Recombinant Enterococcus faecalis ATP synthase gamma chain (atpG)

What is the structure and function of the ATP synthase gamma chain in Enterococcus faecalis?

The ATP synthase gamma chain (atpG) in Enterococcus faecalis is a critical subunit of the F₁F₀-ATP synthase complex, which is essential for bacterial energy metabolism. Structurally, the gamma chain forms an asymmetric central stalk connecting the membrane-embedded F₀ sector with the catalytic F₁ sector. It functions as the rotary element that converts the proton motive force into mechanical energy to drive ATP synthesis.

The gamma chain in E. faecalis shares structural homology with other bacterial ATP synthases, particularly those from other Gram-positive bacteria. The protein contains conserved domains that interact with both the c-ring of the F₀ sector and the alpha/beta subunits of the F₁ sector. During ATP synthesis, proton translocation through the F₀ sector drives rotation of the c-ring, which forces the gamma chain to rotate within the F₁ sector, inducing conformational changes in the catalytic sites that facilitate ATP synthesis .

How does the gamma chain of ATP synthase differ between E. faecalis and other bacterial species?

These sequence differences may affect:

• The efficiency of rotational coupling between F₀ and F₁ sectors

• Interactions with inhibitors and antibiotics targeting ATP synthase

• Thermal stability and pH dependence of enzymatic activity

• Interactions with other bacterial proteins specific to E. faecalis

Such differences are particularly important when considering E. faecalis as an opportunistic pathogen, as these unique features might contribute to its virulence or survival under inflammatory conditions, such as those found in inflammatory bowel diseases (IBD) .

Why is studying recombinant E. faecalis ATP synthase gamma chain important for understanding bacterial energetics?

Studying the recombinant E. faecalis ATP synthase gamma chain is crucial for understanding bacterial bioenergetics for several reasons:

First, E. faecalis is a commensal organism that can become an opportunistic pathogen, particularly in nosocomial infections. Understanding its energy metabolism is key to understanding its survival strategies in different host environments .

Second, as a member of the human intestinal core microbiota, E. faecalis plays a role in gut homeostasis, but its increased abundance has been linked to inflammatory bowel diseases. The ATP synthase, as the powerhouse of cellular energy production, may contribute to the bacterium's ability to thrive in inflammatory conditions .

Third, bacterial ATP synthases represent potential antimicrobial targets. The gamma chain, with its central role in the rotary mechanism, could be targeted by novel antibiotics that disrupt energy production in E. faecalis without affecting human ATP synthases .

Finally, comparative studies of ATP synthase components across different bacterial species provide insights into evolutionary adaptations for energy metabolism in diverse ecological niches, including commensal and pathogenic lifestyles.

What are the most effective expression systems for producing functional recombinant E. faecalis atpG?

The optimal expression systems for producing functional recombinant E. faecalis atpG depend on the research objectives and downstream applications. Based on experimental approaches used for other ATP synthase components, several expression systems have proven effective:

Table 1: Comparison of Expression Systems for Recombinant ATP Synthase Components

For E. faecalis atpG expression, the rhamnose-inducible system has shown promise based on methods used for related ATP synthase components. When using E. coli as an expression host, cultures should be grown to an OD₆₀₀ of approximately 0.4, then shifted to 25°C and induced with 1 mM L-rhamnose and 0.4 mM IPTG. This approach typically allows for 14-16 hours of protein expression before harvesting cells by centrifugation .

Purification is most effectively achieved using metal affinity chromatography with Ni-NTA resin, followed by size exclusion chromatography to obtain homogeneous protein preparations suitable for structural and functional studies .

How can researchers accurately measure the activity of recombinant E. faecalis ATP synthase gamma chain in vitro?

Accurate measurement of recombinant E. faecalis ATP synthase gamma chain activity requires both isolation of the protein and reconstitution with other ATP synthase components to form a functional complex. Several complementary approaches can be employed:

ATP Synthesis Assays:
The gold standard for functional assessment is measuring ATP synthesis activity using inverted membrane vesicles containing reconstituted ATP synthase complexes. This can be accomplished by:

• Preparing inverted membrane vesicles using a French pressure cell at approximately 14,000 lb/in²

• Pre-incubating vesicles with the compounds being tested

• Energizing the membranes with NADH

• Quantifying ATP production using either the luciferin/luciferase system or the glucose-6-phosphate dehydrogenase method

Rotation Assays:
Direct observation of gamma chain rotation can be achieved through single-molecule techniques:

• Attach a fluorescent probe or gold nanoparticle to the gamma chain

• Immobilize the F₁ complex on a glass surface

• Visualize rotation using fluorescence microscopy or dark-field microscopy

• Analyze rotational velocity and step size to assess functional integrity

Binding Studies:
Interaction analysis between the gamma chain and other ATP synthase subunits provides insights into structural integrity:

• Surface plasmon resonance to measure binding kinetics

• Isothermal titration calorimetry to determine binding thermodynamics

• Native gel electrophoresis to assess complex formation

Each of these methods offers complementary information about the functional state of the recombinant gamma chain, with the ATP synthesis assay providing the most direct measure of biological activity.

What mutations in the E. faecalis atpG gene affect ATP synthase function and antimicrobial resistance?

While specific mutations in E. faecalis atpG have not been comprehensively characterized in the provided research, insights can be drawn from studies on related ATP synthase components and other bacterial species.

Key Functional Domains and Potential Mutation Sites:

The gamma chain contains several critical regions where mutations could significantly impact function:

• The C-terminal domain that interacts with the α₃β₃ hexamer

• The coiled-coil central shaft region responsible for torque transmission

• The N-terminal domain that interacts with the c-ring of F₀

Mutations in these regions could affect:

• Rotational coupling efficiency

• ATP synthesis/hydrolysis rates

• Susceptibility to ATP synthase inhibitors

Antimicrobial Resistance Mechanisms:

Studies on the ATP synthase c subunit (atpE) have identified mutations conferring resistance to antimicrobials targeting ATP synthase. The frequency of resistance mutations has been observed at rates of approximately 5.2 × 10⁻⁷ and 8.3 × 10⁻⁷ at 5× MIC in related species . Although these findings pertain to atpE rather than atpG, they suggest that similar resistance mechanisms might emerge in the gamma chain when targeted by antimicrobials.

Table 2: Potential Functional Impacts of atpG Mutations

Researchers investigating atpG mutations should consider combining site-directed mutagenesis with functional assays to systematically characterize the impact of specific amino acid substitutions on ATP synthase activity and antimicrobial susceptibility.

What are the challenges in purifying recombinant E. faecalis ATP synthase gamma chain and how can they be overcome?

Purification of recombinant E. faecalis ATP synthase gamma chain presents several challenges that researchers should address through careful methodology:

Challenge 1: Protein Solubility
The gamma chain contains both hydrophilic and hydrophobic regions, which can lead to aggregation during expression and purification.

Solution: Use a dual-detergent approach with initial solubilization using 1% N-dodecyl β-D-maltoside (as used for subunit c ), followed by buffer exchange to a milder detergent like 0.05% digitonin for long-term stability. Additionally, including 10 mM 2-mercaptoethanol in purification buffers helps prevent disulfide-mediated aggregation .

Challenge 2: Maintaining Structural Integrity
Isolated gamma chain may adopt non-native conformations without its partner subunits.

Solution: Consider co-expression with partial ATP synthase complexes (such as the α₃β₃ subcomplex) as demonstrated for Bacillus PS3 ATP synthase . Alternatively, employ stabilizing agents like glycerol (10-20%) in storage buffers.

Challenge 3: Proteolytic Degradation
The exposed regions of the gamma chain are susceptible to proteolysis during purification.

Solution: Include a comprehensive protease inhibitor cocktail during cell lysis and early purification steps. Process samples quickly and maintain cold temperatures (4°C) throughout purification.

Challenge 4: Verifying Functional State
Confirming that the purified protein retains its native functional properties.

Solution: Implement quality control steps including circular dichroism to assess secondary structure content, analytical ultracentrifugation to verify oligomeric state, and functional reconstitution with other ATP synthase components to confirm activity.

Recommended Purification Protocol:

• Express protein with a polyhistidine tag in E. coli at reduced temperature (25°C)

• Harvest cells and prepare membrane fraction

• Solubilize with 1% N-dodecyl β-D-maltoside for 60 minutes at room temperature

• Perform Ni-NTA metal-affinity chromatography

• Apply sample to size exclusion chromatography (Superdex 200)

• Verify purity using SDS-PAGE and Western blotting

• Assess functional integrity through reconstitution assays

This approach has been successful for related ATP synthase components and can be adapted specifically for E. faecalis atpG .

How can researchers effectively design genetic constructs for studying E. faecalis atpG expression and regulation?

Designing effective genetic constructs for studying E. faecalis atpG requires careful consideration of multiple factors that influence expression, regulation, and experimental utility. The following approaches are recommended:

Vector Selection and Design:

• Shuttle Vectors: Utilize S. aureus/E. coli shuttle vectors similar to pAJ96, which allow for manipulation in E. coli and subsequent transfer to Gram-positive bacteria .

• Inducible Promoters: Incorporate tetracycline-inducible or rhamnose-inducible promoter systems, which provide tight regulation of expression levels .

• Tag Placement: Position affinity tags (such as His₆ or FLAG) at either the N- or C-terminus, with flexible linkers to minimize interference with protein function.

Antisense Strategies:

For studying the physiological importance of atpG, antisense RNA approaches can be particularly valuable. This involves:

• Amplifying the target atpG fragment using primers containing appropriate restriction sites (e.g., KpnI)

• Cloning the fragment in antisense orientation relative to an inducible promoter

• Transforming the construct into E. faecalis

• Controlling expression levels using varying concentrations of inducer (0-300 ng/ml)

Reporter Fusions:

To study atpG regulation, reporter gene fusions provide valuable insights:

• Transcriptional Fusions: Clone the atpG promoter region upstream of reporter genes like lacZ or GFP

• Translational Fusions: Create in-frame fusions of atpG with reporters to monitor both expression and protein localization

CRISPR-Cas9 Modification Systems:

For precise genome editing to introduce mutations or regulatory elements:

• Design guide RNAs targeting specific regions of the atpG gene

• Provide repair templates containing desired mutations

• Screen transformants using appropriate selection markers

Table 3: Comparison of Genetic Construct Approaches for E. faecalis atpG Studies

When designing primers for construct generation, researchers should ensure appropriate restriction sites are incorporated, as demonstrated in the protocol for atpE (using primers containing KpnI sites: 5'-TAGCCAGGTACCGTCAACCAGAAGCACGTGGTC-3') .

What are the best approaches for evaluating interactions between the gamma chain and other ATP synthase subunits in E. faecalis?

Evaluating interactions between the gamma chain and other ATP synthase subunits in E. faecalis requires a multi-faceted approach combining biochemical, biophysical, and genetic techniques:

In Vitro Interaction Studies:

• Co-purification Assays: Express recombinant gamma chain with a His-tag and identify interacting partners through pull-down experiments followed by mass spectrometry.

• Surface Plasmon Resonance (SPR): Immobilize purified gamma chain on a sensor chip and measure binding kinetics of other ATP synthase subunits flowed over the surface.

• Isothermal Titration Calorimetry (ITC): Determine binding thermodynamics (ΔH, ΔS, Kd) between the gamma chain and partner subunits, particularly the α₃β₃ hexamer and c-ring components.

• Cross-linking Studies: Use chemical cross-linkers with different spacer lengths to identify proximity relationships between subunits, followed by proteomic analysis.

Structural Approaches:

• Cryo-electron Microscopy: Reconstruct the structure of the entire ATP synthase complex or subcomplexes containing the gamma chain.

• X-ray Crystallography: Determine high-resolution structures of the gamma chain alone or in complex with partner subunits.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map interaction interfaces by identifying regions protected from solvent exchange when complexes form.

Functional Assays:

• ATP Synthesis/Hydrolysis Measurements: Compare the enzymatic activity of complexes with wild-type gamma chain versus mutated versions to identify functionally important interaction sites.

• Single-Molecule Rotation Assays: Directly visualize the rotational behavior of the gamma chain within the F₁ complex using fluorescence microscopy.

Genetic Approaches:

• Bacterial Two-Hybrid Systems: Adapt bacterial two-hybrid approaches to screen for interactions between the gamma chain and other ATP synthase components.

• Suppressor Mutation Analysis: Identify second-site suppressors that restore function to gamma chain mutants, revealing important functional interactions.

• In Vivo Cross-Linking: Use genetically encoded photo-crosslinkable amino acids incorporated into the gamma chain to capture native interactions in living bacteria.

These complementary approaches provide a comprehensive understanding of how the gamma chain interacts with other ATP synthase subunits in the context of E. faecalis biology and pathogenesis.

How does E. faecalis ATP synthase contribute to bacterial virulence and inflammatory processes?

E. faecalis ATP synthase plays several important roles in bacterial virulence and inflammatory processes, particularly in the context of gastrointestinal inflammation:

Energy Production in Inflammatory Environments:

The ATP synthase complex is crucial for energy production, allowing E. faecalis to thrive in the competitive and often nutrient-limited environment of the inflamed intestine. This may contribute to the observed increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases (IBD) .

Link to Inflammatory Response:

While not directly demonstrated for ATP synthase components, related surface structures in E. faecalis have been shown to trigger inflammatory responses. Cell surface-associated lipoproteins, which require the prolipoprotein diacylglyceryl transferase (Lgt) for proper processing, are essential structures for the colitogenic activity of E. faecalis by mediating innate immune cell activation .

Role in Adaptation to Host Environment:

ATP synthase activity may contribute to E. faecalis adaptation to the host environment during disease progression. In IL-10-deficient mice, E. faecalis causes severe intestinal inflammation limited to the distal colon, characterized by defective resolution of pro-inflammatory gene expression in the intestinal epithelium .

Potential Immunogenic Properties:

As a membrane-associated protein complex, components of ATP synthase may be recognized by the host immune system. While not specifically demonstrated for the gamma chain, other surface structures like the enterococcal polysaccharide antigen (Epa) contribute to bacterial adhesion to mucosal surfaces and intestinal barrier disruption .

Research in IL-10-deficient mice has shown that E. faecalis can induce colitis with a severity score of 7.2±1.2. The colitogenic activity was partially impaired in mutants lacking certain surface structures, highlighting the potential role of membrane-associated complexes like ATP synthase in virulence and inflammatory processes .

What is the potential of E. faecalis ATP synthase gamma chain as a target for novel antimicrobials?

The E. faecalis ATP synthase gamma chain presents a promising target for novel antimicrobials due to several favorable characteristics:

Essential Function:

ATP synthase is essential for energy metabolism in E. faecalis, particularly under aerobic or microaerobic conditions. Inhibiting the gamma chain would disrupt the rotary mechanism of ATP synthesis, compromising bacterial energy production and potentially leading to growth inhibition or cell death.

Unique Structural Features:

Precedent for ATP Synthase Targeting:

Studies with other bacterial species have demonstrated the feasibility of targeting ATP synthase components. For example, investigations with S. aureus have shown that compounds inhibiting ATP synthesis can have antibacterial activity .

Potential Targeting Strategies:

• Interfering with Rotation: Small molecules that bind at the interface between the gamma chain and the α₃β₃ hexamer could block the rotational mechanism

• Disrupting Subunit Assembly: Compounds preventing proper association of the gamma chain with other ATP synthase components could inhibit complex formation

• Allosteric Inhibition: Molecules binding to regulatory sites on the gamma chain could induce conformational changes that impair function

Table 4: Advantages and Challenges of Targeting E. faecalis ATP Synthase Gamma Chain

Biochemical ATP synthesis inhibition assays, similar to those described for S. aureus using inverted membrane vesicles and luciferase-based ATP detection , would be valuable screening tools for identifying compounds targeting the E. faecalis ATP synthase gamma chain.

How can structural studies of E. faecalis atpG inform the development of species-specific inhibitors?

Structural studies of E. faecalis atpG can significantly inform the development of species-specific inhibitors through several key approaches:

Comparative Structural Analysis:

Detailed structural information about E. faecalis atpG, obtained through X-ray crystallography or cryo-electron microscopy, can be compared with structures from human mitochondrial ATP synthase and other bacterial species. This comparison would highlight:

• Unique pockets or binding sites present only in E. faecalis atpG

• Conformational dynamics specific to E. faecalis ATP synthase

• Species-specific surface electrostatic patterns that could be targeted by charged molecules

Structure-Based Drug Design:

High-resolution structural data enables computational approaches to identify and optimize potential inhibitors:

• Virtual Screening: Using the E. faecalis atpG structure to screen virtual libraries of compounds for those that fit uniquely into E. faecalis-specific binding pockets

• Fragment-Based Design: Identifying small molecular fragments that bind to specific sites on the gamma chain and linking them to create high-affinity, selective inhibitors

• Molecular Dynamics Simulations: Exploring the dynamic behavior of the gamma chain to identify transient binding pockets that might not be evident in static structures

Structure-Function Relationships:

Understanding how specific structural elements contribute to function can inform rational inhibitor design:

• Interface Mapping: Identifying critical residues at the interface between the gamma chain and other subunits

• Catalytic Mechanism: Elucidating structural changes during the catalytic cycle that could be targeted to trap the enzyme in inactive conformations

• Species-Specific Elements: Characterizing structural features unique to E. faecalis that contribute to its specific functional properties

Experimental Validation Approaches:

Structural hypotheses can be tested through:

• Site-Directed Mutagenesis: Confirming the functional importance of specific residues identified in structural studies

• Co-crystallization Studies: Obtaining structures of atpG in complex with inhibitor candidates to validate binding modes

• Hydrogen-Deuterium Exchange: Mapping conformational changes induced by inhibitor binding

The methodologies used for studying ATP synthase from Bacillus PS3, including heterologous expression in E. coli, purification using affinity chromatography, and functional reconstitution, provide a valuable framework for similar structural studies with E. faecalis atpG . By applying these approaches specifically to E. faecalis ATP synthase gamma chain, researchers can develop a structural foundation for the rational design of species-specific inhibitors with potential therapeutic applications.