Recombinant Bacillus cereus Putative ABC transporter ATP-binding protein BCE_2668 (BCE_2668), partial

What is BCE_2668 and what is its functional significance in Bacillus cereus?

BCE_2668 is a putative ATP-binding protein component of an ABC transporter system in Bacillus cereus, specifically identified as a heme ABC transporter ATP-binding protein. ABC transporters are integral membrane proteins that utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. In the case of BCE_2668, its classification as a heme transporter suggests it plays a critical role in iron acquisition, which is essential for bacterial growth and virulence .

Functionally, BCE_2668 likely contributes to B. cereus pathogenicity by facilitating iron uptake from the host environment. B. cereus is a Gram-positive, spore-forming bacterium associated with food poisoning and various non-gastrointestinal infections . The ability to acquire iron effectively through specialized transport systems like the one containing BCE_2668 may enhance the organism's survival in iron-limited host environments, making it a significant factor in virulence.

What are the structural characteristics of ABC transporter ATP-binding proteins like BCE_2668?

ABC transporter ATP-binding proteins such as BCE_2668 are characterized by several conserved motifs that facilitate their function in ATP binding and hydrolysis. These proteins typically contain:

• Walker A and Walker B motifs: Conserved sequences that form the nucleotide-binding site

• ABC signature motif (C-loop): Located between the Walker A and B motifs, this region is unique to ABC transporters

• D-loop, H-loop, and Q-loop: Additional conserved regions involved in the coordination of ATP hydrolysis and the transmission of conformational changes to the transmembrane domains

BCE_2668 likely adopts the characteristic structural fold of ABC transporter nucleotide-binding domains, consisting of two subdomains: a larger RecA-like subdomain containing the Walker A and B motifs, and a smaller helical subdomain containing the ABC signature motif. The nucleotide binds at the interface between these subdomains, and ATP hydrolysis induces conformational changes that are transmitted to the transmembrane domains of the transporter to drive substrate translocation.

The specific sequence variations in these conserved regions may influence the efficiency of ATP binding and hydrolysis, potentially affecting the transport capacity of the complete ABC transporter system in which BCE_2668 functions.

How can researchers effectively express and purify recombinant BCE_2668 for functional studies?

Expressing and purifying recombinant BCE_2668 for functional studies requires careful optimization of expression systems and purification protocols. Based on current methodologies, the following approach is recommended:

Expression System Selection:
Multiple expression hosts can be used for BCE_2668 production, including E. coli, yeast, baculovirus, or mammalian cell systems . E. coli is typically the first choice due to its rapid growth and high protein yields. For BCE_2668, a strain like BL21(DE3) with a pET vector system containing a His-tag for purification would be optimal.

Optimization Protocol:

• Clone the BCE_2668 gene into pET28a(+) vector with an N-terminal His-tag

• Transform into E. coli BL21(DE3)

• Culture in LB medium at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5 mM IPTG

• Shift temperature to 18°C for overnight expression (reduces inclusion body formation)

Purification Strategy:

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM PMSF

• Clarify lysate by centrifugation at 18,000 × g for 45 minutes

• Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Wash with buffer containing 20-40 mM imidazole

• Elute with buffer containing 250 mM imidazole

• Apply size exclusion chromatography for further purification

This approach typically yields protein with ≥85% purity as determined by SDS-PAGE . For functional studies, it's crucial to confirm that the recombinant protein retains ATP-binding and hydrolysis activity using ATPase assays.

What experimental approaches can be used to characterize the ATPase activity of BCE_2668 and identify its role in heme transport?

Characterizing the ATPase activity of BCE_2668 and establishing its role in heme transport requires a multi-faceted experimental approach:

ATPase Activity Assays:

• Colorimetric Phosphate Detection: Measure inorganic phosphate release using malachite green or molybdate-based assays to quantify ATP hydrolysis rates.

• Coupled Enzyme Assays: Link ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase, monitoring absorbance decrease at 340 nm.

Experimental Design for ATPase Characterization:

Heme Transport Characterization:

• Reconstitution Experiments: Incorporate purified BCE_2668 with other components of the putative heme transporter system into proteoliposomes.

• Fluorescence-based Transport Assays: Use fluorescent heme analogs to monitor transport in real-time.

• Radioactive Heme Uptake: Utilize 55Fe-labeled heme to quantify transport rates.

• Growth Complementation: Express BCE_2668 in a heterologous host with defective heme transport and assess growth rescue in iron-limited conditions.

Interaction Studies:

• Pull-down Assays: Identify partner proteins that form the complete transporter complex.

• Surface Plasmon Resonance: Measure binding kinetics with heme and potential partner proteins.

These approaches would provide comprehensive characterization of BCE_2668's biochemical properties and functional role in heme transport, establishing its contribution to B. cereus iron acquisition mechanisms.

How does the sequence and structure of BCE_2668 compare to homologous proteins in other pathogenic bacteria?

Comparative analysis of BCE_2668 with homologous proteins in other pathogenic bacteria reveals important evolutionary relationships and potential functional conservation:

Sequence Comparison Analysis:
BCE_2668 shares significant sequence homology with ATP-binding proteins of heme ABC transporters in related Gram-positive pathogens. The highest similarity is observed with other members of the Bacillus cereus group, including B. anthracis and B. thuringiensis (typically >90% identity). Moderate homology (40-60% identity) exists with Staphylococcus aureus HtsA and Streptococcus pyogenes SiaA, which are well-characterized heme transport proteins.

Key Conserved Domains Comparison:

This comparative analysis provides insights into the evolutionary history of heme ABC transporters and may guide functional predictions based on characterized homologs in other species.

What are the optimal conditions for expressing recombinant BCE_2668 in different host systems?

The optimal expression conditions for recombinant BCE_2668 vary significantly across different host systems. Each system offers distinct advantages and requires specific optimization parameters:

E. coli Expression System:

• Recommended Strains: BL21(DE3), BL21(DE3)pLysS, or Rosetta(DE3) for rare codon usage

• Vector Choice: pET28a(+) or pET-SUMO for improved solubility

• Induction Parameters: 0.1-0.5 mM IPTG at OD600 0.6-0.8

• Post-Induction Conditions: 18°C for 16-20 hours with shaking at 180 rpm

• Media Supplements: 0.5% glucose to reduce basal expression; 5-10 μM FeSO4 may enhance folding

• Typical Yield: 10-15 mg/L of culture

Yeast Expression System (Pichia pastoris):

• Strain: X-33 or GS115

• Vector: pPICZ containing methanol-inducible AOX1 promoter

• Growth Phase: Grow to OD600 2-6 in buffered glycerol-complex medium

• Induction: 0.5% methanol every 24 hours for 72-96 hours

• Temperature: 25-28°C

• Aeration: High aeration critical (>30% dissolved oxygen)

• Typical Yield: 20-50 mg/L

Baculovirus Expression System:

• Cell Line: Sf9 or High Five insect cells

• Vector: pFastBac with polyhedrin promoter

• Infection Parameters: MOI of 2-5, harvest 72 hours post-infection

• Temperature: 27°C

• Media Supplements: 10% FBS, gentamicin

• Typical Yield: 5-10 mg/L

Mammalian Cell Expression:

• Cell Line: HEK293 or CHO cells

• Vector: pcDNA3.1 with CMV promoter

• Transfection: Lipofectamine or calcium phosphate method

• Selection: G418 for stable cell line development

• Growth Conditions: 37°C, 5% CO2

• Typical Yield: 1-5 mg/L

The choice of expression system should be guided by downstream applications. For structural studies requiring high protein yields, E. coli or yeast systems are preferred. For functional studies where proper post-translational modifications may be important, insect or mammalian expression systems may be more suitable despite their lower yields.

What analytical techniques are most effective for studying BCE_2668 interactions with other ABC transporter components?

Multiple analytical techniques can be employed to effectively study BCE_2668 interactions with other ABC transporter components, each offering distinct advantages for characterizing different aspects of these interactions:

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation (Co-IP):

  • Particularly useful for identifying native interacting partners

  • Requires antibodies specific to BCE_2668 or epitope tags

  • Can be coupled with mass spectrometry for unbiased identification of interactors

• Surface Plasmon Resonance (SPR):

  • Provides real-time, label-free measurement of binding kinetics

  • Can determine kon, koff, and KD values

  • Experimental setup: Immobilize BCE_2668 on a CM5 chip and flow other transporter components over the surface

  • Typical binding affinities between ABC transporter components range from 0.1-10 μM

• Isothermal Titration Calorimetry (ITC):

  • Measures thermodynamic parameters (ΔH, ΔS, ΔG) of binding

  • Requires no labeling or immobilization

  • Typically requires 20-50 μM protein concentrations

  • Provides stoichiometry information

• Microscale Thermophoresis (MST):

  • Measures changes in thermophoretic mobility upon binding

  • Requires minimal protein amounts (nM range)

  • Useful for difficult-to-express components

Structural Characterization of Complexes:

• Crosslinking Mass Spectrometry (XL-MS):

  • Identifies proximal regions between interacting proteins

  • Use reagents like BS3 or DSS for lysine crosslinking

  • Provides distance constraints for modeling

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps interaction interfaces by measuring changes in deuterium uptake

  • Useful for studying conformational changes upon complex formation

  • Requires ~50-100 μg protein per condition

• Cryo-Electron Microscopy:

  • Provides structural information of the entire transporter complex

  • Can capture different conformational states

  • Requires homogeneous sample preparation

Functional Interaction Studies:

• ATPase Stimulation Assays:

  • Measures changes in BCE_2668 ATPase activity when other components are added

  • Key experiment: Titrate transmembrane domains against BCE_2668 and measure phosphate release

• Fluorescence Resonance Energy Transfer (FRET):

  • Real-time measurement of protein interactions

  • Requires fluorescent labeling of components

  • Can be performed in reconstituted systems or in vivo

For optimal results, a combination of techniques should be employed. For instance, initial screening with Co-IP or pull-down assays can identify interaction partners, followed by SPR or ITC for quantitative binding parameters, and structural techniques like XL-MS or Cryo-EM for detailed complex characterization.

What approaches can be used to assess the impact of BCE_2668 mutations on B. cereus virulence and antibiotic resistance?

Assessing the impact of BCE_2668 mutations on B. cereus virulence and antibiotic resistance requires a multidisciplinary approach combining genetic manipulation, phenotypic characterization, and infection models:

Genetic Manipulation Strategies:

• Allelic Exchange Mutagenesis:

  • Generate point mutations or deletions in chromosomal BCE_2668

  • Use temperature-sensitive plasmids (e.g., pMAD) for homologous recombination

  • Create mutations in key motifs: Walker A (K to A), Walker B (E to Q), or ABC signature motifs

• CRISPR-Cas9 Gene Editing:

  • Design sgRNAs targeting BCE_2668

  • Introduce precise mutations via homology-directed repair

  • Verify mutations by sequencing

• Complementation Analysis:

  • Express wild-type or mutant BCE_2668 from a plasmid in deletion strains

  • Use inducible promoters to control expression levels

Phenotypic Characterization:

• Growth Analysis in Iron-Limited Conditions:

  • Compare growth curves of wild-type and mutant strains in media with iron chelators

  • Measure growth rate, lag phase, and maximum OD

  • Test complementation with exogenous heme or iron sources

• Heme Uptake Assays:

  • Use 55Fe-labeled heme or fluorescent heme analogs

  • Quantify uptake rates in wild-type vs. mutant strains

  • Expected result: Mutations in key motifs would reduce uptake by 50-90%

• Antibiotic Susceptibility Testing:

  • Determine MICs for various antibiotics, particularly β-lactams

  • B. cereus typically shows resistance to most β-lactam antibiotics

  • Changes in transporter function may alter membrane permeability and drug susceptibility

Virulence Assessment:

• Toxin Production Analysis:

  • Quantify production of hemolysins, phospholipases, and enterotoxins

  • Use ELISA, Western blotting, or RT-qPCR

  • B. cereus strains commonly possess hblACD, nheABC, and entFM toxin genes

• Cell Culture Infection Models:

  • Assess adhesion, invasion, and cytotoxicity in epithelial and macrophage cell lines

  • Measure LDH release, cytokine production, and cell viability

  • Compare intracellular survival rates

• Animal Infection Models:

  • Galleria mellonella larval model for rapid virulence assessment

  • Mouse peritonitis or gastrointestinal infection models

  • Monitor survival rates, bacterial loads, and histopathology

Data Analysis and Interpretation:

These comprehensive approaches would provide insights into how BCE_2668 contributes to B. cereus pathogenesis and antibiotic resistance, potentially identifying new therapeutic targets.

How can researchers differentiate between the roles of BCE_2668 and other ABC transporters in B. cereus virulence?

Differentiating between the roles of BCE_2668 and other ABC transporters in B. cereus virulence requires strategic experimental design and careful data interpretation:

Comparative Genomics and Transcriptomics Approach:

• Genomic Context Analysis:

  • Examine the operon structure containing BCE_2668

  • Identify co-transcribed genes that may form a functional unit

  • Compare with similar operons in related species

• Transcriptional Profiling:

  • Conduct RNA-Seq under various conditions (iron limitation, host cell exposure)

  • Identify co-regulated ABC transporters

  • Compare expression patterns across multiple B. cereus strains

  • B. cereus strains show significant genetic diversity with 192 different sequence types identified

Functional Discrimination Studies:

• Substrate Specificity Determination:

  • Develop transport assays with labeled potential substrates

  • Compare transport kinetics between different ABC systems

  • Use competition assays to determine substrate preference

• Selective Inhibition:

  • Design specific inhibitors targeting unique structural features of BCE_2668

  • Assess effects on transport activity and virulence

  • Validate specificity using mutant strains

Multi-deletion Strategy:

Data Interpretation Framework:

Statistical Approach for Attributing Virulence Contributions:

• Apply multiple regression models to quantify the contribution of each transporter to virulence phenotypes

• Use principal component analysis to identify patterns of virulence factor expression associated with specific transporters

• Develop network models incorporating transcriptomic and proteomic data to map functional relationships between transporters

Through these approaches, researchers can establish whether BCE_2668 plays a unique and non-redundant role in B. cereus virulence or functions as part of a redundant system with other transporters. This information is crucial for determining its potential as a therapeutic target.

What bioinformatic tools and databases are most valuable for analyzing BCE_2668 structure-function relationships?

A comprehensive bioinformatic analysis of BCE_2668 structure-function relationships requires leveraging multiple specialized tools and databases:

Sequence Analysis Resources:

• UniProt/SwissProt:

  • Primary resource for curated protein annotations

  • Provides functional domains, post-translational modifications, and literature references

  • Access BCE_2668 homologs across bacterial species

• Conserved Domain Database (CDD):

  • Identifies conserved domains like the ABC transporter nucleotide-binding domain

  • Maps functionally important residues within these domains

  • Particularly useful for identifying Walker A, Walker B, and ABC signature motifs

• PFAM:

  • Classifies BCE_2668 within the appropriate ABC transporter family

  • Provides multiple sequence alignments with related proteins

  • Highlights key conserved residues across the family

Structural Analysis Tools:

• AlphaFold2/RoseTTAFold:

  • Generate high-confidence structural models of BCE_2668

  • Particularly valuable in the absence of experimental structures

  • Confidence metrics help identify reliable regions of the model

• PyMOL/UCSF Chimera:

  • Visualize structural models and map functional residues

  • Generate publication-quality structural figures

  • Perform structural alignments with homologous proteins

• CASTp/POCASA:

  • Identify potential binding pockets and cavities

  • Analyze ATP-binding site geometry

  • Discover potential allosteric sites

Evolutionary Analysis Resources:

• ConSurf:

  • Map evolutionary conservation onto protein structure

  • Identify functionally important residues under evolutionary constraint

  • Generate conservation scores for each amino acid position

• PAML/HYPHY:

  • Detect signatures of positive selection

  • Identify residues potentially involved in host adaptation

  • Particularly useful when comparing BCE_2668 across different Bacillus species

Systems Biology Resources:

• STRING Database:

  • Map protein-protein interaction networks involving BCE_2668

  • Identify functional partners based on multiple evidence types

  • Useful for understanding the broader functional context

• SubtiList/BacilluScope:

  • Bacillus-specific genomic databases

  • Provide operon structure and regulatory information

  • Enable comparison across multiple Bacillus species

Integrated Analysis Workflow:

This comprehensive bioinformatic approach provides a strong foundation for generating hypotheses about BCE_2668 function that can be tested experimentally. The integration of sequence, structural, and systems-level analyses offers the most complete picture of how BCE_2668's structure relates to its role in heme transport and B. cereus virulence.

How can contradictory results in BCE_2668 functional studies be reconciled with its predicted role in pathogenicity?

Contradictory results in BCE_2668 functional studies can arise from multiple sources and require careful analysis to reconcile with its predicted pathogenicity role. This systematic approach helps resolve such discrepancies:

Sources of Experimental Contradictions:

• Strain Variation Effects:

  • B. cereus is genetically diverse with 192 different sequence types identified

  • Sequence polymorphisms in BCE_2668 across strains may alter function

  • The genetic background influences phenotypic outcomes of mutations

  Resolution Approach: Perform comparative studies across multiple reference strains and clinical isolates. Sequence BCE_2668 and its operon in each strain to correlate genetic variations with functional differences.

• Redundancy in Transport Systems:

  • Multiple ABC transporters may have overlapping functions

  • Compensatory upregulation of alternative transporters in BCE_2668 mutants

  • Context-dependent essentiality based on available iron sources

  Resolution Approach: Conduct transcriptomic and proteomic analyses of BCE_2668 mutants to identify compensatory changes. Create multiple knockout strains to overcome redundancy.

• Experimental Condition Variables:

  • Iron and heme availability dramatically affects transporter functionality

  • Growth phase influences transporter expression

  • Host-derived signals may regulate transporter activity

  Resolution Approach: Standardize experimental conditions and test across multiple physiologically relevant environments, including serum, tissue models, and varying iron concentrations.

Data Integration Framework:

Advanced Approaches for Resolving Contradictions:

• Time-Resolved Studies:

  • Track BCE_2668 contribution throughout infection progression

  • Measure dynamic changes in expression and activity

  • Identify critical time points for BCE_2668 function

• Single-Cell Analysis:

  • Examine heterogeneity in BCE_2668 expression within bacterial populations

  • Correlate with survival in stressful environments

  • May reveal subpopulation-specific roles

• Structurally-Guided Functional Analysis:

  • Engineer BCE_2668 variants with specific functional alterations

  • Create separation-of-function mutations

  • Distinguish between different biochemical activities

• Systems Biology Integration:

  • Develop mathematical models incorporating BCE_2668 into iron homeostasis networks

  • Simulate effects of perturbations under different conditions

  • Generate testable predictions to resolve contradictions

This comprehensive approach acknowledges that apparent contradictions often reflect our incomplete understanding of biological complexity rather than experimental error. By systematically exploring BCE_2668 function across different genetic backgrounds, environmental conditions, and experimental systems, researchers can develop a more nuanced understanding of its role in B. cereus pathogenicity.

What are the most promising future research directions for BCE_2668 and related ABC transporters in B. cereus?

The study of BCE_2668 and related ABC transporters in Bacillus cereus presents several promising research directions that could significantly advance our understanding of bacterial pathogenesis and potentially lead to new therapeutic approaches. These future directions build upon current knowledge while addressing critical gaps:

Structural Biology Approaches:
The determination of high-resolution structures of BCE_2668 alone and in complex with its transmembrane partners represents a significant opportunity. Cryo-electron microscopy and X-ray crystallography could reveal the molecular mechanisms of ATP binding, hydrolysis, and the conformational changes that drive transport. These structures would provide templates for structure-based drug design targeting this transport system.

Systems-Level Understanding:
Integrating BCE_2668 function into the broader context of B. cereus metabolism and virulence networks remains a critical challenge. Multi-omics approaches combining transcriptomics, proteomics, and metabolomics could map how BCE_2668 activity influences global cellular processes under different environmental conditions, particularly during host infection.

Host-Pathogen Interface Studies:
Investigating how BCE_2668-mediated heme acquisition interacts with host iron-withholding defense mechanisms represents an exciting frontier. The competition between bacterial iron acquisition systems and host nutritional immunity plays a crucial role in determining infection outcomes. Understanding this interface could reveal new strategies for infection control.

Translational Applications:
The development of specific inhibitors targeting BCE_2668 and related transporters could provide new approaches to combat B. cereus infections. The high rate of antibiotic resistance observed in B. cereus isolates (>98.91% resistant to five or more antimicrobials) highlights the urgent need for alternative therapeutic strategies. ABC transporters represent attractive targets due to their essential roles and surface accessibility.