Recombinant Bacillus cereus UPF0736 protein BCE_1296 (BCE_1296)

What approaches can be used to correctly identify and annotate uncharacterized proteins in Bacillus cereus?

Protein identification in B. cereus often begins with proteogenomic approaches. For example, EntD was initially misannotated as a pseudogene (BC_3716) until proteomics identified 4 peptides that mapped to this locus. Subsequent analysis revealed a frameshift error due to an inserted cytosine in the genomic sequence . This highlights the importance of:

• Combining genomic and proteomic data to verify annotations

• Sequencing PCR products of the gene region to confirm the correct reading frame

• Conducting BLAST analysis against related strains to identify homologous proteins

• Depositing corrected sequences in databases such as NCBI

Protein identification should be validated through multiple approaches, including peptide mapping, transcriptional analysis, and sequence correction when errors are detected .

How can transcriptional expression patterns of Bacillus cereus proteins be determined?

Determining when and how a protein is expressed provides crucial information about its function. For a B. cereus protein, researchers can:

• Perform reverse transcription PCR (RT-PCR) analysis during different growth phases (early exponential, late exponential, and stationary phases)

• Identify transcriptional start sites using 5′PCR techniques

• Analyze upstream regions for promoter elements (e.g., σA type -10 sequences or σD type -35 sequences)

• Identify potential terminator loops that suggest transcriptional units

For EntD, researchers determined that early exponential growth phase supported the highest expression of the gene, identified a transcriptional start site 26 bp from the translational start site, and found a putative housekeeping σA type promoter sequence upstream .

What are the characteristic structural domains found in Bacillus cereus virulence-associated proteins?

Many B. cereus virulence-associated proteins contain specific functional domains that contribute to their biological activity. For example, EntD contains:

• N-terminal signal peptide (first 24 residues) likely cleaved in the mature protein

• Two N-terminus SH3_3 domains (PF08239) involved in protein-protein interactions

• C-terminus putative cell wall binding domain named 3D domain (PF06725)

These domains can be identified using protein family databases such as Pfam (http://pfam.xfam.org/search/sequence) and are often conserved among related proteins. Understanding these domains helps predict protein function and interactions within the bacterial cell system .

How does protein knockout affect cellular and extracellular proteomes in Bacillus cereus strains?

Knocking out a specific protein can have widespread effects on both cellular and extracellular proteomes. In the case of EntD knockout:

• Cellular proteome analysis identified 308 proteins with significant abundance changes between wild-type and ΔentD mutant strains:

  • 154 proteins upregulated in the ΔentD mutant

  • 154 proteins downregulated in the ΔentD mutant

  • Changes affected multiple functional categories including metabolism, cell structure, and virulence

• Exoproteome analysis showed:

  • 82 proteins differentially expressed between wild-type and ΔentD mutant

  • Expression changes were largely growth phase-dependent

  • Only 12 proteins were downregulated and 10 upregulated across all growth phases

Using a label-free proteomic strategy on triplicate biological samples, researchers can identify protein expression changes resulting from gene knockout. Statistical significance should be evaluated using Student's t-test with p < 0.05 considered significant .

What metabolic pathways are affected by virulence-associated proteins in Bacillus cereus?

Virulence-associated proteins can significantly impact central metabolism. For EntD knockout:

• Glycolysis: ΔentD showed decreased abundance of proteins in upper glycolysis (Glk, Pgi) and increased abundance in lower glycolysis (GapA, Eno, Pyk)

• Pyruvate metabolism: All components of the pyruvate dehydrogenase complex (PDC) showed decreased abundance, affecting conversion of pyruvate to acetyl-CoA

• TCA cycle: Citrate synthase increased while fumarate dehydrogenase and malate dehydrogenase decreased, suggesting altered cycle dynamics

• Pentose phosphate pathway: Five proteins showed significant changes, with deoxyribose-phosphate aldolase (DeoC) strongly increased (log2 = 6.6, p < 0.001)

These metabolic changes correlate with phenotypic observations, such as decreased growth rate (approximately 50%) and reduced acetate overflow in the ΔentD mutant .

How do Bacillus cereus proteins contribute to virulence through regulation of toxin production?

B. cereus proteins can significantly impact virulence by regulating toxin production. In the EntD study:

• Exoproteome analysis revealed:

  • None of the 3 Nhe (Non-hemolytic enterotoxin) components were detected in the ΔentD exoproteome

  • CytK (Cytotoxin K) and Hbl (Hemolysin BL) components showed significantly decreased abundance, especially during late exponential and stationary growth phases

  • RT-PCR confirmed that mRNA levels of nhe were strongly decreased (log2 = -16) compared to cytK and hbl (log2 = -0.5)

• Cytotoxicity assays using differentiated Caco-2 cells demonstrated:

  • Reduced cytotoxic effects of ΔentD culture filtrates

  • Over 50% reduction in cell viability with wild-type late exponential and stationary filtrate supernatants, which was not observed with ΔentD filtrates

What techniques can be used to generate and validate gene knockout mutants in Bacillus cereus?

Creating knockout mutants is essential for functional studies. The following methodology was used for EntD knockout:

• PCR amplification of the target gene region:

  • Using primers designed to flank the gene of interest

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

• Insertion of an antibiotic resistance cassette:

  • Digesting the plasmid with appropriate restriction enzymes (e.g., PsiI)

  • Ligating a resistance cassette (e.g., spectinomycin resistance) into the digested site

• Double crossover for allelic exchange:

  • Transferring the construct to a temperature-sensitive shuttle vector (e.g., pMAD)

  • Introducing the plasmid into B. cereus by electroporation

  • Selecting for double crossover events

• Validation of the knockout:

  • PCR verification with primers flanking the insertion site

  • RT-PCR to confirm absence of target gene transcription

  • Proteomic verification of protein absence in cellular and extracellular fractions

What approaches can be used to characterize growth and phenotypic changes in Bacillus cereus mutants?

Phenotypic characterization is crucial for understanding protein function:

• Growth kinetics analysis:

  • Measure growth rate (μmax) in defined media

  • Determine biomass and metabolite production

  • Compare aerobic and anaerobic growth conditions

• Cellular morphology examination:

  • Transmission electron microscopy (TEM) of ultrathin sections

  • Negative staining for flagella visualization

  • Analysis of cell size, shape, and subcellular structures

• Autolytic activity measurement:

  • Harvesting cells at specific growth phases

  • Monitoring optical density decrease over time

  • Expressing results as percentage decrease of OD600 after defined periods (e.g., 72h)

• Motility assessment:

  • Microscopic observation of flagella

  • Quantitative motility assays

Statistical differences between wild-type and mutant strains should be evaluated using appropriate tests (e.g., Student's t-test) .

How can proteomic approaches be optimized for studying Bacillus cereus cellular and extracellular proteins?

Optimized proteomic methodology for B. cereus includes:

• Sample preparation:

  • Collect samples from multiple growth phases (early exponential, late exponential, stationary)

  • Prepare cellular proteome from cells harvested at specific growth rates

  • Prepare exoproteome from culture filtrates

• MS/MS analysis parameters:

  • Employ label-free proteomic strategy on triplicate biological samples

  • Process tens of thousands of MS/MS spectra (e.g., 41,998 for ΔentD and 52,169 for wild-type)

  • Identify proteins with a minimum of two peptides for confidence

• Data analysis:

  • Categorize proteins into functional groups

  • Determine significant abundance changes (p < 0.05) based on spectral counts

  • Calculate log2 ratios of protein abundance between strains

  • Validate results with RT-PCR for selected genes

• Functional correlation:

  • Map changed proteins to metabolic pathways

  • Correlate proteomic changes with phenotypic observations

  • Generate pathway diagrams showing protein changes in context

How can complementation experiments be designed to confirm gene function in Bacillus cereus?

Complementation is essential to confirm that observed phenotypes result from the specific gene knockout:

• Amplification of the intact gene:

  • Design primers to capture the entire gene with upstream and downstream regions

  • PCR amplify the region from wild-type genomic DNA

• Construction of complementation vector:

  • Clone the amplified fragment into a suitable vector (e.g., pHT304)

  • Verify sequence integrity by sequencing

• Introduction into mutant strain:

  • Transform the mutant with the complementation vector

  • Select transformants with appropriate antibiotics

• Validation of complementation:

  • Confirm gene expression by RT-PCR

  • Verify protein production by MS/MS

  • Assess restoration of wild-type phenotype

What cytotoxicity assays can be used to evaluate the virulence of Bacillus cereus strains?

Cytotoxicity assays provide valuable insights into bacterial virulence:

• Cell model selection:

  • Differentiated Caco-2 cells mimic gastrointestinal epithelium

  • Other cell lines may be chosen based on target tissue relevance

• Sample preparation:

  • Collect culture filtrate supernatants from different growth phases

  • Standardize protein concentrations across samples

• Cytotoxicity measurement:

  • MTT cell viability/metabolic assay

  • Incubate cells with bacterial filtrates (e.g., 100 μg proteins for 24 hours)

  • Measure cell viability reduction compared to controls

• Data analysis:

  • Express results as percentage of cell viability reduction

  • Compare effects across growth phases and strains

  • Correlate with toxin protein levels identified in proteomic analysis

This approach allowed researchers to demonstrate that wild-type B. cereus filtrates caused more than 50% reduction in Caco-2 cell viability, while ΔentD filtrates showed significantly reduced cytotoxicity .

How can sequence similarity analysis be used to predict functions of uncharacterized Bacillus cereus proteins?

Sequence similarity analysis is a powerful approach for predicting protein function:

• BLAST analysis against related species:

  • Compare amino acid sequences with other B. cereus group strains

  • Identify homologous proteins with known functions

  • Analyze conservation patterns across species

• Multiple sequence alignment:

  • Align protein sequences of homologs (e.g., EntA, EntB, EntC, and EntD)

  • Identify conserved domains and residues

  • Determine sequence identity percentages (e.g., EntD shares 69%, 63%, and 65% sequence identity with EntA, EntB, and EntC, respectively)

• Domain architecture analysis:

  • Identify functional domains using protein family databases

  • Compare domain organization across related proteins

  • Predict functional similarities based on shared domains

• Phylogenetic analysis:

  • Construct phylogenetic trees of protein families

  • Determine evolutionary relationships

  • Infer potential functions based on closely related proteins

How do mutations in one protein affect the expression and function of related proteins in Bacillus cereus?

The interplay between related proteins provides insights into functional redundancy and compensation:

• Compensatory mechanisms:

  • When EntD was knocked out, the abundance levels of related proteins EntA and EntC significantly increased in late exponential and stationary phase exoproteomes (log2 > 0.8, p < 0.05)

  • This suggests partial functional compensation among family members

• Co-regulation patterns:

  • EntB was not detected in the ΔentD culture filtrate

  • This indicates potential co-regulation between EntD and EntB expression

• Impact on virulence factors:

  • Absence of EntD affected multiple toxin systems (Nhe, Hbl, CytK)

  • Suggests a regulatory network linking Ent family proteins with virulence factors

• Growth phase-dependent effects:

  • Different proteins were affected at different growth phases

  • Only a small subset of proteins showed consistent up- or down-regulation across all growth phases