Recombinant Bacillus cereus UPF0754 membrane protein BCA_0919 (BCA_0919)

What is the primary structure of Bacillus cereus UPF0754 membrane protein BCA_0919?

The BCA_0919 protein consists of 378 amino acids with the following sequence: MNIWLSMLTTTGLGAIIGGFTNHLAIKMLFRPHRPMYIGKFQVPFTPGLIPKRRDELAVQLGKMVVEHLLTPEGIGKKLTNEEFQKGLIHWAQVEVGKVMTNEQSLRHMLEKWDVAHVEKEATEKIEQVIIEKIEAFLEEYYTYTWEQALPHSVHEKIENAIPNVSAFILKRATHFFESEEGKSRLSKMIDDFFASRGALLNLVGMFLGNVSVVDRVQPEVIKFLGQDGTKQLLTDVLQKEWEKLKGRDVKELETFVEKEMIVSSILSAVQVEETVSKFLNQSVQQVCEPVRETIIEKVVPGVVTKGLKWGTENVESILNNLHLAEIVQQEVSTFSTERLEDLVLSITKNELKMITYLGALLGGMIGIVQGLLLLFLK . This hydrophobic profile suggests multiple transmembrane domains, consistent with its annotation as a membrane protein. When analyzing primary structure, researchers should pay particular attention to conserved motifs that may indicate functional domains within the UPF0754 protein family.

What expression systems are recommended for BCA_0919 protein production?

E. coli expression systems have been successfully used for the recombinant production of BCA_0919. The protein can be expressed as a full-length construct (amino acids 1-378) with an N-terminal His tag to facilitate purification . For membrane proteins like BCA_0919, researchers should optimize expression conditions including induction temperature (typically lowered to 16-22°C), inducer concentration, and expression duration to maximize proper folding. Alternative expression systems such as Bacillus subtilis might be considered for proteins from Bacillus species to ensure proper post-translational modifications, though E. coli remains the most commonly used system due to its high yield and ease of genetic manipulation.

How should purified BCA_0919 protein be stored for optimal stability?

The purified BCA_0919 protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt . For working stocks, aliquoting is necessary to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity. Storage at 4°C is recommended for up to one week for working aliquots . The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . When preparing for long-term storage, addition of glycerol to a final concentration of 30-50% is recommended to prevent freeze-thaw damage to the protein structure .

What is the recommended reconstitution protocol for lyophilized BCA_0919?

Before opening the vial containing lyophilized BCA_0919, briefly centrifuge to bring the contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and aliquot for storage at -20°C/-80°C . When reconstituting membrane proteins like BCA_0919, gradual addition of the reconstitution buffer with gentle mixing is recommended to prevent protein aggregation. For functional studies, researchers may need to incorporate the protein into artificial membrane systems or suitable detergent micelles to maintain native conformation.

What approaches are most effective for studying BCA_0919 membrane topology?

For determining BCA_0919 membrane topology, researchers should employ a multi-method approach. Computational prediction tools like TMHMM and Phobius can provide initial models based on hydrophobicity analysis of the amino acid sequence. These predictions should be experimentally validated using techniques such as cysteine scanning mutagenesis combined with membrane-impermeable sulfhydryl reagents. PhoA/LacZ fusion reporter systems can also be valuable, where the reporter enzyme activity depends on its cellular localization. For higher resolution structural information, techniques such as cryo-electron microscopy or X-ray crystallography may be employed, though membrane proteins present significant challenges for these methods. In each case, the His tag on the recombinant BCA_0919 protein can serve as a useful topological marker .

How can potential functions of the UPF0754 protein family be experimentally determined?

UPF0754 (Uncharacterized Protein Family 0754) proteins like BCA_0919 lack experimentally determined functions. A comprehensive approach to functional characterization would include:

• Comparative genomics analysis to identify conserved gene neighborhoods and potential functional associations

• Phenotypic characterization of knockout mutants under various stress conditions

• Protein-protein interaction studies using pull-down assays with the His-tagged BCA_0919

• Transcriptomic analysis comparing wild-type and knockout strains

• Metabolomic profiling to identify potential metabolic pathways affected by BCA_0919 deletion

Given the membrane localization, BCA_0919 may function in transport, signaling, or membrane integrity. Researchers should design experiments that test these hypotheses specifically, perhaps using fluorescent substrates to monitor transport activity or membrane potential-sensitive dyes to assess membrane integrity in wild-type versus mutant strains.

What strategies should be employed for studying protein-protein interactions of BCA_0919?

To investigate protein-protein interactions of BCA_0919, researchers can leverage the N-terminal His tag for pull-down assays . Cross-linking studies with membrane-permeable cross-linkers followed by mass spectrometry can identify proteins in close proximity to BCA_0919 in vivo. For more specific interaction studies, techniques such as bacterial two-hybrid systems or split-GFP complementation assays may be employed, though these require careful design for membrane proteins. Co-immunoprecipitation can be performed using anti-His antibodies, followed by mass spectrometry to identify interacting partners. For confirming direct interactions, in vitro techniques such as surface plasmon resonance or microscale thermophoresis can be used with purified proteins. When designing these experiments, researchers should consider the detergent environment needed to maintain BCA_0919 in its native conformation.

How does BCA_0919 expression relate to Bacillus cereus pathogenicity?

While direct evidence linking BCA_0919 to pathogenicity is limited, researchers investigating this relationship should consider:

• Comparing expression levels of BCA_0919 in pathogenic versus non-pathogenic Bacillus strains

• Analyzing BCA_0919 expression during different growth phases and under infection-relevant conditions

• Creating knockout mutants to assess virulence in appropriate model systems

• Examining the effect of BCA_0919 deletion on known virulence factors, such as diarrheal enterotoxin and emetic toxin

The role of membrane proteins in bacterial pathogenesis often involves adhesion, invasion, or toxin secretion. Experimental designs should include adhesion assays to host cells, invasion assays if relevant, and toxin production/secretion measurements comparing wild-type and BCA_0919 mutant strains. Additionally, researchers could investigate whether BCA_0919 is involved in antibiotic resistance mechanisms, as membrane proteins can function in drug efflux systems.

What structural characterization methods are most suitable for BCA_0919?

Structural characterization of membrane proteins like BCA_0919 presents significant challenges. The following methodological approaches are recommended:

For BCA_0919, initial characterization using CD to confirm proper folding after reconstitution is recommended, followed by more advanced techniques if higher resolution structures are required. The His-tagged version of the protein facilitates purification to the high concentrations needed for structural studies. For membrane proteins, incorporation into nanodiscs or lipid cubic phases may improve success rates for structural determination.

What controls should be included when studying BCA_0919 function?

A robust experimental design for studying BCA_0919 function should include several controls:

• Empty vector controls when overexpressing BCA_0919 to account for expression system effects

• Complementation studies with wild-type BCA_0919 in knockout strains to confirm phenotype specificity

• Site-directed mutagenesis of conserved residues to create functionally compromised variants

• Closely related membrane proteins from the same family as specificity controls

• Heat-inactivated BCA_0919 protein for in vitro functional assays

When assessing phenotypes in Bacillus cereus, researchers should be aware that members of the B. cereus group can exhibit strain-to-strain variation . Therefore, multiple clinical or environmental isolates should be tested to ensure reproducibility. Additionally, when using the His-tagged version of BCA_0919 , control experiments with the tag alone or with the tag in a different position should be considered to ensure the tag isn't affecting protein function.

How can I optimize purification protocols for BCA_0919 to maintain native conformation?

Optimizing purification of membrane proteins like BCA_0919 requires careful consideration of detergent selection and buffer conditions. The His-tagged version facilitates purification via immobilized metal affinity chromatography (IMAC) , but several critical factors should be addressed:

• Screen multiple detergents (e.g., DDM, LMNG, CHAPS) at concentrations just above their critical micelle concentration

• Include stabilizing agents such as glycerol (5-10%) and specific lipids that might be required for function

• Maintain pH stability (pH 7.5-8.0 is typically recommended for Bacillus proteins)

• Consider adding protease inhibitors to prevent degradation during purification

• Validate protein folding after each purification step using analytical techniques like size exclusion chromatography

The purification protocol should be validated by functional assays specific to the protein's predicted activity. For membrane proteins without known function, like BCA_0919, circular dichroism can provide evidence of proper folding by confirming the expected high alpha-helical content typical of transmembrane domains.

What approaches can resolve contradictory data in BCA_0919 localization studies?

When facing contradictory data regarding BCA_0919 subcellular localization, researchers should implement a systematic troubleshooting approach:

• Verify antibody specificity for localization studies using knockout strains as negative controls

• Compare multiple fractionation methods to ensure consistency in membrane fraction isolation

• Use orthogonal techniques such as:

  • Fluorescent protein fusions with live-cell imaging

  • Immunogold electron microscopy for high-resolution localization

  • Protease protection assays to determine topology

• Consider growth conditions, as membrane protein localization may change based on environmental factors

Contradictory results often stem from differences in experimental techniques or growth conditions. Researchers should standardize these variables across experiments and consider that BCA_0919 may have dynamic localization depending on cellular state. Additionally, tag position (N-terminal versus C-terminal) can affect localization results, so both configurations should be tested when possible.

How can BCA_0919 research contribute to understanding Bacillus cereus pathogenesis?

Bacillus cereus is known to cause foodborne illnesses through production of enterotoxins and the emetic toxin . Research on BCA_0919 could contribute to understanding pathogenesis through several avenues:

• Investigation of potential involvement in toxin secretion pathways

• Analysis of BCA_0919's role in membrane integrity under host-relevant stress conditions

• Assessment of BCA_0919's contribution to adhesion to host cells or environmental persistence

• Evaluation of BCA_0919 as a potential vaccine candidate if surface-exposed epitopes are identified

Understanding membrane proteins in pathogenic bacteria provides insights into virulence mechanisms and potential therapeutic targets. If BCA_0919 is found to be essential for virulence or survival under specific conditions, it could represent a target for novel antimicrobial development. Researchers should design experiments comparing virulence factor production and secretion between wild-type and BCA_0919 knockout strains under conditions that mimic the host environment.

What comparative genomics approaches can reveal about BCA_0919 function across bacterial species?

Comparative genomics offers powerful insights into potential BCA_0919 functions by examining evolutionary patterns:

• Phylogenetic analysis of UPF0754 family proteins across bacterial species

• Synteny analysis to identify consistently co-occurring genes that might suggest functional relationships

• Analysis of selection pressure on different protein domains to identify functionally constrained regions

• Identification of species that have lost UPF0754 family proteins and analysis of compensatory mechanisms

Researchers should compile orthologs from diverse bacterial species and employ both sequence-based and structure-based alignment methods. Conservation patterns often reveal functional sites, while co-evolution analysis can identify potential interaction partners. Correlation of UPF0754 presence/absence with specific phenotypes or ecological niches across species may provide functional clues that can be experimentally tested in Bacillus cereus.

How should researchers approach BCA_0919 knockout studies?

Genetic knockout studies of BCA_0919 require careful methodological considerations:

• Design efficient allelic exchange protocols specific for Bacillus cereus, which can be transformation-resistant

• Create both clean deletions and insertional inactivations to verify consistent phenotypes

• Include complementation with the wild-type gene to confirm phenotype specificity

• Consider conditional knockouts if BCA_0919 proves essential under standard conditions

• Evaluate phenotypes under various stress conditions relevant to Bacillus cereus ecology and pathogenesis

When analyzing knockout phenotypes, researchers should employ a systems biology approach incorporating transcriptomics, proteomics, and metabolomics to comprehensively characterize cellular changes. Growth assays should be conducted under various media conditions and stresses to identify specific conditions where BCA_0919 becomes important for bacterial fitness. For pathogenesis studies, appropriate infection models should be selected based on the specific aspects of Bacillus cereus virulence being investigated.

What mass spectrometry approaches are most suitable for studying BCA_0919 post-translational modifications?

Membrane proteins like BCA_0919 may undergo various post-translational modifications (PTMs) that affect their function. The following mass spectrometry approaches are recommended:

• Bottom-up proteomics: Enzymatic digestion followed by LC-MS/MS to identify specific modified residues

• Top-down proteomics: Analysis of intact protein to capture the combination of modifications

• Targeted approaches like Selected Reaction Monitoring (SRM) for quantification of specific modified peptides

• Enrichment strategies for specific PTMs (e.g., phosphopeptide enrichment with TiO2 or IMAC)

Sample preparation is critical for membrane proteins, requiring optimization of solubilization conditions. Detergent removal prior to MS analysis can be achieved using methods such as KDS precipitation or detergent-compatible MS approaches. Researchers should consider that PTMs may be substoichiometric or condition-dependent, necessitating analysis under various growth conditions to capture the full range of BCA_0919 modifications.

How can researchers effectively analyze BCA_0919 expression patterns under different growth conditions?

To analyze BCA_0919 expression patterns comprehensively, researchers should employ multiple complementary approaches:

• qRT-PCR for sensitive quantification of mRNA levels across conditions

• Western blotting with anti-His antibodies for protein-level quantification of the recombinant protein

• Proteomics approaches for unbiased quantification in the native context

• Reporter fusions (e.g., BCA_0919 promoter driving luciferase expression) for real-time monitoring

• Single-cell techniques to capture population heterogeneity in expression

Experimental design should include time-course sampling to capture dynamic expression changes and multiple biological replicates to ensure statistical validity. Growth conditions to test should include various nutrient sources, stress conditions (pH, temperature, oxidative stress), and host-relevant environments if studying pathogenesis. Correlation of expression patterns with specific phenotypes can provide insights into function, while comparison with known regulatory networks can place BCA_0919 in a broader cellular context.