterization, researchers should:

• Determine the isoelectric point through isoelectric focusing

• Assess secondary structure composition using circular dichroism spectroscopy

• Analyze thermal stability through differential scanning calorimetry

• Investigate potential post-translational modifications using mass spectrometry

What expression systems are optimal for recombinant production of BCE33L1278?

For recombinant expression of BCE33L1278, several systems can be considered based on experimental purposes. E. coli-based expression systems typically provide the highest yields for non-toxic bacterial proteins and would be suitable for initial characterization studies. For a small protein like BCE33L1278 (82 aa, 8.8 kDa), a pET vector system with a 6x-His tag allows for straightforward IMAC purification .

Optimization parameters should include:

For structural studies requiring native conformation, Bacillus subtilis expression systems may be preferable since they more closely resemble the native environment of B. cereus proteins and contain appropriate chaperones for proper folding.

How can researchers verify the functional integrity of purified BCE33L1278?

Verifying functional integrity of BCE33L1278 presents a challenge given its uncharacterized nature. A multi-tiered approach is recommended:

• Structural integrity assessment:

  • Circular dichroism to confirm proper secondary structure formation

  • Size-exclusion chromatography to verify monomeric state or expected oligomerization

  • Thermal shift assays to determine stability profiles

• Functional characterization:

  • Pull-down assays to identify potential binding partners

  • Bacterial two-hybrid systems to screen for protein-protein interactions

  • Complementation assays in BCE33L1278 knockout strains of B. cereus

• Comparative analysis:

  • Analyze conservation patterns across UPF0180 family members

  • Compare to functionally characterized orthologs in related species, if any exist

Since B. cereus is a known pathogen that expresses numerous virulence factors and resistance mechanisms , testing for interactions with host immune components or antimicrobial agents may provide insights into BCE33L1278's biological role.

What bioinformatic approaches are useful for predicting BCE33L1278 function?

For an uncharacterized protein like BCE33L1278, comprehensive bioinformatic analysis provides crucial insights:

• Sequence-based analysis:

  • BLAST searches against characterized proteins

  • Multiple sequence alignment with UPF0180 family members

  • Identification of conserved domains and motifs using InterPro or PFAM

  • Prediction of signal peptides using SignalP

• Structural prediction:

  • Secondary structure prediction using PSIPRED

  • Tertiary structure modeling using AlphaFold or I-TASSER

  • Structure-based function prediction using ProFunc

• Genomic context analysis:

  • Examine neighboring genes in the B. cereus genome

  • Analyze potential operons or gene clusters

  • Investigate transcriptional regulation patterns

• Phylogenetic profiling:

  • Construct phylogenetic trees of UPF0180 family proteins

  • Compare distribution across bacterial species, particularly within the B. cereus group

  • Correlate presence/absence with specific phenotypes

Orthologous proteins from other Bacillus species could be highly informative. For instance, the related protein BC3310 from B. cereus ATCC 14579 has been characterized as a multidrug transporter, suggesting potential roles in transport or resistance mechanisms .

How might BCE33L1278 contribute to B. cereus pathogenicity or survival mechanisms?

While direct evidence for BCE33L1278's role in pathogenicity is lacking, several investigative approaches can elucidate potential contributions:

• Gene knockout studies:

  • Create a markerless deletion mutant of BCE33L1278 in B. cereus strain ZK / E33L using homologous recombination techniques similar to those used for BC3310

  • Compare virulence profiles between wild-type and mutant strains in infection models

  • Assess survival under various stress conditions (pH, temperature, antimicrobials)

• Transcriptomic analysis:

  • Analyze expression patterns of BCE33L1278 during different growth phases

  • Compare expression levels between pathogenic and non-pathogenic conditions

  • Identify co-regulated genes to establish potential functional networks

• Protein localization studies:

  • Determine subcellular localization using fluorescent fusion proteins

  • Investigate potential secretion using signal sequence analysis

  • Assess interactions with host cell components

B. cereus produces numerous virulence factors and exhibits resistance to various antimicrobials, including β-lactams . If BCE33L1278 contributes to these mechanisms, comparative susceptibility testing between wild-type and knockout strains would reveal significant differences.

What methodologies are appropriate for investigating BCE33L1278 protein-protein interactions?

Characterizing protein-protein interactions for BCE33L1278 requires a multi-method approach:

• In vitro methods:

  • Pull-down assays using immobilized recombinant BCE33L1278

  • Surface plasmon resonance to determine binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Cross-linking mass spectrometry to capture transient interactions

• In vivo approaches:

  • Bacterial two-hybrid system adapted for B. cereus

  • Co-immunoprecipitation from B. cereus lysates

  • Proximity-dependent biotin labeling (BioID) to capture neighborhood proteins

• Structural biology techniques:

  • X-ray crystallography of BCE33L1278 with binding partners

  • NMR spectroscopy for dynamic interaction studies

  • Cryo-EM for larger complexes

A targeted approach might investigate potential interactions with other proteins of the UPF0180 family or proteins involved in pathogenicity mechanisms. Given the small size of BCE33L1278 (82 aa), it may function as part of a larger protein complex or participate in regulatory interactions within B. cereus.

How can site-directed mutagenesis elucidate functional domains in BCE33L1278?

Site-directed mutagenesis offers powerful insights into protein function. For BCE33L1278, the following systematic approach is recommended:

• Target selection based on sequence analysis:

  • Conserved residues across UPF0180 family members

  • Charged residues that may participate in catalysis or binding

  • Cysteine residues that might form disulfide bonds (positions 39, 40, 41, and 77)

  • Potential phosphorylation or other modification sites

• Mutagenesis strategy:

  • Alanine scanning of conserved regions

  • Conservative substitutions (e.g., D→E, K→R) to test charge requirements

  • Non-conservative substitutions to drastically alter properties

  • Deletion or truncation mutants to identify essential regions

• Functional evaluation:

  • Expression levels and solubility assessment

  • Structural integrity through circular dichroism

  • Complementation assays in BCE33L1278-deficient strains

  • Phenotypic testing for resistance, growth, and virulence

Drawing from studies of similar bacterial proteins like BC3310, where mutation of a conserved aspartate residue (D105) abolished function , identification of essential residues in BCE33L1278 could reveal mechanistic insights. The clustered cysteine residues (C39-C40-C41) present a particularly interesting target for mutagenesis as this unusual arrangement may indicate a metal-binding site or specialized structural element.

What comparative genomic approaches can reveal the evolutionary significance of BCE33L1278?

Understanding the evolutionary context of BCE33L1278 provides valuable insights into its biological importance:

• Phylogenetic analysis:

  • Construct phylogenetic trees of UPF0180 family proteins across bacterial species

  • Analyze selective pressure using dN/dS ratios to identify conserved functional domains

  • Identify co-evolution patterns with other proteins

• Genomic context comparison:

  • Analyze gene neighborhood conservation across Bacillus species

  • Identify syntenic regions that may indicate functional relationships

  • Compare promoter regions to identify conserved regulatory elements

• Distribution analysis:

  • Map presence/absence of BCE33L1278 orthologs across the B. cereus group

  • Correlate with ecological niches and pathogenicity profiles

  • Identify horizontal gene transfer events

The high conservation of proteins within the B. cereus group, as observed with BC3310 (>91% amino acid identity across 225 strains) , suggests essential functions. Similar analyses for BCE33L1278 would reveal whether it belongs to the core genome of B. cereus or represents a strain-specific adaptation.

How does the structural biology of BCE33L1278 inform potential drug targeting strategies?

For researchers interested in BCE33L1278 as a potential therapeutic target, structural biology provides crucial insights:

• Structure determination approaches:

  • X-ray crystallography of purified recombinant BCE33L1278

  • NMR spectroscopy for solution structure and dynamics

  • Cryo-EM for complexes with binding partners

  • Computational modeling based on homologous structures

• Structure-based drug design workflow:

  • Identification of potential binding pockets or active sites

  • Virtual screening of compound libraries

  • Fragment-based drug discovery

  • Structure-activity relationship studies

• Target validation methods:

  • Competitive binding assays with identified compounds

  • Site-directed mutagenesis of predicted binding residues

  • Phenotypic assays measuring B. cereus growth inhibition

  • Specificity assessment against human proteins

B. cereus produces potent β-lactamases and demonstrates resistance to various antimicrobials , making novel targets valuable. If BCE33L1278 proves essential for B. cereus viability or virulence, structural characterization could lead to new therapeutic approaches against this pathogen, which causes serious infections including anthrax-like pneumonia and central nervous system infections .