Recombinant Erwinia carotovora subsp. atroseptica ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Erwinia carotovora subsp. atroseptica?

ATP synthase subunit c (atpE) in E. carotovora is a membrane protein component of the F0 sector of F-type ATP synthase. It forms a ring structure in the bacterial membrane that facilitates proton transport during ATP synthesis. The protein consists of 79 amino acids with the sequence MENLSVDLLYMAAALMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The protein functions as part of the rotary mechanism that couples proton movement across the membrane to ATP synthesis, a process crucial for bacterial energy metabolism.

What expression systems are most effective for producing recombinant E. carotovora atpE?

E. coli is the predominant expression system for recombinant E. carotovora atpE protein production. For optimal expression, researchers should consider the following methodology:

• Vector selection: pET expression systems with T7 promoters offer high-level expression for membrane proteins like atpE

• Strain optimization: E. coli strains C41(DE3) or C43(DE3) are recommended for membrane protein expression to reduce toxicity

• Expression conditions: Induction with lower IPTG concentrations (0.1-0.5 mM) at lower temperatures (16-25°C) improves proper folding

• Purification strategy: His-tag purification followed by size exclusion chromatography yields high purity

The expression produces a recombinant protein with greater than 90% purity as determined by SDS-PAGE . Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0 helps maintain protein stability.

How does the amino acid sequence of E. carotovora atpE compare with other bacterial species?

Sequence alignment analyses reveal high conservation patterns in ATP synthase subunit c across bacterial species, with interesting variations:

These variations may reflect adaptations to different membrane environments while maintaining the core proton-transporting function. Structural studies suggest these differences may influence protein-lipid interactions rather than catalytic properties .

What are the most reliable protocols for isolation and purification of recombinant E. carotovora atpE?

For successful isolation and purification of recombinant atpE, researchers should implement this optimized protocol:

• Cell lysis: Disrupt E. coli cells using a combination of enzymatic (lysozyme) and mechanical (sonication) methods in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol

• Membrane isolation: Separate membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

• Solubilization: Solubilize membrane proteins using 1% n-dodecyl-β-D-maltoside (DDM) or 2% digitonin for 2 hours at 4°C

• Affinity purification: Apply to Ni-NTA resin and wash with buffer containing 20-40 mM imidazole

• Elution: Elute with 250-300 mM imidazole

• Size exclusion: Further purify using size exclusion chromatography

This protocol typically yields >90% pure protein suitable for structural and functional studies . For long-term storage, add 50% glycerol and store at -80°C, as repeated freeze-thaw cycles significantly reduce protein activity.

How can researchers effectively design experiments to study atpE function in membrane environments?

When designing experiments to study atpE function in membrane environments, researchers should consider multiple complementary approaches:

• Reconstitution studies:

  • Incorporate purified atpE into liposomes containing specific lipid compositions

  • Measure proton translocation using pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Validate functional integrity with ATP synthesis assays coupling proton gradient to ATP production

• Site-directed mutagenesis approach:

  • Target conserved residues (particularly the essential carboxyl group that participates in proton transport)

  • Design systematic alanine scanning to identify critical functional residues

  • Quantify effects using proton pumping and ATP hydrolysis/synthesis assays

• Biophysical characterization:

  • Circular dichroism spectroscopy to assess secondary structure in different detergent or lipid environments

  • Thermal stability assays to determine protein stability under varying conditions

  • EPR or NMR studies for structural dynamics insights

These methodological approaches should be performed under controlled temperature conditions, as protein stability has been shown to be temperature-dependent in related systems .

How does E. carotovora atpE function differ from other ATP synthase subunits in energy metabolism?

ATP synthase subunit c (atpE) serves a distinct role in bacterial energy metabolism compared to other ATP synthase subunits:

• Proton channeling: atpE forms the c-ring that translocates protons across the membrane, directly participating in chemiosmotic coupling

• Energy conversion: While the β subunits (atpD) catalyze ATP synthesis/hydrolysis, atpE converts the energy of proton movement into mechanical rotation

• Inhibitor binding: atpE contains binding sites for specific inhibitors like oligomycin and dicyclohexylcarbodiimide (DCCD) that block proton translocation

• Assembly regulation: atpE assembly into the c-ring is often a rate-limiting step in ATP synthase complex formation

Research has demonstrated that the c-ring size (number of c subunits) can vary between species, affecting the bioenergetic efficiency. Experimental evidence suggests that E. carotovora atpE forms rings with 10-12 subunits, requiring translocation of 10-12 protons per ATP synthesized .

What is the significance of studying E. carotovora atpE for understanding bacterial pathogenicity?

Studying E. carotovora atpE provides several insights into bacterial pathogenicity:

• Energy requirements during infection:

  • ATP synthase is essential for providing energy during various stages of host infection

  • Experimental inhibition of ATP synthase significantly reduces virulence factor production

• Stress adaptation mechanisms:

  • Temperature-responsive gene expression studies show atpE is differentially regulated during host adaptation

  • Changes in ATP synthase activity help pathogens adapt to changing host environments

• Novel antimicrobial targets:

  • The essential nature and structural differences from host ATP synthases make atpE a potential antibiotic target

  • Binding site mapping and inhibitor screening can identify compounds that specifically target bacterial ATP synthases

Researchers have demonstrated that Pectobacterium atrosepticum (formerly E. carotovora subsp. atroseptica) adjusts its energy metabolism during plant infection, with ATP synthase genes showing altered expression patterns at different infection stages. Transposon mutagenesis studies have identified several temperature-responsive genetic elements that influence virulence factor production .

How has E. carotovora atpE evolved in comparison with other phytopathogens?

Comparative genomic and evolutionary analyses of atpE across phytopathogens reveal interesting patterns:

• Sequence conservation: Core functional domains in atpE show strong conservation across plant pathogens, particularly in regions involved in proton binding and translocation

• Adaptive variation: Regions exposed to the membrane environment show higher variation rates, possibly reflecting adaptation to different host membrane environments

• Horizontal gene transfer: Phylogenetic analysis suggests limited horizontal transfer of atpE genes compared to virulence factors

A comprehensive evolutionary study of ATP synthase components across 42 bacterial plant pathogens revealed that atpE evolution closely follows species evolution, suggesting it is primarily under purifying selection rather than diversifying selection. The genomic diversity observed in E. carotovora strains (with an average pairwise difference of 2.13% in related genes) suggests adaptation to specific ecological niches like potato plants .

How do structural differences in atpE across bacterial species correlate with functional adaptations?

Structural differences in atpE across bacterial species reveal important functional adaptations:

Researchers studying these variations typically employ a combination of structural biology techniques (X-ray crystallography, cryo-EM) and functional assays to correlate structural differences with functional adaptations. These studies contribute to our understanding of how ATP synthases have evolved to function in different environments and organisms .

What are the major technical challenges in studying E. carotovora atpE structure-function relationships?

Researchers face several significant technical challenges when investigating atpE structure-function relationships:

• Membrane protein crystallization:

  • Difficulty obtaining well-diffracting crystals of membrane proteins

  • Need for specialized detergents and lipid cubic phase methods

  • Limited success with traditional crystallization approaches

• Functional reconstitution:

  • Challenges in maintaining native-like environment for functional studies

  • Variability in reconstitution efficiency between experiments

  • Need for sensitive assays to measure proton translocation

• Assembly studies:

  • Difficulty tracking assembly intermediates of the c-ring

  • Limitations in studying interactions with other ATP synthase components

  • Need for specialized techniques to study membrane protein complexes

• In vivo relevance:

  • Connecting in vitro observations to physiological functions

  • Limited tools for manipulating atpE in E. carotovora

  • Challenges in interpreting phenotypes of genetic modifications

Advanced techniques like cryo-electron microscopy, native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry are increasingly being applied to address these challenges, though each comes with its own technical limitations and requirements for specialized equipment and expertise .

How might engineered variants of E. carotovora atpE be used to study bioenergetic mechanisms?

Engineered variants of E. carotovora atpE offer powerful tools for studying fundamental bioenergetic mechanisms:

• Site-directed mutagenesis applications:

  • Creation of proton-binding site mutants to study proton translocation mechanisms

  • Introduction of reporter groups (fluorescent, spin labels) at strategic positions

  • Engineering of modified c-rings with altered stoichiometry

• Experimental approach for coupling ratio studies:

  • Design chimeric proteins with segments from species with different c-ring sizes

  • Test ATP synthesis efficiency and proton translocation stoichiometry

  • Validate in reconstituted systems using defined proton gradients

• Methodological strategies for inhibitor studies:

  • Engineer resistance or sensitivity to specific inhibitors

  • Perform structure-guided mutations to map inhibitor binding sites

  • Develop high-throughput screening systems for novel inhibitors

These engineered variants can help resolve longstanding questions about the molecular mechanism of proton translocation, the structural basis of rotary catalysis, and the evolutionary adaptation of ATP synthases to different environments. Researchers typically combine molecular biology techniques with biophysical measurements to characterize these engineered variants .

What contradictions exist in current research regarding E. carotovora atpE function and how might they be resolved?

Several unresolved contradictions and knowledge gaps exist in current research on E. carotovora atpE:

• Stoichiometry determination:

  • Different methods yield conflicting results for c-ring stoichiometry

  • Resolution approach: Combined structural (cryo-EM) and functional (ATP/H+ ratio) measurements on the same preparations

• Lipid interactions:

  • Contradictory findings on the role of specific lipids in atpE function

  • Resolution approach: Systematic lipid composition studies with defined reconstitution systems

• Assembly pathways:

  • Competing models for c-ring assembly and integration into ATP synthase

  • Resolution approach: Time-resolved pulse-chase experiments and isolation of assembly intermediates

• Regulatory mechanisms:

  • Conflicting data on regulatory modifications of atpE under stress conditions

  • Resolution approach: Comprehensive post-translational modification mapping under defined conditions

• Growth condition adaptation:

  • Inconsistent findings on how atpE expression/function adapts to different growth conditions

  • Resolution approach: Standardized growth protocols and multi-omics approaches integrating transcriptomics, proteomics, and metabolomics data

These contradictions highlight areas where methodological differences may be contributing to conflicting results. Future research using standardized protocols, improved structural biology techniques, and integrated multi-omics approaches will be essential to resolve these questions .