Recombinant Bacillus cereus UPF0176 protein BCE_1961 (BCE_1961)

What is the structural composition of BCE_1961?

BCE_1961 belongs to the UPF0176 protein family in Bacillus cereus and likely shares structural similarities with other proteins in this family. Like BCE33L1694, it contains approximately 316-319 amino acids with characteristic N-terminal SH3_3 domains (PF08239) involved in protein-protein interactions and a C-terminal cell wall binding domain known as 3D domain (PF06725) . The protein likely contains a signal peptide sequence of approximately 24 residues at the N-terminus that is cleaved in the mature protein . The predicted molecular mass is around 33-34 kDa with an isoelectric point of approximately 8.9, similar to related UPF0176 proteins .

How should BCE_1961 be stored for optimal stability?

The shelf life of recombinant BCE_1961 depends on several factors including storage state, buffer ingredients, and storage temperature. For optimal stability, the following guidelines are recommended:

Repeated freezing and thawing should be avoided as it may compromise protein integrity. Working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw cycles . For long-term storage, addition of 5-50% glycerol (with 50% being typical) as a cryoprotectant is recommended when storing in liquid form .

What is the recommended reconstitution protocol for BCE_1961?

For optimal reconstitution of lyophilized BCE_1961, the following procedure is recommended:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard for most applications)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store at -20°C/-80°C for long-term storage

This protocol helps maintain protein stability and activity while minimizing degradation that could affect experimental outcomes.

What expression systems are suitable for producing BCE_1961?

BCE_1961 can be successfully expressed in E. coli expression systems, similar to other UPF0176 family proteins . When designing expression constructs, researchers should consider:

• Including the full-length sequence (approximately 319 amino acids)

• Choosing appropriate affinity tags that won't interfere with protein function

• Optimizing codon usage for E. coli if necessary

• Selecting expression vectors with appropriate promoters for controlled expression

The tag type should be determined based on the specific experimental requirements, as it may influence protein folding, solubility, and downstream applications .

How does BCE_1961 impact bacterial growth and metabolism?

Based on studies of similar UPF0176 family proteins like EntD, BCE_1961 likely plays a significant role in bacterial growth and metabolism. Disruption of similar genes results in significant growth impairment with approximately 50% reduction in growth rate and altered metabolic profiles . The following metabolic pathways may be affected:

• Glucose uptake and glycolysis - disruption of similar genes decreases glycolytic rate

• Pyruvate metabolism - affected through altered pyruvate dehydrogenase complex functionality

• Acetate overflow - typically reduced by approximately 50% in mutant strains

• TCA cycle interconnection - disrupted due to changes in acetyl-CoA metabolism

These effects appear to be oxygen-independent, suggesting BCE_1961 impacts core metabolic functions rather than specific aerobic or anaerobic pathways .

What proteome changes are associated with BCE_1961 disruption?

Disruption of UPF0176 family proteins like EntD leads to extensive proteome remodeling, affecting approximately 300 proteins across multiple functional categories. Based on proteomic analysis of similar proteins, the following patterns may be observed with BCE_1961 disruption:

The extensive changes suggest BCE_1961 may function as a regulatory protein affecting multiple cellular processes either directly or indirectly through protein-protein interactions facilitated by its SH3_3 domains .

How does BCE_1961 contribute to B. cereus virulence?

BCE_1961, like other UPF0176 family proteins, may play a significant role in B. cereus virulence through multiple mechanisms:

• Regulation of toxin production - disruption of similar genes reduces expression of virulence factors

• Influence on cell morphology - affecting bacterial cell wall structure and integrity

• Impact on motility - through downregulation of flagellar proteins

• Metabolic adaptation - allowing optimal growth in host environments

• Stress response modulation - affecting bacterial survival under host-imposed stress conditions

Cytotoxicity assays with mutant strains lacking similar proteins show reduced pathogenic potential, suggesting BCE_1961 may be a potential target for antimicrobial development .

What is the relationship between BCE_1961 and other Ent family proteins?

BCE_1961 likely shares significant sequence similarity with other Ent family proteins (EntA, EntB, EntC, and EntD), with predicted sequence identity of approximately 60-70% . These proteins contain similar domain architecture with N-terminal SH3_3 domains and C-terminal cell wall binding domains. Functional analysis suggests they may have partially overlapping but non-redundant functions, as disruption of individual genes produces distinct phenotypes . Comparative analysis of expression patterns shows these genes are differentially regulated, with highest expression typically occurring during early exponential growth phase .

How should mutant strains of BCE_1961 be constructed for functional studies?

For precise functional characterization of BCE_1961, gene disruption or deletion mutants can be constructed using the following methodology:

• PCR amplification of the genomic region containing BCE_1961 with appropriate restriction sites

• Cloning into a suitable vector (e.g., pCRXL-TOPO)

• Insertion of an antibiotic resistance cassette (e.g., spectinomycin resistance) at an appropriate restriction site within the gene

• Subcloning into a temperature-sensitive shuttle vector like pMAD

• Introduction into B. cereus by electroporation

• Selection for double crossover events to replace the wild-type allele

• Confirmation of gene disruption by PCR and sequencing

What approaches should be used to assess BCE_1961's impact on bacterial physiology?

A comprehensive assessment of BCE_1961's physiological impact should include:

• Growth curve analysis in various media and conditions (aerobic and anaerobic)

• Measurement of key metabolites (e.g., acetate, glucose consumption) using HPLC or enzymatic assays

• Cellular morphology examination using:

  • Light microscopy for basic morphological changes

  • Transmission electron microscopy (TEM) for ultrastructural analysis

  • Negative staining for flagella visualization

• Motility assays using semi-solid agar plates

• Autolysis assessment by monitoring OD600 decrease in buffer over time (72 hours is recommended)

• Cytotoxicity testing against appropriate cell lines

Statistical comparison between wild-type and mutant strains should be performed using Student's t-test or ANOVA depending on the experimental design.

How can BCE_1961 protein-protein interactions be investigated?

To elucidate BCE_1961's interaction network, the following approaches are recommended:

• Pull-down assays using recombinant tagged BCE_1961 as bait

• Bacterial two-hybrid systems to identify direct protein interactions

• Co-immunoprecipitation followed by mass spectrometry (MS/MS) analysis

• Chemical cross-linking coupled with MS to capture transient interactions

• Label-free proteomic comparison between wild-type and BCE_1961 mutant strains to identify regulated proteins

For MS-based proteomics, a workflow similar to that used for EntD characterization is recommended, which involved:

• Sample preparation from multiple growth phases

• LC-MS/MS analysis on biological triplicates

• Identification of proteins with significantly altered abundance (p < 0.05)

• Functional categorization of affected proteins

This approach identified approximately 300 regulated proteins in related studies, providing a comprehensive view of the protein's functional impact.

What techniques should be used to analyze BCE_1961 transcriptional regulation?

To understand BCE_1961 transcriptional regulation, researchers should:

• Determine the transcriptional start site using 5' RACE or similar techniques

• Identify promoter elements upstream of the transcriptional start site

• Analyze expression levels across different growth phases using reverse transcription PCR or qRT-PCR

• Investigate potential regulators using:

  • Electrophoretic mobility shift assays (EMSA) to detect protein-DNA interactions

  • Reporter gene fusions to monitor promoter activity

  • ChIP-seq to identify transcription factor binding sites genome-wide

For BCE_1961, similar proteins show highest expression during early exponential growth phase, with a putative housekeeping σA type -10 sequence (5′-TATAAT-3′) and a σD type -35 sequence (5′-CTAAA-3′) in their promoter regions . Terminator structures may also be present, suggesting transcription as a single unit rather than as part of an operon.

How should proteomic data for BCE_1961 studies be analyzed?

Proteomic analysis of BCE_1961 function should employ a rigorous statistical approach:

• Perform experiments with at least three biological replicates

• Use label-free quantification based on spectral counts or intensity

• Apply appropriate statistical tests (p < 0.05 threshold is standard)

• Categorize regulated proteins into functional groups

• Apply pathway enrichment analysis to identify affected biological processes

In studies of similar proteins, approximately 40,000-50,000 MS/MS spectra yielded identification of 600-700 proteins, with approximately 300 showing significant abundance changes between wild-type and mutant strains . Researchers should expect similar coverage when studying BCE_1961.

What controls are essential for BCE_1961 functional studies?

• Wild-type strain cultured under identical conditions

• Complemented mutant strain (restoring the gene) to confirm phenotype specificity

• Empty vector controls for complementation studies

• Verification of gene disruption/deletion by:

  • RT-PCR to confirm absence of transcript

  • Proteomic analysis to confirm absence of protein

  • PCR verification of genomic modification

• Growth phase standardization (sampling at equivalent growth phase rather than time points)

Particularly important is the verification that any observed phenotypes are specifically due to BCE_1961 disruption rather than polar effects on adjacent genes .

How can BCE_1961 research contribute to understanding B. cereus pathogenicity?

BCE_1961 research offers several avenues for advancing understanding of B. cereus pathogenicity:

• Identification of virulence regulation networks - BCE_1961 disruption affects numerous proteins involved in pathogenicity

• Novel therapeutic target development - proteins essential for virulence represent potential antimicrobial targets

• Diagnostic marker identification - specific antibodies against BCE_1961 could aid in B. cereus detection

• Strain characterization - BCE_1961 sequence variations may correlate with strain virulence potential

• Host-pathogen interaction studies - understanding how BCE_1961 influences bacterial adaptation to host environments

The extensive proteome remodeling observed with disruption of similar proteins suggests BCE_1961 may be a master regulator of pathogenicity-associated functions, making it particularly valuable for understanding B. cereus virulence mechanisms .

What are the challenges in interpreting BCE_1961 complementation studies?

Complementation studies for BCE_1961 present several challenges that researchers should anticipate:

• Dosage effects - multicopy plasmids may lead to overexpression that fails to restore wild-type phenotypes

• Regulatory context - insertion at non-native locations may alter gene regulation

• Polar effects - disruption may affect adjacent genes, complicating interpretation

• Post-translational modifications - complementation may restore protein but not modifications

• Timing of expression - non-native promoters may alter expression timing during growth phases

Researchers should verify complementation by measuring BCE_1961 expression levels through RT-PCR and protein abundance through MS/MS, comparing to wild-type levels . Alternative complementation strategies using single-copy integration at neutral sites may be necessary if multicopy plasmids fail to restore wild-type phenotypes.

What are the key considerations for designing a comprehensive BCE_1961 research project?

A comprehensive BCE_1961 research project should address:

• Multiple aspects of bacterial physiology (growth, metabolism, morphology, motility)

• Both cellular proteome and exoproteome changes

• Temporal dynamics (multiple growth phases)

• Complementary approaches (genetics, proteomics, microscopy, biochemistry)

• Functional validation through multiple independent methods

• Comparative analysis with other UPF0176 family proteins