Recombinant Bacteroides thetaiotaomicron ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Bacteroides thetaiotaomicron and what role does it play?

The ATP synthase subunit c (atpE) in Bacteroides thetaiotaomicron is a critical component of the F₁F₀-ATP synthase complex, responsible for ATP production through oxidative phosphorylation. This membrane-embedded protein forms the c-ring of the F₀ domain, which functions as a proton channel across the bacterial membrane. In B. thetaiotaomicron, as an obligate anaerobe dominant in the human gut microbiome, the ATP synthase plays a crucial role in energy conservation under the low-oxygen conditions of the intestinal environment.

B. thetaiotaomicron has evolved sophisticated metabolic strategies to thrive in the gut ecosystem, where oxygen levels fluctuate and can cause oxidative stress. Research has demonstrated that B. thetaiotaomicron can enhance its oxidative stress tolerance through metabolic adaptations . The ATP synthase complex, including the atpE subunit, likely contributes to this adaptability by maintaining energy production under changing environmental conditions.

When studying atpE in B. thetaiotaomicron, it's important to consider this bacterium's unique niche as a gut commensal that interacts with the host and other microbial species. These interactions influence energy requirements and metabolic priorities, potentially affecting ATP synthase expression and activity patterns across different growth phases and environmental conditions.

How is the atpE gene organized in the B. thetaiotaomicron genome and what regulatory elements control its expression?

The atpE gene in Bacteroides thetaiotaomicron is typically organized within an operon structure containing other ATP synthase subunit genes. Based on expanded transcriptome analysis of B. thetaiotaomicron, researchers have refined annotations of operon structures and transcriptional units across the genome . The ATP synthase genes, including atpE, are likely part of a coordinated transcriptional unit that ensures stoichiometric production of all subunits needed for functional complex assembly.

Regulatory elements controlling atpE expression include promoters that may respond to energy status and environmental cues. The expanded transcriptome atlas for B. thetaiotaomicron has mapped transcriptional start sites (TSSs) across multiple growth conditions, identifying 4,123 TSSs across the chromosome and plasmid . This detailed mapping helps researchers understand how genes like atpE are regulated under different conditions. The upstream region of the atpE gene likely contains AT-rich sequences that serve as binding sites for Bacteroides-specific transcription factors.

Transcriptional regulation in B. thetaiotaomicron involves complex networks that respond to stress and nutrient availability. Research has demonstrated that oxidative stress significantly alters gene expression patterns in this anaerobe . Since ATP synthase is central to energy metabolism, atpE expression is likely coordinated with the bacterium's response to oxygen exposure and metabolic demands. Understanding these regulatory mechanisms provides insights into how B. thetaiotaomicron adapts its energy production machinery to thrive in the dynamic gut environment.

What expression systems are optimal for recombinant B. thetaiotaomicron atpE production?

Several expression systems have been developed for recombinant protein production in Bacteroides thetaiotaomicron, with specific considerations needed for membrane proteins like atpE. For homologous expression, recent advances in synthetic biology tools for B. thetaiotaomicron provide promising options. Researchers have expanded the genetic toolkit for this organism, enabling sophisticated genetic circuits that can be employed for controlled expression of recombinant proteins .

For homologous expression in B. thetaiotaomicron, plasmid-based systems with tunable promoters offer the best results for atpE production. The development of genetic tools like tunable expression systems for B. thetaiotaomicron is particularly valuable, as they provide control over expression levels. One such system is the engineered riboregulator (ER) system, which achieved repression of reporter gene expression by 4,258-fold in the OFF state and a 69-fold increase in the ON state . This type of control is crucial when expressing membrane proteins like atpE, where overexpression can be toxic.

For heterologous expression in E. coli or other hosts, special considerations are necessary due to the hydrophobic nature of atpE. Expression in E. coli often requires fusion tags (like MBP or SUMO) to enhance solubility and prevent aggregation. Additionally, specialized E. coli strains designed for membrane protein expression (C41/C43) yield better results than standard strains. When using heterologous systems, codon optimization based on the host's preference is essential, as B. thetaiotaomicron has a distinctive AT-rich codon bias that differs from common expression hosts.

What purification protocols are most effective for isolating recombinant B. thetaiotaomicron atpE while maintaining structural integrity?

Purification of recombinant ATP synthase subunit c (atpE) from Bacteroides thetaiotaomicron requires specialized protocols due to its hydrophobic nature and membrane integration. A multi-step purification approach yields the best results, beginning with optimized membrane extraction followed by chromatographic separation. Since atpE is a small, hydrophobic protein, care must be taken at each step to prevent aggregation and maintain its native structure.

The initial membrane extraction is critical and requires gentle solubilization using detergents appropriate for membrane proteins. Mild detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are preferred over harsh detergents like SDS, as they better preserve protein-protein interactions and structural integrity. When expressed with affinity tags (His-tag or Strep-tag), immobilized metal affinity chromatography (IMAC) provides an efficient first purification step. Following affinity purification, size exclusion chromatography (SEC) in the presence of appropriate detergents helps separate the target protein from aggregates and contaminating proteins.

For structural studies or functional assays, reconstitution of purified atpE into lipid nanodiscs or proteoliposomes may be necessary to maintain native-like conformation. This approach is particularly important when studying atpE function, as its proton-conducting activity depends on proper membrane insertion. Research on B. thetaiotaomicron has revealed that the bacterium modifies its membrane composition in response to environmental conditions, including oxygen exposure . Therefore, using lipid compositions that mimic the native B. thetaiotaomicron membrane can enhance protein stability and activity in reconstituted systems.

How can researchers verify the correct folding and functionality of recombinant B. thetaiotaomicron atpE?

Verifying the correct folding and functionality of recombinant ATP synthase subunit c (atpE) from Bacteroides thetaiotaomicron requires multiple complementary approaches. Since atpE functions as part of the larger ATP synthase complex, assessing its folding and functionality presents unique challenges compared to soluble proteins. A comprehensive validation strategy combines structural assessment with functional assays.

Structural assessment typically begins with circular dichroism (CD) spectroscopy to analyze secondary structure content, which for atpE should reveal a predominantly α-helical signature consistent with its transmembrane domains. For more detailed structural validation, NMR spectroscopy can be employed for small membrane proteins like atpE, providing atomic-level information about protein folding in membrane-mimetic environments. Additionally, limited proteolysis assays can provide insights into the accessibility of protease cleavage sites, which differs between properly folded and misfolded membrane proteins.

Functional validation of recombinant atpE requires assessing its ability to form functional c-rings and participate in proton translocation. Reconstitution of purified atpE into proteoliposomes followed by proton translocation assays using pH-sensitive fluorescent dyes provides information about its ion channel functionality. For comprehensive assessment, integration of recombinant atpE into ATP synthase complexes (either purified or in membrane vesicles) followed by ATP synthesis/hydrolysis assays provides the most physiologically relevant functional validation. Recent advancements in synthetic biology tools for B. thetaiotaomicron facilitate genetic complementation approaches, where recombinant atpE can be tested for its ability to restore function in atpE-deficient strains.

How can CRISPR/Cas systems be optimized for modifying atpE in B. thetaiotaomicron?

CRISPR/Cas systems have been successfully adapted for genome editing in Bacteroides thetaiotaomicron, offering powerful tools for atpE modification. Recent advances in B. thetaiotaomicron genetic manipulation include the development of the aTc-inducible CRISPR/FnCas12a system, which has been used to delete large genomic regions and target specific genes in multiple Bacteroides species . For atpE modification, this system can be optimized by careful selection of guide RNAs targeting unique regions of the gene while avoiding off-target effects.

When designing CRISPR-based approaches for atpE modification, several key considerations must be addressed. First, appropriate promoters for expressing Cas proteins and guide RNAs must be selected. Research has expanded the annotation of B. thetaiotaomicron transcriptional start sites and promoters across multiple growth conditions , providing valuable information for selecting promoters with desired expression characteristics. Second, the delivery method must be optimized; conjugation from E. coli S17-1 has proven effective for introducing plasmids into B. thetaiotaomicron , making it suitable for CRISPR components delivery.

For precise editing of atpE, homology-directed repair (HDR) templates can be designed to introduce specific mutations or tags. The efficiency of HDR in B. thetaiotaomicron can be enhanced by optimizing the length of homology arms and using selection markers. Additionally, recent research has characterized invertible DNA regions (invertons) in the B. thetaiotaomicron genome , which should be considered when designing guide RNAs to avoid regions with potential structural variation. Finally, phenotypic verification of atpE modifications should include assessment of ATP synthesis capacity and growth under various conditions, as B. thetaiotaomicron has been shown to adapt its metabolism to environmental stressors .

How does atpE expression and function change in B. thetaiotaomicron under different oxygen conditions?

Bacteroides thetaiotaomicron is an obligate anaerobe that nevertheless exhibits remarkable adaptability to oxygen exposure, with implications for ATP synthase function and expression. Research has demonstrated that B. thetaiotaomicron can tolerate oxygen exposure and subsequently recover anaerobic growth . During this oxygen exposure and recovery cycle, the expression and function of energy metabolism genes, including ATP synthase components like atpE, undergo significant adjustments to maintain cellular energy balance.

When exposed to oxygen, B. thetaiotaomicron initiates a complex stress response involving multiple genes and metabolic pathways. Studies examining the bacterium's response to oxidative stress have revealed that B. thetaiotaomicron cells grown on different carbon sources (glucose versus rhamnose) exhibit different tolerance levels to hydrogen peroxide and oxygen exposure . This suggests that metabolic state influences the bacterium's ability to cope with oxidative stress, which likely involves regulation of energy production systems including ATP synthase.

The transition from anaerobic to aerobic conditions and back presents a significant energetic challenge for B. thetaiotaomicron. During oxygen exposure, the bacterium must adapt its energy metabolism to maintain ATP levels while minimizing oxidative damage. The ATP synthase complex, including atpE, may undergo functional adjustments or expression changes to accommodate these shifting energetic demands. Transcriptomic analysis of B. thetaiotaomicron under various stress conditions has revealed condition-specific gene expression patterns , which likely extend to ATP synthase components. Understanding these adaptive responses provides insights into how this important gut commensal maintains energy homeostasis in the dynamically changing intestinal environment, where oxygen gradients exist and fluctuate with host physiology.

What are the implications of atpE modifications for B. thetaiotaomicron colonization and persistence in the gut microbiome?

Modifications to ATP synthase subunit c (atpE) in Bacteroides thetaiotaomicron could significantly impact its fitness and colonization ability in the competitive gut ecosystem. Research has demonstrated that B. thetaiotaomicron experiences population bottlenecks during initial colonization of the mouse gut, with the probability of successful colonization inversely related to microbiota complexity . Since energy metabolism is crucial for competitive fitness, alterations to atpE that affect ATP production efficiency could influence the bacterium's ability to establish and persist in the intestinal environment.

The gut environment presents unique challenges that likely influence ATP synthase function and importance. Oxygen gradients exist across the intestinal lumen and mucosa, creating microenvironments with varying oxidative stress levels. B. thetaiotaomicron has been shown to adapt metabolically to oxygen exposure , suggesting that ATP synthase activity may be modulated in response to these conditions. Modifications to atpE that alter its oxygen sensitivity or functional efficiency under varying redox conditions could therefore impact the bacterium's ability to occupy specific intestinal niches.

Competition for nutrients in the gut ecosystem places premium value on metabolic efficiency. B. thetaiotaomicron has evolved sophisticated polysaccharide utilization systems, with over 80 polysaccharide utilization loci (PULs) identified in its genome . The energy derived from these diverse carbon sources depends on efficient ATP synthesis, making atpE function critical for competitive success. Additionally, B. thetaiotaomicron undergoes phase variation in its capsular polysaccharide (CPS) expression, with different CPS types dominating under different conditions . Since capsule production is energetically costly, alterations to energy metabolism through atpE modifications could influence CPS expression patterns and thereby affect immune evasion and competitive fitness in the gut environment.

What transcriptomic approaches are most informative for studying atpE expression patterns in B. thetaiotaomicron?

Advanced transcriptomic approaches provide valuable insights into atpE expression patterns in Bacteroides thetaiotaomicron across different physiological conditions. Differential RNA sequencing (dRNA-seq) and conventional RNA-seq have been successfully applied to B. thetaiotaomicron, generating comprehensive transcriptome atlases across multiple growth conditions . These approaches can reveal how atpE expression is coordinated with other ATP synthase subunits and energy metabolism genes under various environmental conditions.

For studying atpE specifically, targeted transcriptomic approaches can provide detailed insights. Time-resolved RNA-seq during transitions between growth conditions—such as the anaerobic-aerobic-anaerobic transitions used to study oxidative stress responses in B. thetaiotaomicron —can reveal dynamic changes in atpE expression. This approach involves sampling at multiple timepoints during environmental transitions, with immediate cell quenching in liquid nitrogen to preserve the transcriptional state, followed by RNA extraction and sequencing . Subsequent bioinformatic analysis can identify co-expressed gene clusters and regulatory networks involving atpE.

Integration of transcriptomic data with other omics approaches enhances the interpretation of atpE expression patterns. The combination of transcriptomics with transposon mutant fitness data has proven valuable for identifying conditionally important genes in B. thetaiotaomicron . Similar integrative approaches could reveal conditions where atpE expression is particularly critical for fitness. Additionally, the development of the Theta-Base web browser (http://micromix.helmholtz-hiri.de/bacteroides/) provides a valuable resource for exploring B. thetaiotaomicron transcriptomic data across multiple conditions , facilitating comparative analysis of atpE expression patterns in the context of global transcriptional responses.

What functional assays can accurately measure ATP synthase activity in recombinant B. thetaiotaomicron systems?

Measuring ATP synthase activity in recombinant Bacteroides thetaiotaomicron systems requires specialized assays that account for the anaerobic nature of this organism and the membrane-embedded location of the enzyme complex. Several complementary approaches provide comprehensive assessment of ATP synthase function, with the choice of method depending on the specific research question and experimental system.

For in vitro assessment of purified recombinant ATP synthase containing atpE, the ATP synthesis assay provides direct measurement of function. This assay involves reconstituting the purified enzyme complex into proteoliposomes, establishing a proton gradient across the membrane, and measuring ATP production using luciferase-based detection systems. For the reverse reaction, ATP hydrolysis activity can be measured using colorimetric assays that detect inorganic phosphate release. When working with recombinant systems, it's crucial to include appropriate controls to distinguish ATP synthase activity from background ATP production or hydrolysis.

For cellular systems expressing recombinant atpE, membrane potential measurements provide insights into proton-pumping activity. Fluorescent probes sensitive to membrane potential, such as DiSC3(5) or TMRM, can be used to monitor changes in proton gradient formation in response to substrate addition or inhibitor treatment. Additionally, respiration rate measurements using oxygen electrodes (for aerobic conditions) or alternative electron acceptor consumption assays (for anaerobic conditions) provide indirect assessment of ATP synthase coupling to electron transport chain activity. For anaerobic organisms like B. thetaiotaomicron, specialized anaerobic chambers or sealed cuvette systems are required for these measurements to maintain appropriate conditions throughout the assay.

How can researchers investigate the interactions between recombinant atpE and other ATP synthase subunits in B. thetaiotaomicron?

Investigating interactions between recombinant ATP synthase subunit c (atpE) and other ATP synthase components in Bacteroides thetaiotaomicron requires sophisticated protein-protein interaction methodologies adapted for membrane protein complexes. Several complementary approaches provide insights into these interactions from different perspectives, enabling comprehensive characterization of complex assembly and dynamics.

Co-immunoprecipitation (Co-IP) combined with MS2 affinity purification techniques that have been successfully applied in B. thetaiotaomicron for RNA-protein interactions can be adapted for protein-protein interaction studies. For atpE interactions, this approach typically involves expressing tagged versions of atpE or other ATP synthase subunits, followed by gentle solubilization with appropriate detergents and affinity purification. Mass spectrometry analysis of co-purified proteins identifies interaction partners and can provide quantitative information about interaction strengths under different conditions.

Crosslinking mass spectrometry (XL-MS) offers valuable insights into the spatial arrangement of atpE relative to other ATP synthase subunits. This technique involves treating intact ATP synthase complexes with crosslinking reagents that form covalent bonds between spatially proximal amino acid residues, followed by proteolytic digestion and mass spectrometry analysis to identify crosslinked peptides. The resulting distance constraints can inform structural models of the B. thetaiotaomicron ATP synthase complex. For membrane proteins like atpE, specialized crosslinkers with appropriate hydrophobicity and spacer arm length are required to access the membrane-embedded regions.

Genetic approaches provide complementary functional evidence for subunit interactions. Synthetic biology tools developed for B. thetaiotaomicron, including CRISPR/Cas systems , enable creation of mutant libraries with alterations in potential interaction interfaces. Suppressor mutation analysis, where secondary mutations in one subunit compensate for defects in another, can reveal functional interactions between ATP synthase components. Additionally, bacterial two-hybrid systems adapted for membrane proteins can screen for interactions in vivo, though these may require modification for use in the anaerobic B. thetaiotaomicron.

How might atpE in B. thetaiotaomicron be engineered to create probiotic strains with enhanced colonization abilities?

Engineering the ATP synthase subunit c (atpE) in Bacteroides thetaiotaomicron represents a promising strategy for developing next-generation probiotics with improved gut colonization capabilities. Since energy metabolism directly impacts competitive fitness in the gut ecosystem, modifications to atpE that enhance ATP production efficiency could confer advantages during establishment and persistence. Recent advances in synthetic biology tools for B. thetaiotaomicron provide the technical foundation for such engineering efforts .

Strategic modifications to atpE could target several aspects of ATP synthase function relevant to gut colonization. Alterations that optimize proton translocation efficiency under the specific pH conditions of the intestinal environment could enhance energy conservation. Additionally, engineering atpE variants with improved stability during exposure to host-derived antimicrobial compounds or fluctuating oxygen levels could enhance survival during transit through the gastrointestinal tract. B. thetaiotaomicron has demonstrated remarkable adaptability to oxidative stress , suggesting that further optimization of energy metabolism under these conditions could enhance colonization potential.

Integration of engineered atpE into broader synthetic biology applications for B. thetaiotaomicron could create multifunctional probiotic strains. Recent research has developed sophisticated genetic circuits in B. thetaiotaomicron that can sense, compute, memorize, and respond to environmental signals . Coupling these sensing and response systems with optimized energy metabolism through atpE engineering could create probiotics that dynamically adjust their colonization strategies based on intestinal conditions. Furthermore, the naturally produced outer membrane vesicles (OMVs) of B. thetaiotaomicron have been engineered as delivery systems for therapeutic proteins , suggesting potential applications where enhanced energy metabolism supports increased production of beneficial payloads.

What are the most promising approaches for using recombinant B. thetaiotaomicron atpE in structural biology studies?

Structural characterization of recombinant ATP synthase subunit c (atpE) from Bacteroides thetaiotaomicron presents unique challenges due to its small size, hydrophobic nature, and native organization into oligomeric rings. Recent advances in structural biology methodologies, particularly for membrane proteins, offer promising approaches for elucidating the structure of B. thetaiotaomicron atpE at atomic resolution, providing insights into its function and potential for engineering.

Cryo-electron microscopy (cryo-EM) has revolutionized structural studies of membrane protein complexes and represents a promising approach for B. thetaiotaomicron ATP synthase. This technique is particularly well-suited for determining structures of intact ATP synthase complexes, providing insights into how atpE organizes into the c-ring and interacts with other subunits. For successful cryo-EM studies, purification protocols must be optimized to maintain complex integrity in appropriate detergents or nanodiscs. The recent expansion of genetic tools for B. thetaiotaomicron facilitates expression of tagged versions of ATP synthase components, aiding in complex purification while maintaining native-like assemblies.

For high-resolution studies of isolated atpE, X-ray crystallography of the c-ring remains challenging but feasible. This approach typically requires crystallization of the purified c-ring in lipidic cubic phases or similar membrane-mimetic environments. Alternatively, solid-state NMR spectroscopy offers advantages for structural studies of membrane proteins like atpE in near-native lipid environments. This technique can provide detailed information about protein dynamics and water-accessible regions, which are particularly relevant for understanding proton translocation through the c-ring. Integration of multiple structural biology approaches, complemented by computational modeling, offers the most comprehensive strategy for elucidating the structure-function relationships of B. thetaiotaomicron atpE.

How can integrative multi-omics approaches advance our understanding of atpE function in B. thetaiotaomicron?

Integrative multi-omics approaches offer powerful strategies for comprehensive understanding of ATP synthase subunit c (atpE) function in Bacteroides thetaiotaomicron within its native gut ecosystem context. By combining transcriptomics, proteomics, metabolomics, and functional genomics, researchers can develop holistic models of how atpE contributes to B. thetaiotaomicron fitness and interactions in the complex gut environment.

The integration of transcriptomic data with transposon mutant fitness data has already proven valuable for identifying conditionally important genes in B. thetaiotaomicron . This approach could be extended to elucidate condition-specific roles of atpE by correlating its expression patterns with fitness contributions across diverse environmental conditions. Additionally, proteomics approaches focusing on protein-protein interactions and post-translational modifications could reveal regulatory mechanisms affecting ATP synthase assembly and activity. Recent advances in RNA sequencing methodologies for B. thetaiotaomicron provide the foundation for these integrative studies by establishing high-resolution transcriptome maps across multiple growth conditions.

Metabolomic analyses can provide functional readouts of ATP synthase activity by measuring cellular energy charge, redox status, and metabolic fluxes. When combined with genetic manipulation of atpE, metabolomics can reveal the consequences of altered ATP synthase function on global metabolism. This is particularly relevant given the sophisticated polysaccharide utilization capabilities of B. thetaiotaomicron and its metabolic adaptations to oxidative stress . Finally, in vivo studies using gnotobiotic animal models with defined microbiota can assess how atpE variants affect B. thetaiotaomicron colonization, persistence, and interactions with other microbiome members. The fitness advantage conferred by different B. thetaiotaomicron capsular polysaccharides varies depending on the resident microbiota , suggesting that similar context-dependent effects might be observed for atpE variants with altered energy metabolism characteristics.