Recombinant Bacteroides fragilis ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Bacteroides fragilis and how is it characterized?

ATP synthase subunit c (atpE) is a critical component of the F₀ domain of bacterial ATP synthase, which functions in the energy metabolism pathway. In B. fragilis, as in other bacteria, this protein forms a ring structure in the membrane that facilitates proton translocation coupled to ATP synthesis. The protein is typically characterized by:

• Small size (approximately 8-10 kDa)

• High hydrophobicity due to its membrane-embedded nature

• Conserved sequence features for ion binding and rotation

• Integration into the larger ATP synthase complex

While specific atpE characterization is not detailed in the available literature, B. fragilis demonstrates sophisticated energy metabolism pathways, including the presence of cytochrome b-dependent electron transport systems where fumarate serves as the terminal electron acceptor . These systems allow B. fragilis to generate approximately 4.5 mol of ATP per mole of glucose when heme is available, compared to only 1.7 mol of ATP per mole of glucose without heme .

How does the ATP synthase function relate to B. fragilis metabolism and survival?

B. fragilis, as an obligate anaerobe, possesses distinctive metabolic adaptations including:

• Dual pathways for energy generation depending on environmental conditions

• Cytochrome-dependent electron transport with fumarate as terminal electron acceptor

• Modified central metabolism that compensates for anaerobic growth

The ATP synthase complex plays a crucial role in these adaptations. Research indicates that B. fragilis has a bimodal system for central metabolism, utilizing different pathways depending on environmental conditions. For instance, when examining Krebs cycle components, B. fragilis has demonstrated the presence of both oxidative and reductive branches for α-ketoglutarate biosynthesis . This metabolic flexibility likely extends to energy conservation mechanisms involving ATP synthase.

This metabolic flexibility suggests that ATP synthase components, including atpE, may play critical roles in B. fragilis adaptation to different growth environments .

What expression systems are most suitable for recombinant production of B. fragilis atpE?

When designing expression systems for B. fragilis atpE, researchers should consider:

• Host selection: E. coli is commonly used, but careful consideration of strain is necessary due to potential toxicity of membrane proteins. C41(DE3) or C43(DE3) strains may be preferable for membrane protein expression.

• Vector design: For membrane proteins like atpE, vectors with moderate to low-level expression control are recommended to prevent aggregation. Consider:

  • pET vectors with tunable expression

  • pBAD vectors with arabinose-inducible promoters

  • Fusion tag strategies to enhance solubility

• Growth conditions: Based on B. fragilis culture approaches, optimized conditions include:

  • Reduced induction temperature (16-25°C)

  • Extended, slow induction periods

  • Anaerobic or microaerobic conditions may improve folding

• Solubilization strategies: Since atpE is a highly hydrophobic membrane protein, appropriate detergents for extraction are critical, typically:

  • Mild detergents like DDM or LMNG

  • Lipid supplementation during purification

Similar approaches have been used successfully for membrane proteins from anaerobic bacteria, including those from the Bacteroidetes phylum .

What safety considerations should be implemented when working with recombinant B. fragilis proteins?

Safety considerations for recombinant B. fragilis protein work should follow established guidelines for recombinant DNA research, which originated from the historic Asilomar Conference recommendations :

• Biosafety level assessment: B. fragilis is typically handled at BSL-2, but recombinant constructs should be evaluated based on:

  • Nature of the inserted DNA sequence

  • Expression host characteristics

  • Potential for biological activity

• Containment procedures:

  • Physical containment (laboratory practices and safety equipment)

  • Biological containment (use of attenuated host strains)

• Vector selection: Use approved vectors with limited host range that meet NIH guidelines for recombinant DNA research . Following the precedent established at Asilomar, self-replicating recombinant DNA molecules should be handled according to guidelines for moderate risk work .

• Institutional oversight: All work should be approved by institutional biosafety committees, following modern implementations of guidelines first established following the 1975 Asilomar Conference .

The original recommendations from the Berg committee and Asilomar Conference remain relevant: "Until the potential hazards of such recombinant DNA molecules have been better evaluated or until adequate methods are developed for preventing their spread, scientists throughout the world should join with the members of this committee in voluntarily deferring" potentially hazardous experiments .

How can structural studies of recombinant atpE contribute to understanding B. fragilis energy metabolism?

Structural studies of recombinant B. fragilis atpE can provide critical insights into the unique aspects of energy metabolism in this anaerobe:

• Comparative structural analysis: Comparing the structure of B. fragilis atpE to homologs from aerobic bacteria could reveal adaptations specific to anaerobic environments. This is particularly relevant given the findings that B. fragilis contains enzymes with unexpected evolutionary relationships, such as their aconitase being more closely related to mitochondrial versions than to typical bacterial forms .

• Proton binding site analysis: Structural determination of the critical residues involved in proton translocation can explain how B. fragilis ATP synthase functions under anaerobic conditions with alternative electron acceptors.

• Subunit interaction mapping: Identifying interaction surfaces between atpE and other ATP synthase components could reveal differences in complex assembly or regulation compared to aerobic systems.

• Inhibitor binding studies: Structural data on inhibitor binding could lead to targeted antimicrobials against pathogenic B. fragilis.

Methodological approaches should include:

• X-ray crystallography of purified atpE rings

• Cryo-EM of the entire ATP synthase complex

• NMR studies of specific protein-protein interactions

• Molecular dynamics simulations to understand proton movement

These approaches would be similar to those used to study other unique metabolic enzymes in B. fragilis, such as the aconitase that shows close phylogenetic relationship to mitochondrial aconitases rather than typical bacterial versions .

How can recombinant atpE be used to investigate B. fragilis adaptation to environmental stresses?

Recombinant atpE can serve as a valuable tool for investigating B. fragilis adaptation to various environmental stresses, building on observations of how this organism responds to stressors like antibiotic exposure :

• Expression pattern analysis: Using purified recombinant atpE as a standard for quantification, researchers can measure native atpE expression levels under various stress conditions through techniques like:

  • Western blotting

  • Mass spectrometry-based quantification

  • RNA-seq correlation

• Mutational studies: Generating site-directed mutations in recombinant atpE can identify residues critical for:

  • Proton binding and translocation

  • Subunit interactions

  • Stability under stress conditions

• In vitro reconstitution: Reconstituting ATP synthase activity with recombinant components can determine functional changes under conditions mimicking environmental stress.

Analogous to the transcriptional investigation of B. fragilis under metronidazole exposure , researchers could examine how atpE expression and ATP synthase function change in response to:

This approach aligns with observations that B. fragilis exhibits persistent changes in gene expression patterns even after removal of stressors like metronidazole , suggesting complex adaptation mechanisms that may involve energy metabolism components like ATP synthase.

What are the main challenges in purifying functional recombinant atpE and how can they be addressed?

Purifying functional recombinant atpE presents several technical challenges:

• Membrane protein solubilization:

  • Challenge: atpE is highly hydrophobic and difficult to extract from membranes

  • Solution: Systematic screening of detergents (DDM, LMNG, digitonin) at varying concentrations; inclusion of lipids during purification to maintain native-like environment

• Maintaining oligomeric ring structure:

  • Challenge: The c-subunit functions as a ring of multiple subunits that can dissociate during purification

  • Solution: Mild solubilization conditions; chemical crosslinking approaches; purification of the entire F₀ domain

• Assessing functionality:

  • Challenge: Isolated atpE is difficult to assay functionally outside the complete ATP synthase

  • Solution: Development of reconstitution systems with other ATP synthase components; proton flux assays in proteoliposomes

• Protein aggregation:

  • Challenge: Tendency to aggregate during concentration steps

  • Solution: Addition of glycerol or arginine to purification buffers; maintaining detergent above critical micelle concentration

The approaches to maintaining protein stability during purification draw from lessons learned in handling other membrane proteins from anaerobic bacteria and could benefit from techniques used to study membrane components in B. fragilis stress response .

How can researchers accurately assess the impact of atpE mutations on ATP synthase function?

Assessing the functional impact of atpE mutations requires a multi-faceted approach:

• In vivo complementation studies:

  • Generate B. fragilis atpE knockout strain (may require conditional approaches if essential)

  • Complement with wild-type or mutant atpE

  • Assess growth under various conditions, particularly those requiring efficient energy metabolism

  • Monitor survival under stress conditions

  This approach is analogous to the mutational analysis performed for the B. fragilis aconitase gene (acnA), where a nonpolar in-frame deletion prevented growth in glucose minimal medium unless heme or succinate was added .

• Biochemical characterization:

  • Purify wild-type and mutant proteins

  • Assess oligomerization and stability using analytical ultracentrifugation or native PAGE

  • Examine proton binding using specialized pH-sensitive probes

  • Measure ATP synthesis rates in reconstituted systems

• Structural analysis:

  • Compare structures of wild-type and mutant proteins using X-ray crystallography or cryo-EM

  • Identify conformational changes that might impact function

• Energy coupling measurements:

  • Reconstitute ATP synthase with wild-type or mutant components in liposomes

  • Measure proton translocation using pH-sensitive fluorophores

  • Assess ATP synthesis/hydrolysis coupling ratios

The detection of function can be challenging in B. fragilis proteins, as demonstrated by the historical misunderstanding of its Krebs cycle components. Early studies indicated the lack of some cycle components, but later research using anaerobically prepared cell extracts revealed previously undetected enzyme activities .

How does the study of recombinant atpE integrate with broader research on B. fragilis metabolism and pathogenesis?

Research on recombinant atpE integrates with broader B. fragilis research in several key ways:

• Metabolic flexibility: B. fragilis demonstrates remarkable metabolic adaptability, with bimodal pathways for critical processes like α-ketoglutarate biosynthesis . ATP synthase likely plays a pivotal role in maintaining energy balance across these metabolic shifts, making atpE study essential for understanding the organism's core metabolism.

• Stress response integration: Transcriptomic studies have revealed that B. fragilis undergoes extensive gene expression changes under stress conditions like antibiotic exposure . Energy generation systems, including ATP synthase, likely participate in these stress responses, making atpE a valuable target for understanding adaptation mechanisms.

• Evolutionary relationships: B. fragilis contains surprising metabolic enzymes with evolutionary relationships closer to eukaryotic/mitochondrial versions than to typical bacterial homologs . Investigating whether ATP synthase components like atpE show similar evolutionary patterns could provide insights into the ancient relationships between bacterial groups and mitochondria.

• Pathogenesis mechanisms: During infection, B. fragilis must adapt to changing nutrient availability and host defense mechanisms. Understanding how ATP synthase function, regulated by atpE, contributes to this adaptation could reveal new therapeutic targets.

These integration points highlight how atpE research connects to fundamental questions about B. fragilis biology that have been explored in previous work on its metabolic pathways and stress responses .

What emerging technologies could advance research on B. fragilis atpE and other membrane proteins?

Several emerging technologies show promise for advancing research on B. fragilis atpE:

• Nanodiscs and SMALPs (Styrene-Maleic Acid Lipid Particles):

  • Allow membrane proteins to be studied in more native-like lipid environments

  • Enable structural and functional studies without conventional detergents

  • Particularly valuable for proteins like atpE whose function depends on lipid interactions

• Cryo-EM advances:

  • Improved resolution for membrane protein complexes

  • Capability to resolve heterogeneous populations

  • Potential to visualize ATP synthase in different conformational states

• Cell-free expression systems:

  • Direct synthesis of membrane proteins into artificial membranes or nanodiscs

  • Avoidance of toxicity issues common in cellular expression systems

  • Potential for incorporating non-natural amino acids for mechanistic studies

• Microfluidics-based assays:

  • High-throughput functional analysis of ATP synthase variants

  • Precise control of pH gradients for measuring proton translocation

  • Integration with fluorescence-based activity detection

• Gene editing in B. fragilis:

  • Improved CRISPR-Cas systems for anaerobic bacteria

  • Creation of conditional mutants for essential genes like atpE

  • In situ tagging for localization and interaction studies

These technologies could help overcome current limitations in studying membrane proteins from anaerobic bacteria, similar to how anaerobic preparation techniques revealed previously undetected enzyme activities in B. fragilis .