Recombinant Escherichia fergusonii ATP synthase subunit c (atpE)

What is the ATP synthase subunit c in E. fergusonii and how does it compare to other bacterial species?

ATP synthase subunit c (also known as subunit III) in E. fergusonii is a small, hydrophobic protein component of the F0 sector of ATP synthase that forms a ring structure embedded in the membrane. This ring plays a critical role in the rotational mechanism that couples proton translocation to ATP synthesis.

While specific data on E. fergusonii ATP synthase subunit c remains limited, comparative analysis suggests similarities to E. coli's subunit c structure. In ATP synthases across species, the c-subunit contains two membrane-spanning α-helices connected by a short polar loop, with a conserved carboxyl residue crucial for proton translocation . The specific amino acid sequence may vary between species, but the functional principles remain conserved across bacterial ATP synthases.

What expression systems have been successfully used for recombinant production of ATP synthase subunit c?

Several expression systems have been validated for recombinant production of ATP synthase subunit c, which can be adapted for E. fergusonii atpE expression:

• E. coli expression systems: Most commonly used due to high yield potential and established protocols. For example, the c-subunit from spinach chloroplast ATP synthase has been successfully expressed in E. coli using the pMAL-c2x vector system with a maltose-binding protein (MBP) fusion tag .

• Co-expression with chaperones: The pOFXT7KJE3 plasmid expressing chaperone proteins DnaK, DnaJ, and GrpE has been shown to substantially increase quantities of recombinant proteins that are otherwise difficult to produce .

• His-tagged constructs: C-terminal hexahistidine tags have been successfully applied to E. coli F0 c-subunit, yielding functional protein that can form proton-translocating complexes .

Table 1: Comparison of Expression Systems for Recombinant ATP Synthase Subunit c

How can one verify the correct secondary structure of purified recombinant ATP synthase subunit c?

Verification of the correct secondary structure of purified recombinant ATP synthase subunit c is crucial for ensuring functionality. Multiple complementary approaches should be employed:

• Circular Dichroism (CD) Spectroscopy: This technique can confirm the alpha-helical secondary structure that is characteristic of ATP synthase subunit c. The expected CD spectrum should show negative bands at 208 nm and 222 nm, typical of alpha-helical proteins .

• Fourier Transform Infrared Spectroscopy (FTIR): Can provide additional confirmation of secondary structure elements.

• Nuclear Magnetic Resonance (NMR): For more detailed structural analysis, especially if investigating specific residues involved in proton translocation.

• Functional Assays: Complementing structural studies with functional assays such as reconstitution into liposomes and measuring proton translocation activity can provide evidence that the protein is correctly folded .

What strategies can overcome the challenges in expressing the highly hydrophobic ATP synthase subunit c of E. fergusonii?

Expressing hydrophobic membrane proteins like ATP synthase subunit c presents significant challenges that require specialized approaches:

• Fusion Partner Selection: The maltose-binding protein (MBP) fusion tag has proven effective for expressing spinach chloroplast ATP synthase subunit c . For E. fergusonii atpE, optimizing the fusion partner may improve expression and solubility.

• Codon Optimization: Design a synthetic atpE gene with codons optimized for the expression host. For E. coli expression systems, tools like Gene Designer software can assist in codon selection to enhance translation efficiency .

• Chaperone Co-expression: The co-expression of chaperone proteins (DnaK, DnaJ, and GrpE) can substantially increase yields of difficult-to-express proteins by facilitating proper folding .

• Membrane Protein Expression Protocols:

  • Use lower temperatures (16-20°C) during induction

  • Control expression rate with lower concentrations of inducer

  • Use specialized E. coli strains designed for membrane protein expression

  • Consider cell-free expression systems for toxic proteins

• Detergent Screening: Systematic screening of detergents for extraction and purification is essential for maintaining the native structure of the hydrophobic c-subunit.

How can one assess the oligomerization and ring formation of recombinant E. fergusonii ATP synthase subunit c?

Assessing oligomerization and ring formation of recombinant ATP synthase subunit c requires specialized techniques to analyze the assembly of this membrane protein:

• Blue Native PAGE: This technique can separate intact membrane protein complexes in their native state, allowing visualization of the c-ring assembly.

• Size Exclusion Chromatography (SEC): Can be used to estimate the molecular weight of the assembled c-ring and assess the homogeneity of the sample.

• Crosslinking Studies: Chemical crosslinking followed by SDS-PAGE analysis can provide information about the proximity of c-subunits in the ring structure.

• Electron Microscopy: Negative staining and cryo-EM can visualize the assembled c-ring structure directly.

• Native Mass Spectrometry: Can provide information about the stoichiometry of the intact c-ring assembly.

• Reconstitution Studies: Functional reconstitution into liposomes followed by proton translocation assays can confirm that the assembled c-ring is capable of its biological function .

What methods can differentiate between the ATP synthase c-subunits of E. fergusonii and other Escherichia species in comparative studies?

Differentiating between ATP synthase c-subunits of E. fergusonii and other Escherichia species requires techniques that can detect subtle differences in sequence, structure, or function:

• Mass Spectrometry-Based Approaches:

  • Peptide Mass Fingerprinting: Tryptic digestion followed by MALDI-TOF or ESI-MS analysis

  • Tandem MS (MS/MS): For sequence determination of specific peptides that differ between species

• Immunological Techniques:

  • Develop antibodies against unique epitopes of E. fergusonii c-subunit

  • Western blotting with species-specific antibodies

  • ELISA-based detection systems

• Genomic Analysis:

  • PCR with species-specific primers targeting unique regions of the atpE gene

  • DNA sequencing to identify single nucleotide polymorphisms (SNPs)

• Bioinformatic Comparison:

  • Sequence alignment of atpE genes across Escherichia species

  • Analysis of conserved and variable regions

  • Prediction of species-specific post-translational modifications

Table 2: Comparative Analysis of ATP Synthase Subunit c in Escherichia Species

How does the stoichiometry of the c-ring in E. fergusonii ATP synthase impact its bioenergetic efficiency?

The stoichiometry of the c-ring (the number of c-subunits in the ring) is a critical factor determining the bioenergetic efficiency of ATP synthase, as it establishes the proton-to-ATP ratio:

• Theoretical Basis: The synthesis of ATP is mechanically coupled to the rotation of the c-ring, which is driven by proton translocation. The ratio of protons translocated to ATP synthesized is determined by the number of c-subunits (n) in the ring, with 3 ATP molecules produced for every n protons .

• Experimental Approaches to Determine Stoichiometry:

  • Cryo-electron microscopy of purified c-rings

  • Atomic force microscopy (AFM) of reconstituted membranes

  • Mass spectrometry of intact c-rings

  • Cross-linking studies followed by SDS-PAGE analysis

• Physiological Implications: If E. fergusonii strains have different c-ring stoichiometries compared to other bacteria, this could reflect adaptations to specific environmental niches with different energy demands.

• Research Opportunities: Investigating whether pathogenic E. fergusonii strains, particularly those from avian sources that show higher antimicrobial resistance , have altered ATP synthase c-ring stoichiometry that might contribute to their fitness or virulence.

What is the optimal purification protocol for recombinant E. fergusonii ATP synthase subunit c?

Based on successful approaches with other ATP synthase c-subunits, the following optimized purification protocol can be adapted for recombinant E. fergusonii atpE:

• Fusion Protein Approach:

  • Express atpE as a fusion with maltose-binding protein (MBP) using the pMAL-c2x vector system

  • Transform into an appropriate E. coli strain (e.g., T7 Express lysY/Iq)

  • Induce expression at lower temperatures (16-20°C) with optimized IPTG concentration

• Initial Purification:

  • Harvest cells and lyse using a combination of enzymatic (lysozyme) and mechanical methods

  • Isolate inclusion bodies if the protein is insoluble

  • For MBP fusions, perform initial purification using amylose resin affinity chromatography

• Tag Removal and Secondary Purification:

  • Cleave the fusion tag using an appropriate protease (e.g., Factor Xa for MBP fusions)

  • Separate the cleaved protein using reverse-phase HPLC

  • Alternative approach: Use a C-terminal His-tag which has been shown to maintain functionality in E. coli c-subunit

• Final Purification and Quality Control:

  • Size exclusion chromatography to ensure homogeneity

  • Circular dichroism to verify correct secondary structure

  • SDS-PAGE and western blot to confirm purity and identity

How can functional reconstitution of E. fergusonii ATP synthase c-subunit be achieved and validated?

Functional reconstitution of the ATP synthase c-subunit is essential for confirming its biological activity, particularly its ability to form proton-conducting channels:

• Reconstitution into Liposomes:

  • Prepare liposomes using a mixture of phospholipids that mimic bacterial membranes

  • Incorporate purified c-subunit using detergent-mediated reconstitution

  • Remove detergent through dialysis or biobeads

• Proton Translocation Assays:

  • Load liposomes with pH-sensitive fluorescent dyes (e.g., ACMA, pyranine)

  • Establish a pH gradient across the liposome membrane

  • Monitor fluorescence changes upon addition of ionophores or establishment of membrane potential

  • Compare with control liposomes lacking the c-subunit

• Full ATP Synthase Reconstitution:

  • For more comprehensive functional studies, reconstitute the c-ring with other components of the F0 complex

  • Add purified F1 sector to test complete ATP synthase activity

  • Measure ATP synthesis driven by artificially imposed proton gradients

• Validation Methods:

  • Demonstrate DCCD-inhibitable proton translocation (DCCD binds to the conserved carboxyl residue in subunit c)

  • For His-tagged constructs, show that the reconstituted complex displays biochemical activities similar to the wildtype enzyme: DCCD-inhibitable ATPase activity, ATP synthase activity, and ATP-dependent proton pumping

How might the study of E. fergusonii ATP synthase subunit c contribute to understanding antimicrobial resistance mechanisms?

E. fergusonii is increasingly recognized as an emerging pathogen with zoonotic potential and a reservoir of antimicrobial resistance . Research on its ATP synthase may reveal important connections to resistance mechanisms:

• Energy-Dependent Resistance Mechanisms: Many antimicrobial resistance mechanisms require energy, which is provided by ATP synthase. Understanding the specific properties of E. fergusonii ATP synthase could reveal adaptations that support resistance phenotypes.

• Research Approaches:

  • Compare ATP synthase efficiency between antimicrobial-resistant and susceptible strains

  • Investigate whether mobile genetic elements carrying resistance genes affect ATP synthase gene expression

  • Examine potential interactions between resistance proteins and ATP synthase components

• Source-Specific Variations: Avian and porcine strains of E. fergusonii carry significantly higher numbers of antimicrobial resistance genes and mobile genetic elements compared to bovine strains . Research should investigate whether these strains also show adaptations in their ATP synthase.

Table 3: Correlation Between Source of E. fergusonii Isolation and Genetic Features

Data adapted from comparative genomic analysis

What are the potential applications of studying E. fergusonii ATP synthase c-subunit in developing targeted antimicrobials?

ATP synthase is essential for bacterial survival and represents a potential target for novel antimicrobials. Studies of E. fergusonii ATP synthase c-subunit could contribute to this field in several ways:

• Target-Based Drug Design:

  • Identification of unique structural features in E. fergusonii ATP synthase c-subunit

  • In silico screening of compounds that selectively bind to E. fergusonii ATP synthase

  • Development of species-specific inhibitors that could target this emerging pathogen

• Screening Approaches:

  • Functional assays using reconstituted E. fergusonii ATP synthase to screen compound libraries

  • Comparison of inhibition profiles between E. fergusonii and human ATP synthase to ensure selectivity

• Mode of Action Studies:

  • Investigation of how existing antimicrobials affect ATP synthase function

  • Identification of potential synergies between ATP synthase inhibitors and conventional antibiotics

• Resistance Evolution Monitoring:

  • Studies of how mutations in the atpE gene might confer resistance to ATP synthase inhibitors

  • Understanding the fitness costs of such mutations

What are common challenges in recombinant expression of ATP synthase subunit c and their solutions?

Researchers frequently encounter specific challenges when working with ATP synthase subunit c, which requires specialized troubleshooting approaches:

• Protein Toxicity and Low Expression:

  • Problem: The hydrophobic nature of subunit c can be toxic to expression hosts

  • Solutions:

    • Use tightly controlled expression systems

    • Try lower induction temperatures (16-20°C)

    • Co-express with chaperones like DnaK, DnaJ, and GrpE

    • Consider cell-free expression systems

• Inclusion Body Formation:

  • Problem: Recombinant subunit c often forms inclusion bodies

  • Solutions:

    • Optimize solubilization using different detergents

    • Develop refolding protocols specific to ATP synthase subunit c

    • Use fusion tags that enhance solubility (MBP has shown success)

• Purification Difficulties:

  • Problem: Membrane proteins are challenging to purify while maintaining structure

  • Solutions:

    • Use gentle detergents suitable for membrane proteins

    • Consider native purification approaches

    • For His-tagged constructs, optimize IMAC conditions to maintain protein integrity

• Tag Interference with Function:

  • Problem: Tags may interfere with oligomerization or function

  • Solutions:

    • Use C-terminal tags rather than N-terminal tags (shown to maintain function in E. coli)

    • Include longer linkers between the protein and tag

    • Develop efficient tag removal protocols