Recombinant Bacillus cereus Putative UPF0397 protein BC_2624 (BC_2624)

What is the basic structure and characteristics of UPF0397 protein BC_2624?

UPF0397 protein BC_2624 is a small, 87-amino acid transmembrane protein from Bacillus cereus with the sequence: MNKLSTKLVVAIGIGAALYGILGLWGFSIAPNTFIKPALAILTVFGALFGPVAGLLIGLI GHTVTDTIAGWGIWWGCYPTLLNCPLH . The protein contains hydrophobic regions consistent with membrane-spanning domains, suggesting it functions as an integral membrane protein. Secondary structure predictions indicate alpha-helical transmembrane segments with short interconnecting loops. The protein belongs to the UPF0397 family, a group of uncharacterized proteins found across various bacterial species, particularly in gram-positive bacteria like Bacillus. While its exact function remains to be fully elucidated, sequence analysis suggests potential roles in membrane organization, small molecule transport, or stress response mechanisms.

How is recombinant BC_2624 protein typically prepared for research applications?

Recombinant production of BC_2624 protein typically employs E. coli expression systems with N-terminal His-tags to facilitate purification . The general methodology involves: (1) Cloning the BC_2624 gene into a suitable expression vector with a His-tag sequence; (2) Transforming into an E. coli expression strain optimized for membrane protein production (such as C41/C43 or BL21); (3) Expression optimization through varying temperature, induction conditions, and media formulations; (4) Cell disruption through sonication or high-pressure homogenization; (5) Membrane fraction isolation via differential centrifugation; (6) Detergent solubilization of membrane proteins; (7) Immobilized metal affinity chromatography (IMAC) purification; and (8) Final purification through size exclusion chromatography. The purified protein is typically provided as a lyophilized powder for stability and can be reconstituted in appropriate buffers containing stabilizing agents like trehalose before experimental use .

What approaches can be used to elucidate the structural properties of BC_2624 and how do they complement each other?

Elucidating the structural properties of BC_2624 requires multiple complementary techniques due to its membrane protein nature. A comprehensive structural investigation would include: (1) Circular dichroism (CD) spectroscopy to determine secondary structure composition and stability under varying conditions; (2) Nuclear magnetic resonance (NMR) spectroscopy, particularly suited for small membrane proteins like BC_2624, using isotope-labeled protein reconstituted in detergent micelles or nanodiscs; (3) X-ray crystallography following successful crystallization trials with various detergents and lipidic cubic phase approaches; (4) Cryo-electron microscopy for visualization in near-native membrane environments; (5) Molecular dynamics simulations based on homology models or experimental structures to predict dynamic behavior; and (6) Cross-linking mass spectrometry to identify potential interaction surfaces.

Each method provides distinct but complementary information: CD gives rapid assessment of secondary structure content; NMR provides atomic-level information on structure and dynamics in solution; crystallography offers high-resolution static structures; cryo-EM captures conformational states in membrane-like environments; simulations predict dynamic behavior and conformational changes; and cross-linking MS identifies protein-protein interaction interfaces. Integration of data from these approaches provides the most comprehensive structural understanding of challenging membrane proteins like BC_2624.

How might BC_2624 contribute to Bacillus cereus pathogenicity, and what experimental approaches could test these hypotheses?

While direct evidence connecting BC_2624 to B. cereus pathogenicity is limited, several hypotheses warrant investigation due to the membrane localization of this protein. B. cereus is known to cause food poisoning through enterotoxin production and has shown resistance to multiple antibiotics as demonstrated in studies of ready-to-eat food isolates . To investigate BC_2624's potential role in pathogenicity, researchers could:

• Generate BC_2624 knockout strains and assess changes in:

  • Virulence gene expression (nheABC, hblACD, cytK, entFM, cesB)

  • Biofilm formation capability

  • Resistance to antimicrobial compounds

  • Adherence to epithelial cells

  • Survival in simulated gastrointestinal conditions

  • Toxin secretion efficiency

• Perform comparative proteomics between wild-type and knockout strains under infection-relevant conditions

• Conduct in vivo infection models using knockout vs. wild-type strains in appropriate animal models

• Investigate protein-protein interactions between BC_2624 and known virulence factors using techniques like bacterial two-hybrid systems, co-immunoprecipitation, or proximity labeling

The observed high prevalence of antibiotic resistance in B. cereus isolates, particularly to β-lactams and rifamycin , raises questions about whether membrane proteins like BC_2624 might contribute to this resistance through membrane permeability regulation or interaction with efflux systems.

What bioinformatic approaches are most effective for predicting potential interaction partners of BC_2624?

Predicting interaction partners for BC_2624 requires sophisticated bioinformatic approaches tailored to membrane proteins. An effective strategy would employ multiple complementary methods:

• Co-evolution analysis: Utilizing methods like Direct Coupling Analysis (DCA) or Statistical Coupling Analysis (SCA) to identify residues that show correlated mutations across species, suggesting functional interactions between proteins. This approach can identify evolutionary constraints consistent with protein-protein interfaces.

• Genomic context analysis: Examining gene neighborhood, gene fusion events, and operonic structure across diverse bacterial genomes to identify consistently co-occurring genes that suggest functional relationships.

• Protein-protein interaction network prediction: Employing tools like STRING, STITCH, or PrePPI that integrate multiple evidence sources (experimental data, pathway knowledge, co-expression patterns) to predict interaction probabilities.

• Structural modeling and docking: Generating homology models of BC_2624 and potential partners, followed by protein-protein docking simulations to assess physical compatibility of interaction interfaces.

• Text mining approaches: Using natural language processing algorithms to extract potential interactions from scientific literature.

The integration of these methods through machine learning approaches can significantly improve prediction accuracy. Results should be prioritized based on confidence scores and biological relevance, leading to a ranked list of candidate interacting partners for experimental validation through techniques like bacterial two-hybrid screening, co-immunoprecipitation, or biolayer interferometry.

How might post-translational modifications affect BC_2624 function, and what methodologies can detect these modifications?

Post-translational modifications (PTMs) could significantly influence BC_2624 function through altered protein localization, stability, interaction capabilities, or activity regulation. For bacterial membrane proteins like BC_2624, relevant PTMs might include phosphorylation, methylation, acetylation, lipidation, and disulfide bond formation. These modifications could regulate BC_2624's membrane insertion, oligomerization state, or interaction with other cellular components.

Detection methodologies for PTMs in BC_2624 would include:

• Mass spectrometry (MS) approaches:

  • Bottom-up proteomics: Digestion followed by LC-MS/MS analysis for PTM mapping

  • Top-down proteomics: Analysis of intact protein to preserve modification relationships

  • Targeted MS approaches using multiple reaction monitoring for specific PTMs

  • Enrichment strategies for specific PTMs (e.g., titanium dioxide for phosphopeptides)

• Western blotting with PTM-specific antibodies (e.g., anti-phosphotyrosine)

• Radiolabeling with 32P or 35S to detect phosphorylation or sulfur-containing modifications

• Chemical labeling approaches for specific PTMs (e.g., click chemistry for lipidation)

• 2D gel electrophoresis to separate protein isoforms differing by PTMs

For comprehensive PTM characterization, researchers should culture B. cereus under various physiological conditions relevant to its lifecycle, including environmental stress, simulated host conditions, and biofilm formation, as modifications may be highly condition-dependent. Parallel analysis of recombinant BC_2624 expressed in E. coli would help identify modification differences between native and recombinant systems.

What are the critical considerations for optimizing expression and purification of BC_2624 for structural studies?

Optimizing expression and purification of BC_2624 for structural studies requires careful consideration of multiple parameters due to its membrane protein nature. Key considerations include:

Expression System Optimization:

• Vector selection: Strong but controllable promoters (e.g., T7 or arabinose-inducible) with appropriate fusion tags (His-tag positioning at N- or C-terminus can significantly affect yield)

• E. coli strain selection: Specialized strains like C41/C43(DE3), BL21(DE3)pLysS, or Rosetta for handling membrane proteins and rare codons

• Growth conditions: Temperature (typically lower temperatures of 16-25°C post-induction), media (minimal vs. rich, supplementation with glucose/glycerol), and induction parameters (concentration, timing, duration)

Membrane Extraction and Solubilization:

• Cell disruption method optimization (sonication vs. homogenization vs. French press)

• Detergent screening using a panel including:

  • Mild detergents (DDM, LMNG, DMNG)

  • Zwitterionic detergents (LDAO, FC-12)

  • Novel amphipols or nanodiscs for improved stability

Purification Strategy:

• IMAC optimization: Imidazole concentration gradient, pH optimization, flow rate

• Secondary purification: Size exclusion chromatography buffer optimization containing appropriate detergent concentration just above CMC

• Protein stability assessment using thermal shift assays or size exclusion chromatography with multi-angle light scattering (SEC-MALS)

Quality Control Metrics:

• Purity assessment: SDS-PAGE, Western blotting, mass spectrometry

• Homogeneity assessment: Dynamic light scattering, negative-stain EM

• Functionality tests: Liposome reconstitution, binding assays if ligands are known

The reconstitution buffer composition is critical and should contain stabilizing agents like trehalose (6%) at physiologically relevant pH (7.5-8.0) . For structural studies, additional screening for stabilizing ligands or lipids may be necessary to improve conformational homogeneity.

What are the most effective approaches for studying BC_2624 localization and membrane topology in Bacillus cereus?

Determining the precise localization and membrane topology of BC_2624 in Bacillus cereus requires multiple complementary approaches:

• Fluorescent Protein Fusion Analysis:

  • Creating translational fusions of BC_2624 with fluorescent proteins (GFP, mCherry)

  • Microscopic visualization of localization patterns during different growth phases

  • Time-lapse imaging to track dynamic relocalization during environmental changes

• Membrane Topology Mapping:

  • Cysteine accessibility method: Introducing cysteine residues at various positions and assessing their accessibility to membrane-impermeable sulfhydryl reagents

  • Reporter fusion approach: Creating fusions with reporter enzymes (PhoA, LacZ) whose activity depends on cellular localization

  • Protease protection assays: Limited proteolysis of membrane fractions followed by mass spectrometry to identify protected regions

• Immunolocalization Techniques:

  • Development of specific antibodies against BC_2624

  • Immunogold electron microscopy for ultrastructural localization

  • Fractionation followed by Western blotting to confirm membrane association

• In vivo Cross-linking:

  • Photo-cross-linking with unnatural amino acid incorporation at specific positions

  • Chemical cross-linking followed by mass spectrometry to identify neighboring proteins

• Computational Prediction Validation:

  • Experimental validation of transmembrane segment predictions using the approaches above

  • Comparison of experimental results with predictions from tools like TMHMM, Phobius, or TOPCONS

These approaches should be combined and performed under various growth conditions and stress responses to develop a comprehensive understanding of BC_2624's dynamic localization and orientation within the bacterial membrane system.

What methodologies are recommended for investigating potential protein-protein interactions involving BC_2624?

Investigating protein-protein interactions (PPIs) involving BC_2624 requires specialized approaches suitable for membrane proteins. A comprehensive strategy would include:

• In vivo Approaches:

  • Bacterial Two-Hybrid (BACTH) system: Fusing BC_2624 to T18/T25 fragments of adenylate cyclase and co-expressing with a library of potential partners

  • Split-GFP complementation: Fusing BC_2624 to one GFP fragment and potential partners to the complementary fragment

  • In vivo cross-linking with formaldehyde or photo-cross-linkers followed by co-immunoprecipitation

  • Proximity-dependent biotin labeling (BioID or TurboID fusions) to identify proximal proteins in the native environment

• In vitro Approaches:

  • Pull-down assays using recombinant His-tagged BC_2624 and B. cereus lysates

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) for direct binding measurements

  • Microscale Thermophoresis (MST) for interaction analysis in solution

  • Isothermal Titration Calorimetry (ITC) for thermodynamic characterization of interactions

• Mass Spectrometry-Based Methods:

  • Co-immunoprecipitation coupled with LC-MS/MS identification

  • Chemical cross-linking MS (XL-MS) to identify interaction interfaces

  • Hydrogen-Deuterium Exchange MS (HDX-MS) to map binding interfaces

  • Label-free quantitative proteomics comparing wild-type and BC_2624 knockout strains

• Structural Biology Approaches:

  • Co-crystallization attempts with identified partners

  • NMR chemical shift perturbation experiments

  • Cryo-EM of complexes in membrane mimetics

To evaluate the biological significance of identified interactions, validation studies should include assessing the effect of site-directed mutations at predicted interaction interfaces and phenotypic analyses of interaction-disrupting mutations. When reporting interactions, researchers should provide quantitative metrics (Kd values, enrichment scores) and assess interaction specificity through appropriate controls.

How should researchers interpret evolutionary conservation patterns of BC_2624 across Bacillus species and beyond?

Interpreting evolutionary conservation patterns of BC_2624 requires systematic comparative analysis across bacterial species with consideration of both sequence and structural features. Researchers should:

• Perform comprehensive sequence alignment analysis:

  • Generate multiple sequence alignments (MSAs) of BC_2624 homologs across diverse bacterial species

  • Calculate sequence conservation scores at each position

  • Identify universally conserved residues versus clade-specific variations

  • Map conservation patterns onto predicted structural features (transmembrane regions, loops)

• Conduct phylogenetic analysis in context:

  • Construct phylogenetic trees of UPF0397 family proteins

  • Compare protein-based phylogeny with species phylogeny to identify potential horizontal gene transfer events

  • Analyze co-evolution with functionally related proteins

• Interpret conservation in functional context:

  • Highly conserved residues across distant species likely indicate functional importance

  • Residues conserved only within pathogenic species may suggest virulence-related functions

  • Conservation patterns in transmembrane regions versus loop regions provide insights into structural versus functional constraints

• Apply rate-shift analysis:

  • Identify positions with accelerated or decelerated evolutionary rates

  • Apply statistical models (e.g., PAML, HyPhy) to detect signatures of positive or purifying selection

• Consider genomic context conservation:

  • Analyze conservation of flanking genes and operon structure

  • Identify co-evolved gene clusters that might share functional relationships

Researchers should be cautious about overinterpreting conservation patterns in the absence of functional data. High conservation could indicate essential cellular functions, structural constraints, or selective pressure maintaining specific protein-protein interactions. The amino acid sequence of BC_2624 (MNKLSTKLVVAIGIGAALYGILGLWGFSIAPNTFIKPALAILTVFGALFGPVAGLLIGLI GHTVTDTIAGWGIWWGCYPTLLNCPLH) shows patterns consistent with membrane integration, but functional interpretation requires integration with experimental evidence rather than conservation analysis alone.

How can researchers effectively compare native BC_2624 with recombinant versions to ensure biological relevance?

Ensuring that recombinant BC_2624 accurately represents native protein behavior is critical for research validity. Researchers should implement a systematic comparative analysis approach:

• Structural and biochemical comparison:

  • Compare post-translational modification profiles using mass spectrometry

  • Analyze secondary structure composition using circular dichroism spectroscopy

  • Evaluate thermal stability profiles through differential scanning calorimetry

  • Assess oligomerization states using size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

• Functional equivalence testing:

  • Develop functional assays based on predicted roles (membrane integrity, stress response)

  • Compare activity metrics between native and recombinant proteins

  • Perform complementation studies using recombinant protein in knockout strains

• Localization and interaction validation:

  • Compare membrane integration patterns

  • Verify that recombinant protein maintains the same protein-protein interactions as native protein

  • Use fluorescently tagged versions to compare localization patterns

• Tag influence assessment:

  • Test multiple tag positions (N-terminal, C-terminal) and types

  • Create tag-free versions through precision protease cleavage

  • Quantitatively compare properties of tagged vs. untagged proteins

• Expression system considerations:

  • Compare E. coli-expressed recombinant protein with protein expressed in Bacillus subtilis (closer to native environment)

  • Assess lipid environment effects using different membrane mimetics

What statistical approaches are most appropriate for analyzing BC_2624 knockout phenotypes in various stress conditions?

• Experimental design considerations:

  • Include biological replicates (minimum n=3, preferably n≥5) for each condition

  • Incorporate technical replicates within each biological replicate

  • Design factorial experiments to examine interaction effects between genotype (WT vs. knockout) and environmental conditions

  • Include complementation strains to confirm phenotype specificity

  • Use multiple knockout strains created with different methods to control for off-target effects

• Appropriate statistical tests:

  • For continuous variables (growth rates, survival percentages, enzyme activities):

    • Two-way ANOVA with post-hoc tests (Tukey's HSD or Bonferroni) for multiple condition comparisons

    • Mixed-effects models for time-series data with repeated measures

    • Non-parametric alternatives (Kruskal-Wallis, permutation tests) when normality assumptions are violated

  • For count data (colony-forming units, transcription events):

    • Generalized linear models with appropriate distributions (Poisson, negative binomial)

    • Chi-square or Fisher's exact tests for categorical outcomes

• Advanced analytical approaches:

  • Multivariate analysis for simultaneous examination of multiple phenotypic variables

  • Principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify patterns across numerous measurements

  • Survival analysis methods for time-to-event data

  • Bayesian hierarchical modeling for complex experimental designs

• Effect size reporting:

  • Include measures of effect size (Cohen's d, odds ratios) beyond just p-values

  • Report confidence intervals for all measurements

  • Consider minimum effect sizes of biological significance

• Multiple testing correction:

  • Apply appropriate corrections (Bonferroni, Benjamini-Hochberg) when testing multiple hypotheses

  • Control family-wise error rate or false discovery rate depending on research goals

A typical analysis workflow might begin with descriptive statistics, proceed to appropriate inferential statistics based on data type and distribution, and conclude with multivariate approaches to integrate findings across conditions. For complex phenotypes like biofilm formation or virulence in animal models, researchers should consider consulting with statistical experts during experimental design phases rather than after data collection.

What are the most promising future research directions for understanding BC_2624 function?

The study of BC_2624 remains in its early stages, with several promising research directions that could significantly advance our understanding of this UPF0397 family protein. The most strategic approaches include:

• Comprehensive functional genomics:

  • Generation of BC_2624 knockout, knockdown, and overexpression strains in Bacillus cereus

  • Parallel phenotypic characterization under diverse environmental conditions

  • Genome-wide synthetic genetic interaction mapping to identify functional relationships

  • Transcriptomic and proteomic profiling to identify regulatory networks

• Structural biology initiatives:

  • High-resolution structure determination using X-ray crystallography, NMR, or cryo-EM

  • Structure-guided functional annotation through comparison with other membrane proteins

  • Dynamic structural studies to capture conformational changes under varying conditions

• Systems biology integration:

  • Metabolomic analysis of knockout strains to identify biochemical pathways affected

  • Integration of multi-omics data to position BC_2624 within cellular networks

  • Mathematical modeling of membrane systems incorporating BC_2624 function

• Translational research applications:

  • Investigation of BC_2624 as a potential antimicrobial target, particularly given B. cereus pathogenicity

  • Exploration of biotechnological applications if unique properties are identified

  • Development of diagnostic tools if BC_2624 proves to have species-specific characteristics

• Evolutionary and ecological perspectives:

  • Investigation of BC_2624 function in the context of B. cereus environmental adaptation

  • Comparative studies across various Bacillus species in different ecological niches

  • Exploration of potential horizontal gene transfer events involving UPF0397 family genes

Each of these research directions would benefit from collaborative approaches combining expertise in molecular microbiology, structural biology, bioinformatics, and systems biology. The small size and potential importance of BC_2624 in B. cereus biology, potentially including pathogenicity aspects, make it an attractive target for fundamental research with potential applied outcomes in food safety and antimicrobial development.

How might current knowledge about BC_2624 contribute to broader understanding of bacterial membrane proteins?

The study of BC_2624 offers valuable opportunities to advance our understanding of bacterial membrane proteins in several ways:

• Model system for small membrane protein characterization:

  • At 87 amino acids, BC_2624 represents an excellent model for small bacterial membrane proteins , which are often overlooked in structural and functional studies

  • Its manageable size makes it amenable to comprehensive mutagenesis and structural determination

  • Lessons learned from BC_2624 characterization could establish methodological workflows applicable to other small membrane proteins

• Insights into UPF0397 protein family functions:

  • BC_2624 research contributes to annotating the functions of the entire UPF0397 family

  • Understanding one member in detail could resolve functions for numerous uncharacterized proteins across multiple bacterial species

  • This addresses a significant gap in bacterial proteome annotation

• Membrane biology in Gram-positive pathogens:

  • Characterization of BC_2624 provides insights into membrane organization in B. cereus and related pathogens

  • This information is valuable for understanding bacterial adaptation to environments encountered during infection

  • Potential connections to antimicrobial resistance mechanisms, particularly given B. cereus resistance patterns to β-lactams and rifamycin

• Technical advances in membrane protein research:

  • Optimization of expression, purification, and structural analysis protocols for BC_2624 enhances the technical toolkit for challenging membrane proteins

  • Development of specialized assays for BC_2624 function could be adapted for other membrane proteins

  • Improved computational prediction tools validated against BC_2624 experimental data

• Evolutionary insights:

  • Understanding of how small membrane proteins like BC_2624 evolve provides perspective on membrane proteome adaptation

  • Insights into essential vs. accessory functions in bacterial membrane systems

  • Potential identification of convergent evolution patterns in membrane proteins across diverse bacterial lineages