Recombinant Staphylococcus saprophyticus subsp. saprophyticus ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c in bacteria?

ATP synthase subunit c is an essential component of the F₀ membrane domain of ATP synthase that participates in transmembrane proton conduction. The subunit c proteins assemble into an oligomeric ring structure (c-ring) within the membrane domain of the ATP synthase complex. This annular architecture has been observed across different bacterial species, creating a rotary element that couples proton movement with ATP synthesis/hydrolysis. The c-ring, together with the central stalk of the soluble F₁ domain, rotates as an ensemble during this process, enabling the conversion of the proton electrochemical gradient into chemical energy in the form of ATP .

How does the amino acid sequence of ATP synthase subunit c influence its assembly and function?

The primary structure of ATP synthase subunit c determines its ability to self-assemble into ring structures. Research has demonstrated that recombinant subunit c expressed in Escherichia coli and purified in non-ionic detergent solutions can self-assemble into annular structures even in the absence of other subunits of the ATP synthase complex . This suggests that the inherent properties of the amino acid sequence directly influence the formation of c-rings. Specific amino acid residues, particularly those involved in intermolecular interactions between adjacent c subunits, are critical for proper assembly. Mutations in the atpE gene can significantly impact both assembly and function, potentially altering ATP production efficiency and sensitivity to inhibitors .

What is the stoichiometry of the c-ring in bacterial ATP synthases?

The stoichiometry of the c-ring varies among different bacterial species. In Escherichia coli, for example, the F₀ membrane domain consists of three different polypeptides in the experimentally determined ratio of a₁b₂c₁₀₋₁₁, indicating that 10-11 c subunits typically form the ring structure . This stoichiometry directly influences the bioenergetic properties of the ATP synthase, as it determines the proton-to-ATP ratio during energy conversion. The exact number of c subunits in the S. saprophyticus ATP synthase has not been definitively established, but based on related Staphylococcal species, it likely follows similar patterns observed in other Bacillales.

What are the optimal expression systems for producing recombinant ATP synthase subunit c?

The most effective expression system for recombinant ATP synthase subunit c production is E. coli. When designing an expression protocol, researchers should consider the following methodological approach:

• Select an appropriate E. coli strain (such as DH5α for initial cloning and BL21(DE3) for protein expression)

• Design a codon-optimized sequence of the atpE gene to enhance expression efficiency

• Clone the atpE gene into a suitable expression vector containing:

  • An inducible promoter (such as T7)

  • An appropriate antibiotic resistance marker

  • A purification tag (His-tag is commonly used)

• Transform the expression host and optimize induction conditions including:

  • IPTG concentration (typically 0.5-1.0 mM)

  • Induction temperature (typically 18-30°C)

  • Induction duration (4-18 hours)

Overexpression of membrane proteins like atpE can be challenging due to potential toxicity and inclusion body formation. Therefore, parameters such as growth temperature, inducer concentration, and expression duration should be carefully optimized .

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

Purifying recombinant ATP synthase subunit c requires specific approaches due to its hydrophobic nature and membrane integration. A recommended purification protocol includes:

• Cell lysis using sonication or French press in buffer containing protease inhibitors

• Membrane fraction isolation through differential centrifugation

• Membrane protein solubilization using non-ionic detergents (such as n-dodecyl-β-D-maltoside or Triton X-100)

• Affinity chromatography using the engineered tag (e.g., His-tag purification using Ni-NTA resin)

• Size exclusion chromatography to separate monomeric and oligomeric forms

• Purity assessment using SDS-PAGE and Western blotting

Researchers should note that the choice of detergent is critical as it significantly impacts the structural integrity and self-assembly properties of the c subunits. Non-ionic detergents are preferred as they help maintain native-like conditions that support the formation of c-rings .

How can I verify the proper folding and oligomerization of recombinant ATP synthase subunit c?

Verification of proper folding and oligomerization of recombinant ATP synthase subunit c can be achieved through multiple complementary techniques:

• Size exclusion chromatography (SEC): To determine the oligomeric state of the protein in detergent solution

• Blue native PAGE: To analyze the intact c-ring complex under non-denaturing conditions

• Circular dichroism (CD) spectroscopy: To assess secondary structure content (predominantly α-helical structure is expected)

• Transmission electron microscopy (TEM): To directly visualize the ring structures formed by purified c subunits

• Mass spectrometry: To confirm the exact molecular weight and potential post-translational modifications

Additionally, functional assays such as reconstitution into liposomes followed by proton transport measurements can provide evidence of proper assembly and functionality of the c-ring complex .

What approaches can be used to generate site-directed mutations in the atpE gene?

Generation of site-directed mutations in the atpE gene can be accomplished through several molecular biology techniques. A recommended methodological workflow includes:

• PCR-based site-directed mutagenesis:

  • Design primers containing the desired mutation

  • Perform PCR using a high-fidelity DNA polymerase

  • Digest the template DNA with DpnI to remove methylated parental DNA

  • Transform into competent E. coli cells

  • Verify mutations by DNA sequencing

• CRISPR-Cas9 genome editing (for chromosomal mutations):

  • Design guide RNAs targeting the atpE locus

  • Construct a repair template containing the desired mutation

  • Co-transform cells with the CRISPR-Cas9 system and repair template

  • Screen transformants for successful editing

  • Verify mutations using PCR and sequencing

When designing mutations, researchers should focus on conserved residues that are likely involved in proton binding or c-ring assembly. Mutations at these key positions can provide valuable insights into structure-function relationships .

How can I analyze the effects of atpE mutations on ATP synthase function?

Analysis of the effects of atpE mutations on ATP synthase function requires a multi-faceted approach:

• ATP synthesis assays:

  • Prepare inverted membrane vesicles from cells expressing wild-type or mutant atpE

  • Measure ATP production upon energization with NADH or succinate

  • Quantify ATP using luciferase-based luminescence assays

• Proton transport measurements:

  • Reconstitute purified ATP synthase into liposomes

  • Monitor pH changes using fluorescent probes (e.g., ACMA or pyranine)

  • Calculate proton transport rates under various conditions

• Growth phenotype analysis:

  • Assess growth rates in media requiring oxidative phosphorylation

  • Determine minimum inhibitory concentrations (MICs) of ATP synthase inhibitors

  • Analyze growth under different pH and energy source conditions

• Structural analysis:

  • Examine c-ring assembly using native PAGE or electron microscopy

  • Investigate structural changes using circular dichroism or NMR spectroscopy

When analyzing data from these experiments, it's important to consider that mutations in atpE might impact multiple aspects of ATP synthase function, including assembly, proton binding, rotational coupling, and interaction with other subunits of the complex .

What is the relationship between atpE mutations and antibiotic resistance in Staphylococcal species?

The relationship between atpE mutations and antibiotic resistance represents an important area of research for Staphylococcal species. Studies on S. aureus have demonstrated that mutations in the atpE gene can confer resistance to certain antibiotics that target ATP synthase, such as tomatidine and its derivatives. The mechanism involves:

• Target site modification: Specific mutations in atpE alter the binding site for ATP synthase inhibitors, reducing their affinity and effectiveness

• Functional adaptation: Resistant mutants often show altered ATP synthesis capabilities, suggesting a trade-off between resistance and normal energy metabolism

• Cross-resistance patterns: Some atpE mutations may confer cross-resistance to multiple ATP synthase inhibitors

Research has shown that when S. aureus strains with mutations in atpE were analyzed, they exhibited high-level resistance to tomatidine (TO) with MICs increasing from 0.06 μg/ml to >64 μg/ml. Interestingly, these resistant mutants also showed further reduced ATP production compared to the parental strain, indicating that the mutations that confer resistance also impact the normal functioning of ATP synthase .

For experimental validation of resistance mechanisms, researchers can:

• Overexpress wild-type or mutant atpE genes in susceptible strains

• Measure MICs against various ATP synthase inhibitors

• Perform ATP synthesis assays to quantify the functional impact of mutations

This approach has been demonstrated with S. aureus, where overexpression of the atpE gene in a susceptible background provided resistance to inhibitors like tomatidine and its derivative FcM .

What methods can be used to assess ATP synthase activity in bacterial membrane preparations?

Assessment of ATP synthase activity in bacterial membrane preparations can be performed using several complementary approaches:

• ATP synthesis assay:

  • Prepare inverted membrane vesicles from bacterial cells

  • Energize the vesicles with electron transport chain substrates (NADH or succinate)

  • Quantify ATP production over time using luciferase-based luminescence assays

  • Calculate synthesis rates under various conditions (pH, inhibitor concentrations)

• ATP hydrolysis assay:

  • Measure phosphate release from ATP using colorimetric methods (e.g., malachite green)

  • Monitor NADH oxidation coupled to ATP hydrolysis in a regenerating system

  • Calculate hydrolysis rates and inhibition profiles

• Proton pumping assay:

  • Use fluorescent pH indicators (ACMA, pyranine) to monitor proton movement

  • Measure fluorescence changes upon energization or ATP addition

  • Quantify proton translocation rates and stoichiometry

A detailed protocol for the ATP synthesis assay using inverted membrane vesicles includes:

• Bacterial cell culture and harvesting by centrifugation

• Cell lysis by French press or sonication in appropriate buffer

• Differential centrifugation to isolate membrane vesicles

• Energization of vesicles with NADH (typically 0.5-1 mM)

• Reaction in synthesis buffer containing ADP and Pi

• Sampling at timed intervals and ATP quantification

• Data analysis and calculation of synthesis rates

This methodology has been successfully applied to study ATP synthase activity in various bacterial species, including S. aureus where mutations in atpE led to significantly reduced ATP production in resistant strains .

How can the interaction between ATP synthase inhibitors and the c-ring be characterized?

Characterizing the interaction between ATP synthase inhibitors and the c-ring requires a combination of biochemical, biophysical, and computational approaches:

• Inhibition assays:

  • Determine IC₅₀ values using ATP synthesis or hydrolysis assays

  • Generate dose-response curves with wild-type and mutant enzymes

  • Analyze inhibition kinetics to distinguish between competitive, non-competitive, or uncompetitive modes

• Binding studies:

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Surface plasmon resonance (SPR) to determine association/dissociation kinetics

  • Fluorescence-based binding assays using labeled inhibitors or proteins

• Structural analyses:

  • X-ray crystallography or cryo-EM of inhibitor-bound c-rings

  • NMR spectroscopy to map binding interfaces

  • Molecular docking and molecular dynamics simulations

• Resistance profiling:

  • Generate and characterize resistant mutants

  • Map resistance mutations to the c-ring structure

  • Correlate structural changes with altered inhibitor sensitivity

Research with S. aureus has demonstrated that mutations in the atpE gene conferring resistance to tomatidine and its derivatives result in significantly higher IC₅₀ values (>512 μg/ml compared to much lower values in wild-type strains). This indicates altered binding or interaction between the inhibitor and its target site on the c-ring .

How does the ATP synthase c subunit differ across bacterial species, and what are the evolutionary implications?

The ATP synthase c subunit shows both conservation and variation across bacterial species, with important evolutionary implications:

• Sequence conservation and variability:

  • Highly conserved motifs within the transmembrane domains, particularly those involved in proton binding and translocation

  • Variable regions that may reflect adaptation to different environmental conditions

  • Species-specific differences in the number of c subunits per ring (ranging from 8-15)

• Structural implications:

  • The core structure of hairpin-like arrangement of two transmembrane helices is preserved

  • The conserved proton-binding site typically includes a critical glutamate or aspartate residue

  • The c-ring diameter varies with the number of c subunits, affecting the H⁺/ATP ratio

• Functional consequences:

  • Different c-ring stoichiometries result in different H⁺/ATP ratios

  • Environmental adaptations (pH, temperature, salt) may select for specific variants

  • Species-specific inhibitor sensitivities reflect structural differences

• Evolutionary analysis approaches:

  • Multiple sequence alignment to identify conserved and variable regions

  • Phylogenetic tree construction to understand evolutionary relationships

  • Ancestral sequence reconstruction to infer evolutionary pathways

For Staphylococcal species, the ATP synthase subunit c shows significant sequence conservation within the Bacillales order. This conservation explains why inhibitors like tomatidine show a narrow yet specific spectrum of activity against the Small Colony Variants (SCVs) of Bacillales. The specificity is attributed to conserved amino acid sequences in the ATP synthase subunit c across species of this order .

What methods are recommended for studying c-ring assembly in vitro and in vivo?

Studying c-ring assembly requires complementary in vitro and in vivo approaches:

In vitro methods:

• Detergent-mediated reconstitution:

  • Purify recombinant c subunits in appropriate detergents

  • Analyze self-assembly using size exclusion chromatography

  • Verify ring formation using electron microscopy or native PAGE

  • Investigate the effects of lipids, pH, and ionic conditions on assembly

• Lipid bilayer reconstitution:

  • Incorporate purified c subunits into liposomes or nanodiscs

  • Assess functional properties through proton translocation assays

  • Investigate the impact of lipid composition on assembly and function

• Crosslinking studies:

  • Use chemical crosslinkers to stabilize assembled c-rings

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

  • Identify intersubunit contacts that drive assembly

In vivo methods:

• Fluorescent protein fusions:

  • Generate functional fusions of c subunits with fluorescent proteins

  • Monitor localization and assembly using fluorescence microscopy

  • Employ FRET to study subunit interactions

• Genetic approaches:

  • Use site-directed mutagenesis to identify residues critical for assembly

  • Screen for assembly-defective mutants

  • Employ suppressor analysis to identify compensatory mutations

• Inducible expression systems:

  • Control the timing and level of c subunit expression

  • Monitor assembly kinetics following induction

  • Investigate the role of assembly factors

Research has demonstrated that recombinant subunit c can form rings in the absence of other ATP synthase subunits when purified in non-ionic detergent solutions. This indicates that the primary structure of subunit c contains all necessary information for self-assembly into the characteristic ring structure .

How can molecular modeling and simulation contribute to understanding ATP synthase function?

Molecular modeling and simulation provide powerful tools for understanding ATP synthase function at atomic resolution:

• Homology modeling:

  • Generate 3D models of S. saprophyticus ATP synthase components based on homologous structures

  • Refine models using energy minimization and molecular dynamics

  • Validate models against experimental data

• Molecular dynamics simulations:

  • Simulate c-ring behavior in lipid bilayers

  • Investigate proton translocation pathways and mechanisms

  • Analyze conformational changes during rotation

• Quantum mechanics/molecular mechanics (QM/MM):

  • Study proton binding and transfer at a quantum mechanical level

  • Calculate energetics of proton translocation

  • Investigate the role of key residues in proton coordination

• Docking and virtual screening:

  • Predict binding modes of known inhibitors

  • Screen virtual compound libraries for potential inhibitors

  • Design improved inhibitors based on structure-activity relationships

• Integration with experimental data:

  • Use simulation to interpret experimental results

  • Generate testable hypotheses for experimental validation

  • Refine models based on new experimental findings

A comprehensive approach would include:

• Building a homology model of the S. saprophyticus c-ring based on related structures

• Embedding the model in a lipid bilayer mimicking bacterial membranes

• Running extensive molecular dynamics simulations to analyze stability and dynamics

• Simulating the effects of mutations identified in experimental studies

• Calculating the energetics of inhibitor binding and the impact of resistance mutations

These computational approaches complement experimental studies and provide insights into mechanisms that may be challenging to access experimentally, such as the precise pathway of proton movement through the c-ring or the atomic details of inhibitor interactions .

What is the potential of ATP synthase subunit c as a target for novel antimicrobials?

ATP synthase subunit c represents a promising target for novel antimicrobial development for several compelling reasons:

• Essential cellular function:

  • ATP synthase is critical for energy metabolism in bacteria

  • In Bacillales, ATP synthase function is particularly important for survival, especially in Small Colony Variants (SCVs) with altered respiratory chains

  • Complete deletion of ATP synthase in Bacillus subtilis severely affects growth, indicating its essential nature

• Structural uniqueness:

  • Bacterial ATP synthase c subunits differ significantly from their mammalian counterparts

  • These structural differences enable highly selective inhibition (selectivity index >10⁵ for some compounds)

  • Conserved sequences within bacterial orders allow for spectrum-specific targeting

• Demonstrated druggability:

  • Compounds like tomatidine and its derivatives effectively target ATP synthase

  • Specific binding sites on the c-ring have been identified through resistance mutations

  • Structure-activity relationships can guide further optimization

• Effectiveness against persistent infections:

  • Small Colony Variants (SCVs) of Staphylococcal species are often implicated in persistent infections

  • These variants show increased susceptibility to ATP synthase inhibitors

  • Targeting energy metabolism is particularly effective against slow-growing, persistent bacteria

• Reduced resistance development:

  • Some ATP synthase inhibitors like FC04-100 prevent high-level resistance development

  • Resistance mutations often come with fitness costs (further reduced ATP production)

  • Combining ATP synthase inhibitors with other antimicrobials may reduce resistance development

Research on S. aureus has demonstrated that targeting the ATP synthase is a viable approach for antibacterial development, particularly against persistent forms like SCVs. The narrow yet specific spectrum of activity of compounds like tomatidine against Bacillales suggests similar potential for targeting S. saprophyticus ATP synthase .

How can the selection and characterization of resistant mutants inform inhibitor development?

Selection and characterization of resistant mutants provide valuable insights for inhibitor development through the following methodological approaches:

• Selection of resistant mutants:

  • Expose bacteria to increasing concentrations of inhibitors (step-wise selection)

  • Use chemical mutagenesis to increase mutation frequency

  • Select mutants on media containing inhibitor concentrations above the MIC

  • Verify stability of resistance phenotype through multiple passages

• Genetic characterization:

  • Sequence the atpE gene and the entire ATP synthase operon

  • Perform whole-genome sequencing to identify compensatory mutations

  • Create gene knockout and complementation strains to confirm the role of identified mutations

  • Overexpress mutant atpE genes in susceptible backgrounds to validate their contribution to resistance

• Biochemical characterization:

  • Determine MICs of various inhibitors against resistant mutants

  • Prepare membrane vesicles and measure ATP synthesis inhibition (IC₅₀ values)

  • Compare ATP production capacity between wild-type and resistant strains

  • Evaluate fitness costs through growth rate analysis

• Structural analysis:

  • Map resistance mutations onto structural models

  • Identify binding sites and interaction networks

  • Predict cross-resistance patterns based on structural information

• Application to inhibitor development:

  • Design inhibitors that bind to conserved regions less prone to mutations

  • Develop inhibitors that maintain activity against resistant mutants

  • Create combination strategies targeting multiple sites

Research with S. aureus has shown that in vitro-generated tomatidine-resistant SCVs carried mutations in the atpE gene. Sequence analysis and structural modeling revealed the consequences of these mutations, while functional studies demonstrated that these mutations further impaired ATP production. This comprehensive characterization informed the development of derivatives like FC04-100, which prevents high-level resistance development in prototypic strains and limits resistance in SCVs .

This table demonstrates how introducing specific atpE mutations or overexpressing atpE can confer resistance to ATP synthase inhibitors, providing crucial information for inhibitor optimization .

What experimental design considerations are critical when evaluating ATP synthase inhibitors in vitro and in vivo?

When evaluating ATP synthase inhibitors, careful experimental design is essential for generating reliable and translatable results:

In vitro evaluation:

• Assay selection and validation:

  • Include both target-based (ATP synthase) and phenotypic (growth inhibition) assays

  • Validate assays using known inhibitors and proper controls

  • Ensure reproducibility through multiple independent experiments

  • Design dose-response studies covering a wide concentration range

• Species and strain selection:

  • Test against multiple relevant Staphylococcal species and strains

  • Include both standard laboratory strains and clinical isolates

  • Compare activity against normal and SCV phenotypes

  • Consider testing related species to determine spectrum of activity

• Resistance development assessment:

  • Perform serial passage experiments under inhibitor pressure

  • Determine frequency of resistance

  • Characterize resistant mutants genetically and phenotypically

  • Assess cross-resistance to other antimicrobials

• Cytotoxicity and selectivity evaluation:

  • Test against mammalian cell lines to determine safety margins

  • Calculate selectivity indices (ratio of mammalian to bacterial inhibition)

  • Assess effects on mitochondrial function in mammalian cells

  • Investigate potential off-target effects

In vivo evaluation:

• Model selection:

  • Choose infection models relevant to clinical presentations

  • Consider both acute and chronic/persistent infection models

  • Include models that reflect the physiological niches of the pathogen

  • Design models to test specific hypotheses about inhibitor action

• Study design:

  • Determine appropriate sample sizes using power calculations

  • Include proper control groups (vehicle, standard-of-care antibiotics)

  • Blind investigators to treatment assignments when possible

  • Randomize animals to treatment groups

• Pharmacokinetic/pharmacodynamic (PK/PD) considerations:

  • Characterize pharmacokinetics in relevant tissues

  • Determine PK/PD indices that correlate with efficacy

  • Design dosing regimens based on PK/PD principles

  • Monitor drug levels during efficacy studies

• Outcome measures:

  • Include microbiological endpoints (bacterial burden)

  • Measure relevant clinical endpoints (survival, clinical scores)

  • Assess potential development of resistance during treatment

  • Consider long-term follow-up to detect relapse

Statistical analysis should employ appropriate methods considering the experimental design. This might include ANOVA for comparing multiple groups, survival analysis for time-to-event data, and regression models for dose-response relationships. Proper experimental design requires constructing the design (including randomization and determining required replicates), executing the plan to collect data, determining appropriate models, fitting models to data, and interpreting results meaningfully .