Recombinant Bacillus anthracis ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Bacillus anthracis?

ATP synthase subunit c (atpE) in B. anthracis is a small, hydrophobic membrane protein that forms part of the F0 portion of the F1F0-ATP synthase complex. Multiple copies of this protein assemble into a ring structure (c-ring) that functions as a proton channel through the bacterial membrane. The rotation of this c-ring, driven by proton translocation, is mechanically coupled to ATP synthesis in the F1 portion of the complex.

This protein contains two transmembrane α-helices connected by a short hydrophilic loop, with a conserved carboxylic acid residue that is essential for proton translocation. The atpE gene in B. anthracis is part of the atp operon, which encodes the various subunits of the ATP synthase complex. While ATP synthase function is universal across bacteria, the specific structural features of B. anthracis atpE may present opportunities for selective targeting in research and therapeutic development.

How does atpE expression vary across different growth conditions in B. anthracis?

ATP synthase subunit c expression in B. anthracis demonstrates significant variability across different growth conditions, reflecting the bacterium's adaptation to diverse environments. During aerobic growth with abundant nutrients, atpE expression is typically high to support oxidative phosphorylation. Under anaerobic or microaerobic conditions, expression patterns shift to accommodate altered energy metabolism requirements.

During the transition from environmental saprophytic growth to pathogenic lifestyle in mammalian hosts, B. anthracis undergoes substantial metabolic reprogramming. Research has shown that major central metabolism genes, including those involved in TCA cycle, glycolysis, and pentose phosphate pathway, show differential regulation across growth phases and in response to virulence factor expression . As ATP synthase represents the terminal complex in respiratory energy generation, atpE expression likely follows similar regulatory patterns.

The master virulence regulator AtxA influences numerous chromosomal genes beyond the plasmid-encoded virulence factors . This regulatory network may include ATP synthase components, creating connections between virulence activation and energy metabolism. Additionally, the cyclic di-AMP (c-di-AMP) signaling pathway affects potassium homeostasis and toxin expression , suggesting another mechanism by which membrane energetics (which involve ATP synthase) may be integrated with virulence regulation.

What methods are most effective for detecting atpE gene expression in B. anthracis samples?

Detecting atpE gene expression in B. anthracis samples requires techniques that accommodate the challenges of working with this pathogen while providing reliable quantitative data. The methods below represent the most effective approaches for different research contexts:

Quantitative RT-PCR (RT-qPCR):

• Offers high sensitivity and specificity for atpE mRNA detection

• Requires careful primer design to ensure specificity within the B. anthracis atp operon

• Needs rigorous normalization against stable reference genes (rpoB or 16S rRNA)

• Provides relative quantification but can be adapted for absolute quantification with standards

RNA-Seq Analysis:

• Enables genome-wide transcriptional profiling that includes atpE in context with other genes

• Can reveal operon structure and potential regulatory RNAs affecting expression

• Similar approaches have been used successfully to identify AtxA-controlled small RNAs (XrrA and XrrB) in B. anthracis

• Particularly valuable for comparing expression across different growth conditions or mutant strains

Protein-Based Detection Methods:

• Western blotting with antibodies specific to AtpE protein

• Mass spectrometry-based proteomics for quantitative protein measurement

• Blue-native PAGE for analyzing intact ATP synthase complexes

Reporter Gene Fusions:

• Construction of atpE promoter-reporter fusions (GFP, luciferase)

• Allows real-time monitoring of expression in living cells

• Useful for studying regulatory factors affecting atpE expression

When working with environmental or clinical samples, additional considerations include effective DNA/RNA extraction methods that can overcome the challenges posed by B. anthracis spores. Protocols similar to those described for environmental detection of B. anthracis can be adapted for expression studies .

How conserved is the atpE gene across different B. anthracis strains?

The atpE gene exhibits remarkable conservation across different B. anthracis strains, reflecting its essential role in cellular energy metabolism. Sequence analysis reveals >99% nucleotide identity among diverse B. anthracis isolates, making it one of the most stable genetic elements in this species. This high conservation stems from strong functional constraints on ATP synthase structure and operation.

Within the broader Bacillus cereus group (which includes B. anthracis, B. cereus, and B. thuringiensis), atpE shows approximately 95-98% sequence identity. The few variable positions are primarily synonymous substitutions that don't alter the amino acid sequence, with non-synonymous changes largely restricted to non-critical residues outside the functional domains.

The conserved regions include:

• The ion-binding site containing the essential carboxylic acid residue

• Transmembrane helix interfaces critical for c-ring assembly

• Contact surfaces with other ATP synthase components

This conservation pattern has several research implications:

• atpE can serve as a reliable genetic marker for B. anthracis identification

• Functional studies on atpE from lab strains likely translate across clinical isolates

• Evolutionary analyses suggest atpE could help resolve phylogenetic relationships within the B. cereus group

• The high conservation makes atpE a potential target for species-specific detection methods

The stability of atpE across B. anthracis strains contrasts with the greater variability observed in virulence-associated genes, particularly those located on the pXO1 and pXO2 plasmids, which show more evidence of horizontal gene transfer and recombination events.

What is the role of ATP synthase in the B. anthracis lifecycle?

ATP synthase plays dynamic but distinct roles across the different phases of the B. anthracis lifecycle, with its function particularly critical during the transition between environmental persistence and pathogenesis. This enzyme complex adapts to the dramatically different energy requirements of dormant spores, vegetative growth, and infection.

During environmental persistence, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination . In vegetative cells growing in soil or decomposing animal remains, ATP synthase provides the primary source of ATP through oxidative phosphorylation. As nutrients become depleted and sporulation initiates, ATP synthase activity supplies the considerable energy required for spore formation, but the assembled complex is largely disassembled in mature spores.

Upon germination, whether in environmental settings or a mammalian host, ATP synthase components must be rapidly reassembled to support the energy-intensive transition to vegetative growth. Research on the master virulence regulator AtxA shows that B. anthracis follows different growth patterns in saprophytic versus pathogenic lifestyles , suggesting differential regulation of energy metabolism, including ATP synthase function.

During infection, B. anthracis enters a vegetative growth phase without sporulation . ATP synthase activity supports the high energy demands of rapid replication, toxin production, and capsule synthesis. Recent research demonstrates connections between metabolism and virulence in B. anthracis, particularly through the c-di-AMP signaling pathway which links potassium homeostasis to toxin expression . Since membrane energetics (which involve ATP synthase) and ion transport are functionally interconnected, ATP synthase likely participates in coordinating metabolism with virulence expression.

How does recombinant expression of atpE affect bacterial metabolism?

Recombinant expression of B. anthracis atpE significantly impacts bacterial metabolism through several mechanisms, with effects that depend on expression levels, host systems, and experimental conditions. These metabolic alterations have implications for both basic research and biotechnological applications.

When atpE is overexpressed within B. anthracis (homologous expression), the stoichiometric balance of ATP synthase components becomes disrupted. This imbalance can lead to incomplete or dysfunctional enzyme assemblies, as the c-subunit must integrate with other ATP synthase components in precise ratios to form functional complexes. Similar metabolic disruptions were observed in studies of protective antigen (PA) overexpression, where altered expression of numerous metabolic genes correlated with reduced growth rates in B. anthracis .

The effects on energy metabolism include:

Heterologous expression in other bacterial species presents additional challenges, as B. anthracis AtpE may not properly integrate with the host's ATP synthase components. This incompatibility can create more severe membrane disruption and energy deficits than homologous expression.

The stress imposed by atpE overexpression typically activates multiple cellular response pathways. Research on PA-producing B. anthracis strains revealed increased transcription of stress-related genes including sigB, the general stress transcription factor, and its regulators rsbW and rsbV . These stress responses further modify cellular metabolism, creating complex adaptive patterns.

What structural characteristics distinguish B. anthracis ATP synthase subunit c from other bacterial homologs?

B. anthracis ATP synthase subunit c shares the fundamental architecture common to bacterial c-subunits while possessing subtle structural features that distinguish it from homologs in other species. These unique characteristics have implications for enzyme function, assembly, and potential targeted inhibition.

The protein's core structure consists of two transmembrane α-helices connected by a small hydrophilic loop, with approximately 80-90 amino acids total length. Computational structural analysis based on homology modeling with other Bacillus species indicates the following distinctive features:

• Ion-binding site variations: B. anthracis AtpE contains a conserved carboxylic acid residue (aspartate) in the second transmembrane helix that is essential for proton translocation. The microenvironment around this residue shows subtle species-specific variations in hydrophobicity and side chain orientation that influence proton affinity and transport kinetics.

• C-ring stoichiometry: The number of c-subunits forming the complete ring (c-ring) appears to be 10 in Bacillus species, smaller than the 11-15 subunits found in some other bacteria. This stoichiometry affects the bioenergetic parameters of ATP synthesis by determining the proton:ATP ratio.

• Intersubunit contact interfaces: The surfaces where adjacent c-subunits interact contain species-specific residues that influence ring stability and assembly characteristics. These interfaces represent potential targets for species-selective inhibitors.

• Lipid interaction surfaces: The exterior surface of the c-ring interacts with membrane lipids, with B. anthracis AtpE showing distinctive patterns of hydrophobic and aromatic residues that may reflect adaptation to the specific membrane composition of this pathogen.

• Interface with other ATP synthase components: The regions of AtpE that contact other subunits (particularly the a-subunit and the central stalk) contain subtle sequence variations that ensure proper assembly with cognate partners.

These structural characteristics influence not only the functional properties of B. anthracis ATP synthase but also present opportunities for selective targeting in research and potential therapeutic applications.

How does the master virulence regulator AtxA influence ATP synthase expression in B. anthracis?

The master virulence regulator AtxA in B. anthracis has well-established roles in controlling plasmid-encoded virulence factors, but emerging evidence suggests its regulatory influence extends to chromosomal genes involved in metabolism, potentially including ATP synthase components. This connection represents an important nexus between virulence and energy metabolism regulation.

The relationship between AtxA and ATP synthase expression appears to involve several mechanisms:

• Direct regulation: While not definitively established, AtxA may bind to regions upstream of ATP synthase genes to directly influence their transcription under specific conditions.

• sRNA-mediated regulation: The AtxA-controlled sRNAs XrrA and XrrB may include ATP synthase mRNAs among their targets, creating post-transcriptional regulation .

• Growth phase coordination: AtxA activity varies across growth phases, with research showing that "major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases" . This pattern suggests coordination between virulence factor expression and energy metabolism.

• Environmental sensing integration: AtxA responds to environmental cues relevant to pathogenesis, including CO2/bicarbonate levels, temperature, and carbohydrate availability. These same signals influence ATP synthase expression, potentially through shared regulatory pathways.

Understanding the relationship between AtxA and ATP synthase expression could provide valuable insights into how B. anthracis coordinates virulence and metabolism during infection, potentially revealing new targets for intervention strategies.

What purification methods yield the highest quality recombinant B. anthracis AtpE protein?

Purifying recombinant B. anthracis ATP synthase subunit c (AtpE) presents significant challenges due to its small size, extreme hydrophobicity, and tendency to form aggregates. Multiple purification strategies have been developed to address these challenges, each with specific advantages and limitations as outlined in the following comparative analysis:

The detergent-based extraction method typically involves:

• Membrane isolation from expression host bacteria

• Solubilization with mild detergents (DDM, LDAO)

• Immobilized metal affinity chromatography using a His-tag

• Size exclusion chromatography for final purification

The organic solvent extraction approach includes:

• Direct extraction of cell pellets with chloroform:methanol (2:1)

• Precipitation with diethyl ether

• Resolubilization in formic acid or TFA

• Reverse-phase HPLC purification

For fusion protein strategies, the protocol generally follows:

• Expression of AtpE fused to a soluble partner (MBP, SUMO)

• Purification under native conditions

• Specific protease cleavage

• Separation of AtpE from the fusion partner

Quality control metrics should include:

• SDS-PAGE analysis with silver staining

• Mass spectrometry confirmation of protein identity

• Circular dichroism to assess secondary structure

• Functional reconstitution assays when applicable

The optimal purification strategy depends on the intended application. For structural studies requiring high purity but not native conformation, organic solvent extraction provides the best results. For functional studies where native folding is critical, detergent-based methods with careful reconstitution into proteoliposomes are preferred.

How does c-di-AMP signaling affect ATP synthase function in B. anthracis?

The relationship between c-di-AMP signaling and ATP synthase function in B. anthracis represents an emerging area of research that connects second messenger signaling, ion homeostasis, energy metabolism, and virulence regulation. Recent findings establish several important connections between these pathways.

Research has demonstrated that c-di-AMP in B. anthracis "binds to a series of receptors involved in potassium uptake" and "inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch" . Importantly, this study established that "decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake" . This connection between potassium homeostasis and toxin expression has significant implications for ATP synthase function.

The relationship functions through several interconnected mechanisms:

• Membrane potential integration: ATP synthase activity depends on the proton motive force, which consists of both the pH gradient (ΔpH) and the electrical potential (Δψ) across the membrane. The electrical component is significantly influenced by potassium transport, creating a direct bioenergetic link between c-di-AMP regulated potassium uptake and ATP synthase driving force.

• Energy status sensing: In many bacteria, c-di-AMP levels respond to energy stress and cellular ATP concentrations. This creates a potential feedback loop where ATP synthase activity influences c-di-AMP production, which in turn affects potassium homeostasis and membrane energetics.

• Coordinated virulence regulation: The finding that c-di-AMP affects toxin expression through potassium transport suggests a regulatory network that coordinates metabolism, ion homeostasis, and virulence. ATP synthase, as a central component of energy metabolism, is likely integrated into this network.

The mathematical relationship between these parameters can be expressed as:

$\text{PMF} = \Delta\psi - 2.3 \frac{RT}{F} \Delta\text{pH}$

Where c-di-AMP influences Δψ through regulation of K+ transport, thereby affecting the driving force for ATP synthesis.

Understanding this relationship provides novel insights into how B. anthracis coordinates its metabolic state with virulence expression during infection.

How do mutations in the atpE gene affect antimicrobial resistance in B. anthracis?

Mutations in the atpE gene encoding ATP synthase subunit c can significantly impact antimicrobial resistance in B. anthracis, particularly against drugs that target this specific component. These mutations represent an important but understudied mechanism of resistance with implications for both natural and engineered antimicrobial compounds.

The most direct relevance of atpE mutations to antimicrobial resistance involves drugs that specifically target subunit c, such as diarylquinolines (e.g., bedaquiline) and certain oligomycin derivatives. Specific mutations in the following regions have been associated with resistance:

• Ion-binding site: Mutations affecting the conserved aspartate residue or neighboring amino acids can alter drug binding while preserving sufficient proton translocation for ATP synthesis.

• Helix-helix interface: Changes at the interface between transmembrane helices can modify the binding pocket for certain inhibitors without disrupting c-ring assembly.

• Subunit-subunit interface: Alterations where adjacent c-subunits interact can affect the binding of drugs that exploit the interfaces in the assembled c-ring.

Beyond direct resistance to ATP synthase inhibitors, atpE mutations can have broader effects on antimicrobial susceptibility through several mechanisms:

The development of atpE-mediated resistance typically involves trade-offs, as mutations must maintain sufficient ATP synthase function for bacterial survival while altering drug binding. This balance creates a fitness landscape that constrains the evolution of resistance, potentially providing opportunities for designing antimicrobials with reduced resistance potential.

Understanding the molecular basis of these resistance mechanisms requires integration of structural biology, bioenergetics, and microbial physiology approaches, with potential applications in developing new strategies to overcome antimicrobial resistance in B. anthracis.

What experimental models best represent the in vivo expression and function of AtpE during B. anthracis infection?

Developing experimental models that accurately represent the in vivo expression and function of AtpE during B. anthracis infection requires approaches that capture the complex host-pathogen interactions while enabling detailed molecular analysis. The following hierarchy of models provides increasing biological relevance but with trade-offs in experimental accessibility:

Cell Culture Models:

• Macrophage infection models (RAW264.7, J774, primary macrophages)

• Lung epithelial cell lines for inhalational anthrax studies

• Endothelial cell models for vascular dissemination phase

• Advantages: Controlled conditions, amenable to real-time imaging, gene expression analysis

• Limitations: Lack tissue complexity and systemic responses

Ex Vivo Tissue Models:

• Precision-cut lung slices for respiratory infection

• Blood-brain barrier models for late-stage infection

• Skin explant cultures for cutaneous anthrax

• Advantages: Maintains tissue architecture and cellular diversity

• Applications: AtpE expression in tissue microenvironments

Animal Infection Models:

• Mouse models with defined genetic backgrounds

• Guinea pig models (more susceptible than mice)

• Rabbit models (closely mimic human disease progression)

• Non-human primate models (gold standard but highly restricted)

• Applications: In vivo expression patterns, mutant phenotypes

For molecular analysis of AtpE expression and function across these models, several methodological approaches have proven effective:

• Reporter Systems: AtpE promoter fusions with fluorescent proteins or luciferase enable real-time monitoring of expression in living systems. Similar approaches have been used successfully to study expression of virulence factors in B. anthracis.

• Inducible Expression Systems: Controlled modulation of atpE expression during infection can reveal its role in different infection phases. Systems responsive to non-toxic inducers allow manipulation without disturbing the host-pathogen interaction.

• Tissue-specific Transcriptomics: RNA isolation from infected tissues followed by B. anthracis-specific sequence analysis can reveal atpE expression patterns in different anatomical sites. This approach has been used to study AtxA-regulated genes during infection .

• Metabolic Labeling: Incorporation of stable isotopes can track ATP synthase activity during infection, revealing how energy metabolism adapts to the host environment.

The most informative approach often combines multiple models, moving from simplified systems for mechanistic studies to more complex models for validation of biological relevance.

How can researchers distinguish between host and bacterial ATP synthase activity in infection models?

Distinguishing between host and B. anthracis ATP synthase activity in infection models presents significant methodological challenges but is achievable through several complementary approaches. This differentiation is crucial for understanding the specific contribution of bacterial energy metabolism to infection processes.

Biochemical Discrimination Strategies:

• Differential inhibitor sensitivity: Certain compounds show selectivity between bacterial and eukaryotic ATP synthases:

  • Oligomycin strongly inhibits mitochondrial ATP synthase with minimal effect on bacterial enzymes

  • Diarylquinolines preferentially target bacterial ATP synthases

  • N,N'-dicyclohexylcarbodiimide (DCCD) binds both types but with different kinetics

• pH-dependent activity profiles: Bacterial and mammalian ATP synthases have different pH optima for activity:

  • B. anthracis ATP synthase typically shows optimal activity at pH 7.0-7.5

  • Mammalian mitochondrial ATP synthase functions optimally at pH 8.0-8.5

  • Assays conducted across pH ranges can help differentiate their contributions

Molecular Detection Approaches:

• Species-specific antibodies: Antibodies that specifically recognize B. anthracis ATP synthase components allow immunological detection:

  • Western blotting of tissue lysates from infection models

  • Immunohistochemistry for spatial localization

  • Flow cytometry for quantification in cell culture models

• Mass spectrometry techniques: Targeted proteomics can detect species-specific peptides:

  • Selected reaction monitoring (SRM) for quantification

  • Peptide mass fingerprinting for identification

  • Can detect specific B. anthracis AtpE tryptic peptides even in complex samples

Genetic and Expression-based Methods:

• Reporter systems: Engineering B. anthracis with reporters linked to atpE expression:

  • Fluorescent proteins for microscopic visualization

  • Luciferase for non-invasive bioluminescence imaging

  • Can be combined with tissue clearing techniques for 3D visualization

• RNA-based detection: RNA sequencing with species-specific mapping:

  • Discriminates between host and pathogen transcripts

  • Can reveal atpE expression relative to other bacterial and host genes

  • Similar approaches have been used to study AtxA-regulated genes in B. anthracis

Each approach has specific advantages and limitations, with the optimal strategy depending on the particular infection model and research questions being addressed. Combining multiple complementary methods provides the most robust differentiation between host and bacterial ATP synthase activities.

What are the challenges in developing ATP synthase inhibitors that specifically target B. anthracis?

Developing ATP synthase inhibitors that specifically target B. anthracis presents multiple challenges spanning structural biology, medicinal chemistry, pharmacokinetics, and practical considerations for biodefense applications. These challenges must be systematically addressed to create effective agents against this high-priority pathogen.

Selectivity Challenges:

• Structural conservation: ATP synthase is highly conserved across species, with approximately 80% sequence similarity between bacterial and human mitochondrial versions of subunit c. Achieving sufficient selectivity requires targeting subtle structural differences or bacterial-specific interfaces.

• Bacillus cereus group similarity: B. anthracis shares >95% sequence identity in atpE with non-pathogenic members of the B. cereus group, complicating selective targeting within this bacterial family and raising potential off-target effects on beneficial soil bacteria.

• Binding site architecture: The most druggable sites in ATP synthase are formed by multiple subunits in the assembled complex. High-resolution structural data specific to B. anthracis ATP synthase is lacking, making structure-based design challenging.

Pharmacological Barriers:

• Penetration issues: B. anthracis has a complex cell envelope with a thick peptidoglycan layer and, in virulent strains, a poly-γ-D-glutamic acid capsule. Inhibitors must penetrate these barriers to reach the membrane-embedded ATP synthase.

• Spore ineffectiveness: During the spore state, B. anthracis has minimal metabolic activity and disassembled ATP synthase complexes, making ATP synthase inhibitors ineffective against the environmentally persistent form of the pathogen.

Resistance Considerations:

• Mutation potential: As discussed in question 2.6, mutations in atpE can confer resistance while maintaining sufficient enzyme function. The potential resistance mechanisms must be anticipated in inhibitor design.

• Metabolic bypass: Under certain conditions, B. anthracis can rely on fermentation for ATP production, potentially allowing escape from growth inhibition by ATP synthase-targeted compounds.

Development Strategies:

Despite these challenges, several promising approaches for developing B. anthracis-specific ATP synthase inhibitors include:

• Targeting unique interfaces between subunits rather than conserved catalytic sites

• Developing prodrugs activated by B. anthracis-specific enzymes

• Combination approaches with compounds that increase susceptibility to ATP synthase inhibition

• Focusing on allosteric sites that may show greater species variation

• Designing compounds that selectively accumulate in B. anthracis through pathogen-specific uptake mechanisms

The unique threat posed by B. anthracis as both a natural pathogen and potential bioterrorism agent justifies continued exploration of ATP synthase as a therapeutic target despite these significant challenges.

How does environmental adaptation affect ATP synthase assembly and function in B. anthracis?

B. anthracis encounters diverse environments during its lifecycle, from soil to mammalian hosts, and its ATP synthase assembly and function demonstrate remarkable adaptability to these changing conditions. This environmental responsiveness involves coordinated modifications at multiple levels, from gene expression to enzyme assembly and activity regulation.

Soil Environment Adaptation:
In soil habitats, B. anthracis experiences fluctuating nutrient availability, temperature, pH, and moisture conditions. ATP synthase adapts through:

• Modulation of expression levels in response to energy demand

• Adjustments in c-ring assembly to optimize proton:ATP ratios for specific conditions

• Membrane lipid composition changes that affect ATP synthase activity

• Integration with sporulation initiation as resources become limited

Host Adaptation During Infection:
Upon transitioning to mammalian hosts, B. anthracis encounters more stable temperature and pH but faces immune defenses and changing nutrient profiles. Adaptation involves:

• Coordinated regulation with virulence pathways through AtxA-dependent mechanisms

• Increased expression to support rapid vegetative growth and toxin production

• Potential modifications in response to host-derived antimicrobial peptides

• Integration with potassium homeostasis systems influenced by c-di-AMP signaling

The relationship between c-di-AMP signaling and ATP synthase function is particularly significant for environmental adaptation. Research demonstrates that c-di-AMP "binds to a series of receptors involved in potassium uptake" and influences osmotic stress responses . Since membrane potential (which drives ATP synthase) and potassium homeostasis are interconnected, this represents a mechanism by which B. anthracis coordinates ATP synthase function with environmental osmotic conditions.

The stress-responsive transcription factor SigB, which shows increased expression in certain B. anthracis growth conditions , likely influences ATP synthase expression during stress adaptation. Additionally, the differential regulation of chaperones observed in B. anthracis strains suggests specific protein folding and assembly requirements for membrane proteins like ATP synthase components during environmental transitions.

Understanding these adaptation mechanisms provides insights into how B. anthracis maintains energy homeostasis across diverse environments and may reveal vulnerabilities that could be exploited for targeted interventions.