Recombinant Arcobacter butzleri ATP synthase subunit c (atpE)

What is the genomic context of the atpE gene in Arcobacter butzleri?

The atpE gene in A. butzleri is part of the ATP synthase operon, which is essential for energy generation in this bacterium. While the search results don't specifically mention atpE, examining the complete genome of A. butzleri strain RM4018 reveals the presence of genes required for energy production . The atpE gene, which encodes the c subunit of ATP synthase, would likely be part of this energy metabolism network, similar to other Epsilonproteobacteria.

Based on comparative analysis with related organisms like Campylobacter jejuni and Helicobacter pylori, the ATP synthase genes in A. butzleri would be expected to maintain the general organization found in the epsilon subdivision of Proteobacteria. The genomic analysis of A. butzleri shows that many of its metabolic genes share higher similarity with Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, rather than with closer taxonomic relatives .

What expression systems are most effective for producing recombinant A. butzleri ATP synthase subunit c?

For optimal expression of recombinant A. butzleri ATP synthase subunit c, E. coli-based expression systems represent the standard approach due to their versatility and high yield potential. Based on the genomic analysis of A. butzleri, which shows a G+C content of 27-30%, specialized E. coli strains optimized for low G+C content gene expression would be most suitable .

For effective expression, consider the following methodological approach:

• Gene synthesis with codon optimization for E. coli

• Cloning into a vector with an inducible promoter (T7 or tac)

• Addition of a purification tag (His6, GST, or MBP)

• Transformation into expression strains capable of membrane protein expression

Table 1: Comparison of Expression Systems for A. butzleri atpE

What purification strategies yield highest purity for recombinant A. butzleri atpE protein?

Purification of recombinant A. butzleri ATP synthase subunit c requires specialized techniques due to its hydrophobic nature as a membrane protein. Based on the physicochemical properties of similar proteins, a multi-step purification approach is recommended:

• Membrane isolation through differential centrifugation

• Selective solubilization using mild detergents (DDM, LDAO, or C12E8)

• Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

• Size-exclusion chromatography for final polishing

The critical parameter is detergent selection, which must maintain protein stability while effectively solubilizing the membrane-embedded c-subunit. Drawing from research on the ABC transporters in A. butzleri, where the YbhF protein has a molecular weight of 63.58 kDa and a theoretical isoelectric point of 5.97 , similar biophysical considerations should be applied when developing purification protocols for atpE.

How stable is recombinant A. butzleri atpE protein under different storage conditions?

The stability of recombinant A. butzleri ATP synthase subunit c varies significantly depending on storage conditions. Based on similar membrane proteins, the following stability profile can be established:

Table 2: Stability Profile of Recombinant A. butzleri atpE

Maintaining detergent concentration above the critical micelle concentration (CMC) is crucial for stability. The addition of glycerol helps prevent freeze-thaw damage. For experimental validation of protein stability, circular dichroism spectroscopy and functional assays should be performed periodically.

What structural features distinguish A. butzleri atpE from other bacterial ATP synthase c-subunits?

While specific structural data on A. butzleri atpE is not directly provided in the search results, comparisons can be made based on the bacterium's evolutionary position and environment. A. butzleri belongs to the Epsilonproteobacteria, sharing evolutionary history with Campylobacter and Helicobacter species, but showing unique adaptations .

The ATP synthase subunit c typically forms a ring structure in the membrane, with the number of c-subunits varying among species. Based on the adaptation of A. butzleri to diverse environmental conditions , its atpE protein likely contains structural modifications that optimize function across varying pH and temperature ranges.

Drawing parallels from the analysis of the YbhF ABC transporter in A. butzleri, which contains conserved sequence motifs including Walker A, Q-loop, and Walker B domains , the atpE protein would also be expected to maintain highly conserved functional domains while potentially showing variation in regions that confer environmental adaptability.

How does the unique metabolism of A. butzleri influence the function of its ATP synthase?

A. butzleri possesses a unique central metabolism with several adaptations that would directly impact ATP synthase function. The genomic analysis reveals that A. butzleri lacks the SucCD succinyl-CoA synthetase and the SdhABCD succinate dehydrogenase enzymes that are typically part of the TCA cycle . Instead, it encodes proteins with high similarities (71-85%) to the fumarate reductase FrdABC found in C. jejuni, H. hepaticus, H. pylori, and W. succinogenes .

This metabolic organization suggests that A. butzleri's ATP synthase operates under a distinct proton motive force (PMF) environment. The bacterium demonstrates fumarate reduction but no succinate oxidation, with this activity increasing under anaerobic conditions . This metabolic flexibility likely requires corresponding adaptations in the ATP synthase complex, particularly in the c-subunit ring that directly interacts with the PMF.

Research methodologies to investigate this relationship should include:

• Measurement of ATP synthesis rates under varying electron donor/acceptor conditions

• Determination of the H+/ATP ratio in isolated ATP synthase

• Site-directed mutagenesis of key residues in atpE to assess their role in coupling efficiency

• Comparative analysis of atpE sequences from A. butzleri strains from different environments

What experimental approaches can resolve the molecular mechanism of proton translocation in A. butzleri ATP synthase?

Investigating the proton translocation mechanism in A. butzleri ATP synthase requires sophisticated biophysical and biochemical techniques. Based on studies of ATP-binding proteins in A. butzleri, such as the YbhF component of the ABC transporter , similar approaches can be applied to atpE research:

• High-resolution structural analysis:

  • X-ray crystallography of the purified c-ring

  • Cryo-electron microscopy of the intact ATP synthase complex

  • NMR spectroscopy of isotopically labeled atpE protein

• Functional analysis:

  • Reconstitution into liposomes for proton pumping assays

  • Site-directed mutagenesis of conserved proton-binding residues

  • Patch-clamp electrophysiology of reconstituted ATP synthase

• Computational approaches:

  • Molecular dynamics simulations of the c-ring in a lipid bilayer

  • Quantum mechanics/molecular mechanics calculations of proton transfer energetics

  • Comparative genomics across Epsilonproteobacteria to identify conserved functional elements

Table 3: Critical Residues for Investigation in A. butzleri atpE

How can recombinant A. butzleri atpE be utilized to study antimicrobial resistance mechanisms?

ATP synthase represents a promising antimicrobial target, and recombinant A. butzleri atpE can be leveraged to study resistance mechanisms. Drawing parallels from research on the ABC efflux system YbhFSR in A. butzleri , several methodological approaches can be developed:

• Binding studies with known ATP synthase inhibitors:

  • Diarylquinolines (e.g., bedaquiline)

  • Oligomycin and venturicidin

  • Novel synthetic compounds

• Selection of resistant mutants:

  • In vitro passage of A. butzleri in subinhibitory concentrations of inhibitors

  • Whole-genome sequencing to identify mutations in atpE and related genes

  • Site-directed mutagenesis to confirm causative mutations

• Structure-function studies:

  • Generation of atpE variants with predicted resistance mutations

  • Biochemical characterization of inhibitor binding affinity

  • Structural analysis of inhibitor-bound and resistant forms

The research on A. butzleri resistance demonstrates that efflux pumps significantly contribute to antimicrobial resistance . Similar investigations into ATP synthase inhibitor resistance would provide valuable insights into potential resistance mechanisms that could emerge in clinical settings.

What role does ATP synthase play in A. butzleri adaptation to environmental stresses?

A. butzleri is remarkably adaptable to diverse environmental conditions, suggesting specialized roles for its ATP synthase. The genomic analysis reveals a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with numerous respiration-associated proteins .

To investigate the role of ATP synthase in environmental adaptation, the following methodological approaches are recommended:

• Comparative genomics:

  • Analysis of atpE sequence diversity across A. butzleri strains from different environments

  • Identification of environment-specific variants

• Expression analysis:

  • qRT-PCR of ATP synthase genes under various stressors (acid, oxidative, osmotic)

  • Proteomic analysis to quantify ATP synthase subunit expression levels

• Functional assays:

  • Measurement of ATP synthesis rates under environmental stress conditions

  • Assessment of proton permeability of membrane vesicles from stressed cells

• Mutational analysis:

  • Generation of atpE point mutants to assess environmental tolerance

  • Complementation studies in ATP synthase-deficient strains

Studies of the YbhFSR transporter in A. butzleri showed no significant impact on resistance to oxidative stress but demonstrated a role in resistance to human serum . Similar comprehensive phenotypic analyses would be valuable for understanding ATP synthase's role in stress response.

How does the c-ring stoichiometry of A. butzleri ATP synthase compare to other bacterial species, and what are its functional implications?

The c-ring stoichiometry (number of c-subunits forming the ring) varies among species, typically ranging from 8-15 subunits, and directly affects the H+/ATP ratio and bioenergetic efficiency of the ATP synthase. While the exact stoichiometry for A. butzleri is not reported in the search results, methods to determine this parameter include:

• Structural determination:

  • Atomic force microscopy of isolated c-rings

  • Cryo-electron microscopy of intact ATP synthase

  • Mass spectrometry of chemically cross-linked c-rings

• Functional approaches:

  • Measurement of H+/ATP ratio in reconstituted systems

  • Comparison of ATP synthesis rates at varying PMF values

• Computational predictions:

  • Homology modeling based on related Epsilonproteobacteria

  • Molecular dynamics simulations of c-ring assembly

Table 4: Comparative c-ring Stoichiometry in Bacterial ATP Synthases

The metabolic flexibility of A. butzleri, which can grow under both aerobic and anaerobic conditions , suggests its ATP synthase may have evolved a c-ring stoichiometry that optimizes energy conversion efficiency across varying environmental conditions.