Recombinant Bacillus cereus UPF0754 membrane protein BC_0879 (BC_0879)

What is the basic characterization of Bacillus cereus UPF0754 membrane protein BC_0879?

The UPF0754 membrane protein BC_0879 is a 378-amino acid membrane protein from Bacillus cereus strain ATCC 14579. Its amino acid sequence suggests a predominantly hydrophobic profile consistent with integral membrane proteins, featuring multiple transmembrane regions. The full amino acid sequence is:

MNIWLNMLTTTGLGAIIGGYTNHLAIKKMLFRPHRPIYIGKFQVPFTPGLIPKRRDELAVQLGKMVVEHLLTPEGIGIITNEQSLRHMLEKNVAHVEEEATRKIEHVITEKIHAFLAEYYTYTWEQALPHSVNEKVENAIPNVSAFILERGISFFESEEGKARLSKMIDDFFASRGTLLNLVGMFLGNVSVVDRVQPEVIKFLGQDATKQLLTDVLQKEWEKLKGRDVKELEAFVEKEMIVSSVLSAVKVEETVSKFLNQSVQQVCEPVRETIIEKVVPSAVAKGLKWGTENVESILNNLHLAEIVQQEVSTFSTERLEDLVLSITKNELKMITYLGALLGGMIGLVQGLLLLFLR

The protein belongs to the UPF0754 family of uncharacterized proteins, with a predicted molecular weight of approximately 42 kDa based on its amino acid composition.

What expression systems are recommended for producing recombinant BC_0879?

For recombinant expression of BC_0879, researchers should consider:

• E. coli expression systems: For initial studies, BL21(DE3) strains with pET or pBAD vectors can be used with appropriate fusion tags (His6, MBP, or SUMO) to enhance solubility.

• B. subtilis expression systems: For more native-like post-translational modifications, Bacillus-based expression systems may provide advantages when expressing Bacillus proteins.

• Cell-free expression systems: For difficult membrane proteins, cell-free systems supplemented with lipid nanodiscs or detergents can improve yields.

Methodology should include optimization of induction parameters (temperature, inducer concentration, and duration) specifically for membrane proteins. Lower temperatures (16-20°C) and longer induction periods often yield better results for membrane proteins compared to standard protocols. Expression levels should be monitored using Western blot analysis with antibodies against the fusion tag, similar to methods used in tracking GerRB-SGFP2 fusion proteins in B. cereus studies .

What purification strategies work best for BC_0879?

Purification of BC_0879 membrane protein requires specialized approaches:

• Membrane extraction: Isolate membrane fractions using differential centrifugation followed by selective detergent solubilization. Test multiple detergents (DDM, LDAO, or Triton X-100) at various concentrations to optimize solubilization while maintaining protein structure.

• Affinity chromatography: For His-tagged constructs, use immobilized metal affinity chromatography (IMAC) with careful detergent maintenance throughout all buffers.

• Size exclusion chromatography: Critical for removing aggregates and ensuring monodispersity, using buffer conditions that maintain protein stability.

Protein purity should be assessed using SDS-PAGE and Western blotting, while functionality can be validated through activity assays or binding studies as appropriate. This approach aligns with methodologies used for other B. cereus membrane proteins where detergent selection significantly impacts protein stability .

How can researchers assess the structural characteristics of BC_0879?

Structural characterization of BC_0879 should follow a multi-technique approach:

• Secondary structure analysis:

  • Circular Dichroism (CD) spectroscopy in the far-UV range (190-260 nm)

  • FTIR spectroscopy for complementary secondary structure information

• Tertiary structure predictions:

  • Computational modeling using homology-based approaches

  • Ab initio modeling for regions lacking homologous templates

• Experimental structure determination:

  • X-ray crystallography (challenging for membrane proteins)

  • Cryo-electron microscopy for higher molecular weight complexes

  • NMR spectroscopy for dynamic regions

For initial assessment, researchers can use transmembrane prediction algorithms (TMHMM, Phobius) to identify potential membrane-spanning regions. The protein's predominantly hydrophobic profile with alternating hydrophilic segments suggests multiple transmembrane domains typical of integral membrane proteins. This is similar to analytical approaches used for other B. cereus membrane proteins .

What is the putative role of BC_0879 in B. cereus physiology and virulence?

While the specific function of BC_0879 remains uncharacterized, several approaches can elucidate its role:

• Gene knockout studies: Creating a Δbc_0879 mutant using methods similar to those employed for the ΔentD mutant would allow comprehensive phenotypic characterization including:

  • Growth kinetics in various media

  • Cell morphology analysis using transmission electron microscopy

  • Stress response assays (oxidative, osmotic, acid)

  • Virulence factor production

  • Biofilm formation capacity

• Transcriptomic analysis: RNA-Seq comparing wild-type and Δbc_0879 mutant would identify genes with altered expression, potentially revealing pathways involving BC_0879.

• Proteomics comparison: Similar to the EntD study , comparative proteomics of cellular and exoproteome fractions between wild-type and mutant strains could identify interacting proteins and affected pathways.

Given its membrane localization, BC_0879 may participate in:

• Transport processes

• Signal transduction

• Cell wall synthesis

• Spore formation

• Stress response mechanisms

A systematic analysis of these possibilities using targeted assays would help establish its functional role within B. cereus biology.

How does BC_0879 compare to homologous proteins in other Bacillus species?

Comparative analysis of BC_0879 with homologs requires:

• Homology identification:

  • BLAST searches against non-redundant protein databases

  • Profile-based searches using HMM models of the UPF0754 family

• Multiple sequence alignment:

  • Identification of conserved residues and motifs

  • Evolutionary analysis using phylogenetic trees

• Functional conservation testing:

  • Cross-species complementation studies

  • Domain swapping experiments

A preliminary analysis indicates that homologs exist across the Bacillus genus with varying degrees of conservation. Proteins with similar domain architecture may be found in B. subtilis, B. anthracis, and B. thuringiensis, potentially with shared functions. Conservation patterns could provide insights into functional regions, similar to how germinant receptor proteins show conservation across Bacillus species with retention of functional domains .

What methods can be used to investigate protein-protein interactions involving BC_0879?

For membrane proteins like BC_0879, specialized approaches for protein-protein interaction studies include:

• Co-immunoprecipitation with membrane solubilization:

  • Use crosslinking agents suitable for membrane proteins (DSS, BS3)

  • Employ gentle detergent solubilization

  • Validate with reverse co-IP

• Bacterial two-hybrid systems adapted for membrane proteins:

  • BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system

  • Split-ubiquitin membrane yeast two-hybrid system

• Proximity-based labeling:

  • BioID or TurboID fusions expressed in B. cereus

  • APEX2-based proximity labeling

  • Mass spectrometry identification of labeled proteins

• Fluorescence-based methods:

  • Förster resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Fluorescence microscopy co-localization studies

The identification of interaction partners can provide significant insights into function, similar to how GerR protein interactions help understand germinosome complex formation in B. cereus spores . When designing fusion constructs, researchers should consider both N- and C-terminal fusions, as membrane topology may affect accessibility.

What approaches can determine the subcellular localization and dynamics of BC_0879?

Determining the precise subcellular localization of BC_0879 requires:

• Fluorescent protein fusions:

  • C-terminal or N-terminal SGFP2 fusions (depending on topology)

  • Expression under native promoter

  • Live-cell imaging with appropriate membrane stains

• Immunolocalization:

  • Generation of specific antibodies against BC_0879

  • Immuno-electron microscopy for high-resolution localization

  • Immunofluorescence microscopy with membrane markers

• Membrane fractionation:

  • Separation of inner and outer membrane fractions

  • Western blot analysis of fractions

  • Activity assays in specific fractions

• Dynamic studies:

  • Fluorescence recovery after photobleaching (FRAP)

  • Single-molecule tracking

  • Super-resolution microscopy

These approaches can determine whether BC_0879 localizes uniformly throughout the membrane or concentrates in specific microdomains or complexes. Drawing from established protocols used to visualize germination proteins in B. cereus , researchers should optimize fixation conditions specifically for membrane proteins and include appropriate controls to distinguish specific from background signals.

How can researchers address challenges in functional characterization of an uncharacterized membrane protein like BC_0879?

Functional characterization of uncharacterized membrane proteins presents unique challenges that can be addressed through:

• Phenotypic microarrays:

  • Compare wild-type and knockout strains across hundreds of growth conditions

  • Identify conditions where the protein becomes essential

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and mutant strains

  • Identify metabolic pathways affected by the protein's absence

• Transporter function testing:

  • Liposome reconstitution with purified protein

  • Substrate screening using fluorescent reporters

  • Electrophysiological measurements if applicable

• Structure-guided functional studies:

  • Identify potential binding pockets or catalytic sites

  • Site-directed mutagenesis of conserved residues

  • Activity assays with predicted substrates

• Systematic interaction mapping:

  • Protein-protein interaction network analysis

  • Integration with known pathways in B. cereus

An integrated approach combining multiple lines of evidence is often necessary for functionally annotating uncharacterized membrane proteins. This strategy has proven effective in characterizing other B. cereus proteins such as EntD, where proteomic approaches identified its role in numerous cellular processes including metabolism, cell structure, oxidative stress response, and motility .

What strategies can overcome low expression yields of recombinant BC_0879?

Low expression yields are common with membrane proteins and can be addressed through:

• Codon optimization:

  • Adjust codons to match expression host preferences

  • Remove rare codons that might cause translational pausing

• Expression strain optimization:

  • Test strains with enhanced membrane protein expression (C41/C43)

  • Consider strains with reduced proteolytic activity

• Fusion partner screening:

  • Test multiple fusion tags (His, MBP, SUMO, Trx)

  • Evaluate both N-terminal and C-terminal tag positions

• Expression parameter optimization:

  Parameter	Variables to Test	Typical Optimal Conditions
  Temperature	16°C, 20°C, 25°C, 30°C	16-20°C for membrane proteins
  Inducer concentration	0.1-1.0 mM IPTG	0.1-0.2 mM IPTG
  Media	LB, TB, 2xYT, M9	TB or 2xYT with supplements
  Induction time	4h, 8h, 16h, 24h	Overnight at lower temperatures
  Additives	Glycerol, sucrose, betaine	0.5-1% glycerol often helpful

• Alternative expression systems:

  • Cell-free systems with added lipids/detergents

  • Mammalian or insect cell expression for complex proteins

Experience from expressing other B. cereus membrane proteins suggests that lowering expression temperature and extending induction time significantly improves the yield of correctly folded protein .

How can researchers validate the proper folding and functionality of recombinant BC_0879?

Validating proper folding and functionality requires multiple approaches:

• Physical characterization:

  • Size-exclusion chromatography to assess monodispersity

  • Thermal stability assays (DSF/nanoDSF) with various buffers and additives

  • Circular dichroism to confirm secondary structure content

• Ligand binding assays:

  • Thermal shift assays with potential ligands

  • Microscale thermophoresis for binding studies

  • Surface plasmon resonance with immobilized protein

• Functional reconstitution:

  • Liposome reconstitution for transport or activity assays

  • Proteoliposome-based functional tests

  • Complementation of knockout phenotypes

• Structural integrity assessment:

  • Limited proteolysis to assess compact folding

  • Negative-stain electron microscopy

  • Native PAGE analysis

Proper folding validation is essential before proceeding to functional studies, as misfolded membrane proteins often show non-specific effects or aggregation. This approach aligns with methods used to validate the functionality of other B. cereus membrane proteins .

What considerations are important when designing site-directed mutagenesis experiments for BC_0879?

When designing mutagenesis experiments for BC_0879:

• Target selection based on:

  • Sequence conservation across homologs

  • Predicted functional motifs or domains

  • Membrane topology predictions

  • Charged residues within transmembrane regions (often functionally important)

• Mutation strategy:

  • Conservative substitutions for initial functional mapping

  • Alanine scanning of predicted functional regions

  • Charge reversal for electrostatic interaction sites

  • Cysteine substitutions for accessibility studies

• Experimental validation:

  • Expression level and localization checks for each mutant

  • Stability assessment to distinguish folding from functional effects

  • Comprehensive phenotypic characterization

  • Structural impact assessment where possible

• Systematic approach:

  Mutation Type	Purpose	Examples
  Alanine substitutions	Remove side chain without affecting backbone	R120A, D245A
  Conservative substitutions	Maintain chemical properties	K→R, D→E, L→I
  Non-conservative substitutions	Test importance of specific properties	K→E, D→K, G→P
  Truncations	Define essential regions	C-terminal truncations
  Domain swapping	Test functional conservation	Replace domains with homologous sequences

This structured approach to mutagenesis has proven effective in characterizing functional regions in other membrane proteins from B. cereus .

How does BC_0879 research relate to studies on B. cereus pathogenicity and virulence?

While direct connections between BC_0879 and virulence remain to be established, researchers should consider:

• Pathogenicity context:

  • B. cereus causes food poisoning and opportunistic infections

  • Membrane proteins often contribute to adhesion, invasion, and toxin secretion

  • Potential role in adaptation to host environments

• Integration with known virulence mechanisms:

  • Investigate relationships with known secretion systems

  • Assess impacts on toxin production or regulation

  • Examine potential roles in adhesion or biofilm formation

• Infection model studies:

  • Compare wild-type and Δbc_0879 strains in infection models

  • Evaluate tissue adhesion, invasion, and persistence

  • Assess immune response modulation

• Comparative studies with clinical isolates:

  • Sequence variation analysis across pathogenic strains

  • Expression level assessment during infection

  • Correlation with virulence profiles

Similar to how EntD was found to regulate multiple virulence-associated functions in B. cereus, including metabolism, cell structure, antioxidative ability, cell motility, and toxin production , BC_0879 may play multifunctional roles in bacterial physiology that collectively contribute to pathogenicity.

How might BC_0879 relate to B. cereus spore formation and germination processes?

Connections between BC_0879 and sporulation/germination could be explored through:

• Expression pattern analysis:

  • Temporal expression profiling throughout sporulation stages

  • Spatial localization during spore formation

  • Expression changes during germination

• Phenotypic characterization of mutants:

  • Sporulation efficiency of Δbc_0879 strains

  • Spore resistance properties (heat, chemicals, radiation)

  • Germination response to various germinants

• Potential interactions with known sporulation/germination proteins:

  • Co-localization with germinant receptors

  • Protein-protein interaction studies with GerR complex components

  • Investigation of potential germinosome association

• Comparative analysis with spore membrane proteome:

  • Presence in dormant spore inner membrane

  • Dynamic changes during germination

  • Potential structural roles in spore membrane

Recent research has established the existence of germinosomes (clusters of germination proteins) in B. cereus spores , and as a membrane protein, BC_0879 might interact with these complexes or play complementary roles in spore biology.

What bioinformatic approaches can predict functional partners and pathways for BC_0879?

Advanced bioinformatic analyses can provide functional insights:

• Genomic context analysis:

  • Examination of gene neighborhood conservation

  • Operon structure prediction

  • Regulatory motif identification in promoter regions

• Co-expression network analysis:

  • Mining transcriptomic datasets for co-expressed genes

  • Correlation analysis across various conditions

  • Identification of expression clusters

• Protein-protein interaction prediction:

  • Structure-based docking with potential partners

  • Coevolution analysis to identify interacting surfaces

  • Interface prediction algorithms

• Pathway enrichment and functional clustering:

  • Gene Ontology enrichment of predicted partners

  • KEGG pathway mapping

  • Protein domain co-occurrence patterns

• Cross-species functional inference:

  • Phenotype analysis of homologs in model organisms

  • Literature mining for functional information on homologs

  • Conservation pattern analysis across bacterial phyla

Integration of multiple bioinformatic approaches with experimental validation has proven effective for characterizing previously unknown proteins in B. cereus, as demonstrated in the EntD study .

What emerging technologies offer new opportunities for studying BC_0879?

Cutting-edge technologies that can advance BC_0879 research include:

• AlphaFold2 and other AI-driven structure prediction:

  • Generate high-confidence structural models

  • Identify potential ligand-binding sites

  • Guide rational mutagenesis and functional studies

• Cryo-electron tomography:

  • Visualize BC_0879 in its native membrane environment

  • Identify associated protein complexes

  • Determine in situ structural arrangement

• Native mass spectrometry:

  • Analyze intact membrane protein complexes

  • Identify associated lipids and small molecules

  • Determine stoichiometry of interaction partners

• CRISPR-based approaches:

  • CRISPRi for controlled gene repression

  • CRISPR-based imaging of genomic loci

  • High-throughput screening of genetic interactions

• Single-cell techniques:

  • Single-cell RNA-seq for heterogeneity assessment

  • Microfluidics-based single-cell analysis

  • Super-resolution microscopy for localization studies

These emerging technologies can overcome traditional challenges in membrane protein research and provide unprecedented insights into the structure, function, and interactions of BC_0879.

How should researchers approach comprehensive functional characterization of BC_0879?

A systematic approach to BC_0879 functional characterization should include:

• Hierarchical experimental design:

  • Begin with bioinformatic predictions to guide hypotheses

  • Perform genetic knockout studies for phenotypic assessment

  • Conduct targeted biochemical and structural experiments

  • Develop comprehensive interaction maps

  • Validate in physiologically relevant conditions

• Multi-disciplinary integration:

  • Combine genetic, biochemical, and structural approaches

  • Integrate systems biology data (transcriptomics, proteomics)

  • Apply computational modeling to synthesize diverse data

  • Validate predictions through focused experiments

• Collaborative framework:

  • Establish collaborations across specialties

  • Share reagents and methodologies

  • Develop standardized protocols for reproducibility

  • Create centralized data repositories

• Translational perspectives:

  • Evaluate potential as a drug target or diagnostic marker

  • Assess conservation in clinical isolates

  • Investigate interactions with host factors

  • Consider biotechnological applications