Recombinant Bacillus cereus subsp. cytotoxis ATP synthase subunit c (atpE)

What is Bacillus cereus subsp. cytotoxis and how does it relate to other B. cereus group members?

Bacillus cereus subsp. cytotoxis is a thermotolerant member of the Bacillus cereus group that reliably harbors the coding gene for cytotoxin K-1 (CytK-1). This toxin is a highly cytotoxic variant that was initially recovered from a diarrheal foodborne outbreak that caused three fatalities . The B. cereus group encompasses several closely related species including B. cereus, B. anthracis, and B. thuringiensis, all sharing similar genetic characteristics but with distinct virulence profiles.

B. cytotoxicus has notable genetic differences from other B. cereus group members, particularly in its virulence factors. Unlike typical B. cereus strains, B. cytotoxicus lacks the Hbl genes but harbors a novel variant of Nhe genes . Complete genome sequencing of B. cereus strains has revealed unique metabolic pathways not previously identified in the B. cereus group, such as urease and xylose utilization, as well as potential mechanisms for antigenic variability in surface structures .

The cytotoxicity of B. cytotoxicus strains shows significant variation, with some strains displaying high cytotoxicity while others show minimal effects toward cell lines . This variability has prompted research into the specific genetic factors contributing to pathogenicity, with CytK-1 identified as playing a major role in cytotoxicity.

What is the structure and function of ATP synthase subunit c (atpE) in bacteria?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F₀F₁-ATP synthase complex responsible for ATP production in bacteria. This membrane protein forms the c-ring structure within the F₀ portion of the enzyme complex, which is embedded in the bacterial cell membrane. The c-ring plays a crucial role in the rotary mechanism of ATP synthase, participating in proton translocation across the membrane that drives ATP synthesis.

The structure of subunit c typically consists of two transmembrane α-helices connected by a small hydrophilic loop. Multiple c subunits (typically 8-15, depending on the species) assemble into a ring structure that forms the proton channel within the membrane domain. Each c subunit contains a conserved carboxylic acid residue (usually aspartate or glutamate) that is essential for proton binding and translocation during the catalytic cycle.

In bacterial energy metabolism, ATP synthase functions as the final enzyme in the oxidative phosphorylation pathway, utilizing the proton gradient established across the cell membrane to generate ATP. This process is fundamental for bacterial survival, especially under aerobic conditions, making ATP synthase components potential targets for antimicrobial development.

How do researchers identify and characterize ATP synthase genes in bacterial genomes?

Identification and characterization of ATP synthase genes, including atpE, in bacterial genomes typically follows a systematic approach combining bioinformatic and experimental methods. The ATP synthase genes in bacteria are generally organized in an operon structure (atpBEFHAGDC), with atpE encoding the c subunit.

For bioinformatic identification, researchers employ:

• Homology-based searches using BLAST against known atpE sequences from related species

• Identification of conserved motifs characteristic of F-type ATP synthase c subunits

• Analysis of genomic context to locate the complete ATP synthase operon

• Prediction of transmembrane domains and secondary structure features

Experimental characterization typically involves:

• PCR amplification using primers designed from conserved regions

• Cloning and sequencing of the amplified gene

• Expression analysis using RT-PCR or RNA-seq to examine transcription patterns

• Protein detection using antibodies against conserved epitopes

Complete genome sequencing has facilitated the identification of ATP synthase genes in numerous bacterial species, including members of the B. cereus group . Comparative genomic analyses have revealed variations in these genes that may correlate with metabolic adaptations to different ecological niches.

What are the optimal expression systems for recombinant production of B. cytotoxicus atpE?

Expressing recombinant ATP synthase subunit c presents significant challenges due to its hydrophobic nature and membrane-associated properties. Based on successful approaches with similar bacterial membrane proteins, several expression systems offer distinct advantages.

Cell-free protein synthesis systems have emerged as particularly valuable for producing membrane proteins like ATP synthase subunits. These systems allow for the direct application of synthesized proteins in cell-based assays without prior purification steps, as they lack cytotoxic compounds such as endotoxins typically present in bacterial systems . Cell-free systems offer remarkable flexibility, enabling:

• Individual synthesis of subunits in separate reactions

• Co-expression of multiple subunits simultaneously

• Sequential addition of components to study assembly dynamics

• Incorporation of membrane mimetics or microsomes for proper folding

For conventional in vivo expression, E. coli remains the most widely used system, with specialized strains such as C41(DE3) and C43(DE3) designed specifically for membrane protein expression. Key considerations for E. coli expression include:

• Using low induction temperatures (16-25°C) to improve folding

• Employing fusion tags (MBP, SUMO) to enhance solubility

• Adding detergents or lipids to the culture medium

• Using weak promoters to prevent toxic accumulation

For challenging membrane proteins like atpE, the choice of expression system should be guided by the specific research objectives and downstream applications.

What purification strategies are most effective for recombinant ATP synthase subunit c?

Purifying recombinant ATP synthase subunit c requires specialized approaches due to its hydrophobic nature and tendency to form oligomeric structures. A systematic purification strategy should include:

1. Membrane extraction and solubilization:

• Isolate membrane fractions through differential centrifugation

• Solubilize using mild detergents such as n-dodecyl-β-D-maltoside (DDM), LDAO, or digitonin

• Maintain detergent concentration above critical micelle concentration (CMC)

2. Affinity chromatography:

• Utilize N- or C-terminal affinity tags (His6, Strep-tag II)

• Perform binding and washing steps in the presence of detergent

• Consider on-column detergent exchange if needed

3. Additional purification steps:

• Size exclusion chromatography to separate monomeric and oligomeric forms

• Ion exchange chromatography for further purification

• Consider detergent screening to optimize stability

When working with cell-free synthesized proteins, the translation mixture can be fractionated into soluble and microsomal fractions, simplifying initial purification steps . This approach has been successful for other multi-component bacterial proteins, allowing separation of membrane-associated forms from soluble variants.

For structural studies, consider transitioning from detergents to more stabilizing environments such as nanodiscs, amphipols, or reconstitution into liposomes. These approaches better mimic the native membrane environment and can improve protein stability and functionality for downstream analyses.

How can researchers verify the proper folding and functionality of recombinant atpE?

Verifying the proper folding and functionality of recombinant ATP synthase subunit c requires multiple complementary approaches that assess both structural integrity and functional capacity. The following methodological strategies are recommended:

Structural verification methods:

• Circular dichroism (CD) spectroscopy to confirm secondary structure content (expected high α-helical content)

• Size exclusion chromatography to assess oligomeric state

• Limited proteolysis to evaluate structural compactness

• Thermal stability assays to compare with native protein

• NMR spectroscopy for structural assessment at atomic resolution

Functional assessment approaches:

• Reconstitution into liposomes with other ATP synthase subunits

• Proton translocation assays using pH-sensitive fluorescent dyes

• ATP synthesis activity measurements in reconstituted systems

• Binding assays with other ATP synthase components

• Electrophysiology to measure proton conductance

For proteins produced in cell-free systems, researchers can directly assess membrane integration by analyzing the distribution between soluble and microsomal fractions . This approach has been validated for other bacterial membrane proteins and provides valuable insights into membrane association properties.

When analyzing multicomponent systems like ATP synthase, it's also informative to study the assembly process by combining individually synthesized subunits and monitoring complex formation through techniques like native PAGE, analytical ultracentrifugation, or FRET-based interaction assays.

How can researchers design knockout studies of atpE in B. cytotoxicus?

Designing knockout studies for atpE in B. cytotoxicus requires careful consideration given the essential nature of ATP synthase for bacterial energy metabolism. Based on successful knockout approaches used for other genes in B. cytotoxicus, the following methodological framework is recommended:

1. Design of knockout constructs:

• Create a construct containing a selectable marker (e.g., kanamycin resistance gene) flanked by homologous sequences adjacent to the atpE gene

• For essential genes like atpE, consider conditional knockout strategies:
  a) Inducible promoter systems
  b) Antisense RNA approaches
  c) CRISPR interference (CRISPRi) for tunable repression

2. Transformation strategies:

• Electroporation has been successfully used for B. cytotoxicus transformations

• Filter mating conjugation can efficiently transfer mutations between strains

• Heat shock transformation may require optimization of conditions for thermotolerant strains

3. Verification of mutants:

• PCR confirmation of the desired genetic modification

• RAPD profiling to confirm the genetic background remains unchanged

• Sequencing to verify the precise modification

• Phenotypic confirmation through growth under different conditions

A study of cytK-1 knockout in B. cytotoxicus successfully created viable mutants using a kanamycin resistance marker and verified the transformants using RAPD profiling . This approach demonstrated that disruption of the cytK-1 gene reduced cytotoxicity by more than 90%, confirming its role in pathogenicity . Similar methodologies could be adapted for atpE studies, though conditional approaches may be necessary given its likely essential function.

What cell models are appropriate for studying the function of recombinant atpE?

Selecting appropriate cell models for studying recombinant atpE function depends on the specific research questions being addressed. The following experimental systems offer distinct advantages for different aspects of ATP synthase research:

1. Bacterial expression hosts:

• E. coli ATP synthase deletion strains can be complemented with recombinant atpE

• B. subtilis as a closer relative to B. cytotoxicus

• Bacterial membrane vesicles for bioenergetic studies

2. Cell-free systems:

• Eukaryotic cell-free systems with microsomal fractions provide membranes for integration

• Bacterial cell-free systems supplemented with lipids or nanodiscs

• Hybrid cell-free systems combining bacterial and eukaryotic components

3. Reconstituted systems:

• Liposomes with purified ATP synthase components

• Nanodiscs for single-molecule studies

• Planar lipid bilayers for electrophysiological measurements

4. Eukaryotic cell lines (for toxicity or functional studies):

• Caco-2 cells have been successfully used to assess B. cytotoxicus toxicity

• HEK293 cells for heterologous expression of bacterial proteins

• Specialized cell lines with reduced endogenous ATP synthase activity

For cytotoxicity studies related to B. cytotoxicus, intestinal epithelial cell models like Caco-2 have proven valuable in assessing the effects of bacterial proteins . The tetrazolium salt (MTT) method has been effectively used to quantify cytotoxicity in these models . For functional studies of atpE specifically, reconstituted systems or bacterial complementation approaches may provide the most direct assessment of ATP synthase activity.

How do researchers design primers for PCR amplification of the atpE gene?

Designing effective primers for PCR amplification of the atpE gene from B. cytotoxicus requires careful consideration of several factors. While the specific primer sequences would depend on the particular strain and sequence, the following methodological approach is recommended:

1. Sequence analysis and primer design strategy:

• Retrieve the atpE gene sequence from closely related B. cereus group members

• Identify conserved regions suitable for primer binding

• Consider the ATP synthase operon structure to identify reliable flanking regions

• Design primers with the following parameters:
  a) Length: 18-30 nucleotides
  b) GC content: 40-60%
  c) Melting temperature (Tm): 55-65°C
  d) Avoid secondary structures and primer-dimer formation

2. Inclusion of molecular cloning features:

• Add appropriate restriction sites for subsequent cloning

• Include 3-6 extra bases at the 5' end for efficient restriction enzyme cutting

• Consider adding tags for detection or purification if needed

• For expression constructs, ensure proper reading frame and codon usage

3. Verification and optimization:

• Perform in silico PCR to check for potential off-target amplifications

• Validate primer specificity through BLAST analysis

• Consider gradient PCR to determine optimal annealing temperature

• For GC-rich regions, include DMSO (2-5%) or specialized polymerases

4. Alternative approaches for challenging templates:

• Nested PCR for increased specificity

• Touchdown PCR to reduce non-specific amplification

• Whole genome amplification followed by specific PCR

• Long-range PCR for capturing the entire ATP synthase operon

When designing primers for B. cytotoxicus, researchers should consider that the B. cereus group shows genomic diversity with unique genomic features in different strains . This diversity may necessitate strain-specific primer optimization to ensure successful amplification.

What techniques can be used to study the structure of recombinant atpE?

Determining the structure of recombinant ATP synthase subunit c requires specialized techniques due to its membrane-associated nature. The following methodological approaches have proven effective for similar membrane proteins:

1. Spectroscopic methods:

• Circular dichroism (CD) spectroscopy for secondary structure assessment

• Fourier-transform infrared spectroscopy (FTIR) for secondary structure in membrane environments

• Nuclear magnetic resonance (NMR) for detailed structural information in solution

• Electron paramagnetic resonance (EPR) with site-directed spin labeling for topological information

2. High-resolution structural techniques:

• X-ray crystallography of purified c-rings (challenging but has been achieved for ATP synthase c-rings)

• Cryo-electron microscopy (cryo-EM) for structures of larger ATP synthase complexes

• Solid-state NMR for membrane-embedded structures

• Mass spectrometry with cross-linking for interaction interfaces

3. Biophysical characterization methods:

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) for oligomeric state determination

• Analytical ultracentrifugation for assembly properties

• Native mass spectrometry for complex composition

• Differential scanning calorimetry for thermal stability

4. Computational approaches:

• Homology modeling based on known ATP synthase c-ring structures

• Molecular dynamics simulations to study conformational dynamics

• Coevolutionary analysis for predicting structural contacts

• Ab initio structure prediction with experimental constraints

For challenging membrane proteins like atpE, combining multiple techniques often provides the most comprehensive structural information. Recent advances in cryo-EM have revolutionized membrane protein structural biology, making it particularly valuable for ATP synthase components, especially when studying the intact complex rather than isolated subunits.

How can researchers assess the role of atpE in bacterial energy metabolism?

Assessing the role of ATP synthase subunit c (atpE) in bacterial energy metabolism requires approaches that can measure both ATP synthesis and the proton translocation function of the protein. The following methodological strategies provide comprehensive insights:

1. Bioenergetic measurements:

• Oxygen consumption rate measurements in intact cells or membrane vesicles

• Membrane potential monitoring using fluorescent probes (e.g., DiSC3(5))

• pH gradient measurements using pH-sensitive fluorophores

• ATP synthesis assays with artificial proton gradients

2. Genetic approaches:

• Conditional knockdown or depletion of atpE

• Site-directed mutagenesis of key residues

• Complementation studies in ATP synthase-deficient strains

• Growth phenotyping under different energy source conditions

3. Biochemical assays:

• ATPase activity measurements with purified enzyme

• Proton pumping assays with reconstituted ATP synthase

• ATP-driven rotation assays for single-molecule studies

• Binding studies with inhibitors and other ATP synthase components

4. Proteomic and metabolomic approaches:

• Quantitative proteomics to assess compensatory changes

• Metabolomic profiling to detect energy metabolism alterations

• Flux analysis to measure carbon and energy flow

• Protein-protein interaction studies to identify regulatory partners

For B. cytotoxicus, which has adapted to specific environmental niches, understanding the role of atpE in energy metabolism may provide insights into its ecological adaptation and potentially its pathogenicity. The thermotolerant nature of this organism suggests possible adaptations in its energy-generating systems that could be reflected in the structure or function of ATP synthase components.

What is the relationship between ATP synthase and bacterial toxin production?

While the direct relationship between ATP synthase and toxin production in B. cytotoxicus has not been specifically addressed in the provided search results, several potential connections can be identified based on general principles of bacterial physiology and the information available about B. cytotoxicus toxins:

1. Energy requirements for toxin production:

• Toxin synthesis and secretion are energy-intensive processes

• ATP synthase function directly impacts cellular energy availability

• Energy status affects the expression of virulence factors in many bacteria

2. Regulatory connections:

• Both CytK-1 and Nhe toxins in B. cytotoxicus are regulated by the PlcR system

• Metabolic and energy sensors often cross-regulate virulence gene expression

• Environmental conditions affecting ATP synthesis may simultaneously signal toxin production

3. Experimental approaches to investigate this relationship:

• Measure toxin production under conditions that affect ATP synthase activity

• Assess CytK-1 expression in strains with modified atpE expression

• Perform transcriptomic analysis comparing wild-type and ATP synthase-modified strains

• Use metabolic inhibitors to assess the correlation between energy status and toxin production

The high cytotoxicity observed in certain B. cytotoxicus strains has been attributed to the production of CytK-1 toxin, with knockout studies demonstrating that CytK-1 is responsible for most of the cytotoxicity toward Caco-2 cells . The remaining toxicity in cytK-1 knockout mutants may be attributed to other factors such as Nhe toxins . Understanding how cellular energy metabolism affects the expression and secretion of these toxins represents an important area for future research.

How can atpE be targeted for potential antimicrobial development?

ATP synthase has emerged as a promising target for antimicrobial development due to its essential role in bacterial energy metabolism. For targeting atpE in B. cytotoxicus or related pathogens, the following research strategies are relevant:

1. Target validation approaches:

• Confirm essentiality through conditional gene silencing or depletion

• Identify structural differences between bacterial and human ATP synthase c subunits

• Assess the impact of ATP synthase inhibition on growth and virulence

• Validate target accessibility in the bacterial membrane

2. Drug discovery strategies:

• Structure-based virtual screening against bacterial c-ring models

• Fragment-based drug discovery using biophysical methods

• High-throughput screening of chemical libraries

• Natural product screening (e.g., derivatives of known ATP synthase inhibitors)

3. Lead compound classes to consider:

• Diarylquinolines (similar to bedaquiline)

• Oligomycin derivatives with enhanced bacterial selectivity

• Synthetic peptides targeting the c-ring assembly

• Small molecules disrupting c-ring/a-subunit interface

4. Assessment methodologies:

• ATP synthesis inhibition assays

• Growth inhibition assays under fermentative vs. respiratory conditions

• Membrane potential monitoring in treated bacteria

• Cytotoxicity testing in mammalian cells to assess selectivity

While B. cytotoxicus is primarily a food safety concern rather than a clinical pathogen, antimicrobials targeting its ATP synthase could potentially be developed as food preservatives or decontamination agents. The thermotolerant nature of B. cytotoxicus may require special consideration when developing inhibitors, as compounds must maintain activity at elevated temperatures where this organism thrives.

What potential biotechnological applications exist for recombinant B. cytotoxicus atpE?

Recombinant ATP synthase components, including atpE from thermotolerant organisms like B. cytotoxicus, have emerging applications in biotechnology and bioengineering. The following innovative applications represent promising research directions:

1. Bioenergy applications:

• Development of ATP-generating biocatalytic systems

• Construction of artificial photosynthetic devices

• Creation of ATP-regenerating systems for coupled enzymatic reactions

• Biomimetic energy conversion technologies

2. Nanobiotechnology:

• ATP synthase-based molecular motors

• Development of nanoscale rotary devices

• Integration of functional c-rings into synthetic membranes

• Energy-harvesting components for nanomachines

3. Biosensor development:

• ATP synthase-based sensors for proton gradients

• Detection systems for compounds affecting membrane potential

• High-throughput screening platforms for ATP synthase inhibitors

• Environmental monitoring devices

4. Protein engineering applications:

• Creating chimeric ATP synthases with novel properties

• Engineering temperature-stable ATP synthase variants

• Development of pH-resistant energy-generating systems

• Customizing proton/ion specificity

The thermotolerant properties of B. cytotoxicus may make its ATP synthase components particularly valuable for applications requiring stability at elevated temperatures. Cell-free protein synthesis systems have demonstrated utility for producing membrane proteins like ATP synthase components , offering a promising platform for generating recombinant atpE for these biotechnological applications.

How does the atpE gene differ between pathogenic and non-pathogenic Bacillus strains?

Comparative genomic analyses of Bacillus species have revealed significant differences between pathogenic and non-pathogenic strains, although specific comparisons of atpE are not detailed in the provided search results. Based on patterns observed in bacterial pathogens and the B. cereus group, several potential differences and research approaches are relevant:

1. Potential differences in atpE between pathogenic and non-pathogenic strains:

• Sequence variations affecting ATP synthase efficiency or regulation

• Differences in gene expression control elements

• Adaptations related to function in host environments (pH tolerance, temperature adaptability)

• Variations in operon structure and co-transcribed genes

2. Comparative genomic approaches:

• The B. cereus group shows genomic diversity, with pathogenic strains possessing unique metabolic pathways and virulence factors

• Complete genome sequencing has revealed strain-specific adaptations within the B. cereus group

• Plasmid-encoded factors can significantly impact strain virulence and metabolism

3. Methodological approach for comparison:

• Whole-genome sequencing of multiple strains spanning pathogenic and non-pathogenic isolates

• Comparative analysis of the ATP synthase operon structure and sequence

• Transcriptomic analysis to identify expression differences

• Functional characterization of atpE variants from different strains

The B. cereus ATCC 10987 genome revealed metabolic pathways not previously identified in the B. cereus group, including urease and xylose utilization pathways, as well as mechanisms for antigenic variability . Similar specializations may exist in energy metabolism genes like atpE, particularly in adaptations to thrive in specific ecological niches or host environments.