Recombinant Bacillus cereus UPF0344 protein BCE_1257 (BCE_1257)

How was BCE_1257 initially identified and classified as an UPF0344 family protein?

BCE_1257 was annotated during the genome sequencing of Bacillus cereus strains. The UPF0344 designation indicates it belongs to a family of "Uncharacterized Protein Family" proteins with similar sequence patterns across multiple bacterial species. The protein was classified based on sequence homology and predicted structural features, though its precise function remains under investigation. Genome sequencing efforts, particularly those focusing on B. cereus strain ATCC 14579 and related strains, contributed to its identification and preliminary classification .

What are the optimal expression systems for recombinant production of BCE_1257?

For successful expression of BCE_1257, E. coli is the preferred heterologous host system. The protein has been successfully expressed as a recombinant full-length fusion protein with an N-terminal His-tag, facilitating purification through affinity chromatography. Optimal expression conditions include:

The recombinant protein is typically expressed as a full-length protein (1-121 amino acids) and can be obtained with >90% purity using standardized purification protocols .

What purification strategies yield the highest purity of recombinant BCE_1257?

A multi-step purification protocol is recommended for obtaining high-purity BCE_1257:

• Initial capture by Ni-NTA affinity chromatography (utilizing the His-tag)

• Buffer exchange to remove imidazole and prevent protein aggregation

• Secondary purification via size exclusion chromatography

• Optional ion-exchange chromatography for removing remaining contaminants

This approach consistently yields >90% pure protein as determined by SDS-PAGE analysis. For applications requiring ultra-high purity, additional chromatographic steps may be incorporated. The protein is typically recovered in Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which enhances stability during storage .

What are the critical considerations for maintaining BCE_1257 stability during storage and handling?

Maintaining BCE_1257 stability requires careful attention to storage conditions:

The protein exhibits greatest stability when stored in Tris/PBS-based buffer containing 6% trehalose (pH 8.0) with 50% glycerol. For reconstitution of lyophilized protein, researchers should use deionized sterile water to a concentration of 0.1-1.0 mg/mL. Research indicates that repeated freeze-thaw cycles significantly decrease protein activity and should be avoided through proper aliquoting strategies .

What is known about the functional role of BCE_1257 in Bacillus cereus biology?

The specific biological function of BCE_1257 remains largely uncharacterized, representing a significant research gap. Based on bioinformatic analyses and sequence features:

• The protein contains multiple transmembrane domains, suggesting a membrane-associated function

• It may participate in stress response mechanisms common to B. cereus group organisms

• It could potentially function in small molecule transport or signaling processes

• Expression patterns suggest possible involvement in adaptation to environmental conditions

Current research on B. cereus pathometabolism indicates that many previously uncharacterized proteins play roles in adaptation mechanisms, particularly in food environments or during host interaction. BCE_1257 may belong to a class of proteins involved in these processes, though direct experimental evidence for its specific function is limited .

How does BCE_1257 compare to homologous proteins in related Bacillus species?

Comparative genomic analyses reveal BCE_1257 homologs across the Bacillus cereus group:

The high conservation of this protein across pathogenic and non-pathogenic Bacillus species suggests it may serve a fundamental physiological role rather than direct involvement in virulence. Multi-locus sequence typing (MLST) studies have positioned BCE_1257 within conserved genetic elements of the B. cereus group .

What experimental approaches are most effective for investigating BCE_1257 function?

To elucidate BCE_1257 function, a multi-faceted research strategy is recommended:

• Gene Knockout Studies:

  • Generate BCE_1257 deletion mutants using CRISPR-Cas9 or traditional allelic exchange methods

  • Analyze phenotypic changes under various stress conditions (pH, temperature, osmotic stress)

  • Compare growth characteristics in different media compositions

• Protein Localization:

  • Utilize GFP-fusion constructs to determine subcellular localization

  • Conduct fractionation studies followed by Western blotting to confirm membrane association

• Interactome Analysis:

  • Employ co-immunoprecipitation coupled with mass spectrometry to identify protein-protein interactions

  • Use bacterial two-hybrid systems for targeted interaction studies

• Expression Profiling:

  • Analyze transcription patterns under various environmental conditions using RT-qPCR

  • Perform RNA-seq to understand co-expressed gene networks

These approaches have proven effective in characterizing previously unknown bacterial proteins and would likely yield valuable insights into BCE_1257 function .

How might BCE_1257 contribute to Bacillus cereus pathogenicity or stress adaptation?

While BCE_1257's specific role in pathogenicity remains unconfirmed, several hypotheses warrant investigation:

• Membrane Stress Response: The protein's transmembrane domains suggest potential involvement in membrane integrity during environmental stress. B. cereus adapts to diverse environments, and BCE_1257 may contribute to membrane homeostasis during stress conditions.

• Potential Role in Biofilm Formation: Many uncharacterized membrane proteins in the B. cereus group participate in biofilm formation, which enhances survival in food matrices and environmental surfaces.

• Connection to Virulence Regulation Networks: Research has shown that multiple redox regulators in B. cereus (Fnr, ResD, Rex, and OhrR) coordinate the expression of both metabolic and virulence genes. BCE_1257 may participate in these regulatory networks, potentially influencing virulence indirectly .

• Association with Mobile Genetic Elements: Genome analyses of B. cereus strains have identified mobile genetic elements near various uncharacterized genes. BCE_1257 could potentially be part of horizontally transferred genetic material that contributes to strain-specific adaptations .

What are the recommended approaches for analyzing potential post-translational modifications of BCE_1257?

Analysis of potential post-translational modifications (PTMs) in BCE_1257 requires specialized methodologies:

Recent studies on B. cereus proteins have revealed that methionine residues often act as ROS scavengers. BCE_1257 contains multiple methionine residues that may be susceptible to oxidation under different growth conditions. Analyzing these modifications could provide insights into potential roles in redox homeostasis. Mass spectrometry-based approaches coupled with appropriate enrichment strategies are essential for comprehensive PTM analysis .

How has BCE_1257 evolved within the Bacillus cereus group, and what does this suggest about its function?

Evolutionary analyses of BCE_1257 reveal interesting patterns:

• Sequence Conservation: The high degree of conservation (>90% sequence identity) across the B. cereus group suggests functional importance, despite its uncharacterized status.

• Genetic Context: The genomic neighborhood of BCE_1257 is relatively conserved across strains, often associated with genes involved in membrane processes or stress responses.

• Selective Pressure Analysis: Examination of non-synonymous to synonymous substitution ratios (dN/dS) across BCE_1257 homologs suggests purifying selection, further supporting functional importance.

• Domain Architecture: The UPF0344 domain has been maintained throughout evolution in the B. cereus group, though subtle variations in transmembrane regions exist between species.

These evolutionary patterns suggest BCE_1257 likely plays a role in core cellular processes rather than strain-specific adaptations. Comparative genomic approaches, particularly those examining gene neighborhoods and co-evolution patterns, may provide additional functional insights .

What analytical techniques are most appropriate for determining BCE_1257 structure-function relationships?

Determining structure-function relationships for BCE_1257 requires a combination of computational and experimental approaches:

• Computational Structural Prediction:

  • Employ modern AI-based structure prediction tools (AlphaFold2, RoseTTAFold)

  • Conduct molecular dynamics simulations to evaluate stability and potential binding pockets

  • Perform in silico docking studies with potential ligands

• Experimental Structure Determination:

  • X-ray crystallography (challenging for membrane proteins)

  • Cryo-electron microscopy for larger complexes

  • NMR spectroscopy for specific domains or peptide fragments

• Structure-Guided Mutagenesis:

  • Generate point mutations at conserved residues

  • Assess functional changes using phenotypic assays

  • Map functional residues to predicted structural features

• Biophysical Interaction Studies:

  • Isothermal titration calorimetry

  • Surface plasmon resonance

  • Microscale thermophoresis for potential binding partners

These approaches have proven successful in characterizing other bacterial membrane proteins with previously unknown functions and would likely provide valuable insights into BCE_1257's structure-function relationships .

What are the primary technical challenges in studying BCE_1257, and how can they be addressed?

Researching BCE_1257 presents several technical challenges:

• Membrane Protein Solubility: As a predicted transmembrane protein, BCE_1257 presents solubility challenges during purification and characterization.

  • Solution: Utilize specialized detergents (DDM, LMNG) or nanodiscs for maintaining native structure.

• Functional Assay Development: Without known function, designing appropriate assays is difficult.

  • Solution: Employ phenotypic microarrays, chemical genetic approaches, and comparative metabolomics to identify conditions where BCE_1257 function becomes apparent.

• Genetic Manipulation: Creating precise genetic modifications in B. cereus can be challenging.

  • Solution: Leverage emerging CRISPR-Cas9 systems optimized for Gram-positive bacteria.

• Protein-Protein Interaction Detection: Membrane protein interactions are notoriously difficult to capture.

  • Solution: Implement proximity labeling approaches (BioID, APEX) combined with MS to identify neighboring proteins in the native context.

These technical approaches, combined with appropriate controls and validation strategies, will help overcome the inherent challenges of studying an uncharacterized membrane protein .

What interdisciplinary approaches might yield new insights into BCE_1257 function?

Understanding BCE_1257 function may benefit from interdisciplinary approaches:

• Systems Biology: Integrating transcriptomics, proteomics, and metabolomics data to position BCE_1257 within cellular networks.

• Evolutionary Biology: Comparative genomics across diverse bacteria to identify co-evolving genes and conserved genetic neighborhoods.

• Structural Biology and Biophysics: Advanced structural techniques combined with computational modeling to predict functional domains and interaction surfaces.

• Food Microbiology: Examining BCE_1257 expression patterns during growth in various food matrices to understand potential roles in adaptation to food environments.

• Host-Pathogen Interaction Studies: Investigating BCE_1257 expression during infection models to determine potential roles in virulence or host adaptation.

These interdisciplinary approaches have proven valuable in characterizing proteins of unknown function in other bacterial systems and would likely contribute significantly to understanding BCE_1257's biological role .