Recombinant Verminephrobacter eiseniae ATP synthase subunit c (atpE)

What is Verminephrobacter eiseniae and why is its ATP synthase subunit c of research interest?

Verminephrobacter eiseniae is a Gram-negative, flagellated, heterotrophic, catalase-negative, rod-shaped bacterium that exists as a symbiont in the nephridia (excretory organs) of earthworms, particularly Eisenia foetida. It belongs to the betaproteobacterial group and is phylogenetically related to the genus Acidovorax, though it forms a distinct clade with other earthworm symbionts . The ATP synthase subunit c (atpE) from this organism is of particular interest because it comes from a specialized symbiotic bacterium that has coevolved with earthworms for over 100 million years yet maintains genomic characteristics that differ from typical long-term symbionts . This makes its ATP synthase components potentially valuable for comparative studies of protein evolution and adaptation in symbiotic relationships.

What are the structural characteristics of the atpE protein?

The atpE protein from Verminephrobacter eiseniae is a subunit c of ATP synthase with 89 amino acids. Its full amino acid sequence is: MEIILGFVALACGLIVGLGAIGASIGIGLMGGKFLESSARQPELINELQTKMFILAGLIDAAFLIGVAIALMFAFANPFVSTLLANMPK . As a typical ATP synthase subunit c, it functions as part of the F0 sector of ATP synthase (also called F-type ATPase), which forms the membrane-embedded proton channel. The protein is highly hydrophobic, as evidenced by its amino acid composition rich in hydrophobic residues, making it well-suited for its role as a lipid-binding membrane protein .

What expression systems are used for recombinant production of this protein?

Recombinant Verminephrobacter eiseniae ATP synthase subunit c (atpE) can be expressed in multiple heterologous systems including E. coli, yeast, baculovirus, and mammalian cell expression systems . Each system offers different advantages depending on research requirements. The choice of expression system may affect protein folding, post-translational modifications, and functional properties of the recombinant protein. For researchers seeking high purity (≥85% as determined by SDS-PAGE), commercial sources offer the protein expressed in these various systems . The expression region typically encompasses amino acids 1-89, representing the full-length protein .

How does the unique evolutionary history of Verminephrobacter affect its ATP synthase structure and function?

Despite cospeciation with earthworm hosts for more than 100 million years, Verminephrobacter symbionts remarkably lack the genome reduction and AT bias typically observed in long-term vertically transmitted symbionts . This genomic stability may extend to the conservation of ATP synthase components, including the atpE subunit. Research suggests that this unusual evolutionary trajectory is likely due to biparental transmission of the symbionts, which enables genetic mixing and relieves evolutionary bottlenecks .

This unique evolutionary history presents an intriguing research opportunity to compare the structure and function of Verminephrobacter ATP synthase components with those from both free-living bacteria and obligate symbionts with reduced genomes. Scientists investigating the molecular evolution of essential energy metabolism proteins might find atpE particularly valuable as a model for understanding how symbiotic relationships influence protein conservation versus adaptation.

What are the functional implications of the lipid-binding properties of atpE in the context of ATP synthesis?

The atpE protein (ATP synthase subunit c) is described as a lipid-binding protein , which is consistent with its role in the membrane-embedded F0 sector of ATP synthase. The protein's hydrophobic regions facilitate its interaction with membrane lipids, crucial for the proper assembly and function of the ATP synthase complex. In the context of ATP synthesis, the c-subunit ring plays a central role in coupling proton translocation across the membrane to the rotary mechanism that drives ATP synthesis.

For researchers exploring structure-function relationships in ATP synthases, the specific lipid-binding properties of Verminephrobacter eiseniae atpE could provide insights into how these enzymes operate in specialized symbiotic bacteria that must adapt to the unique environment of earthworm nephridia. This adaptation might involve specific lipid interactions that optimize ATP synthase function under the low oxygen conditions preferred by Verminephrobacter eiseniae .

How can recombinant atpE be used in studies of bioenergetics in symbiotic relationships?

Recombinant Verminephrobacter eiseniae ATP synthase subunit c (atpE) provides a unique tool for investigating bioenergetics in symbiotic relationships. Since Verminephrobacter eiseniae thrives in low oxygen concentrations while maintaining the ability to grow in fully aerated media , its ATP synthase may possess adaptations for efficient energy production under varying oxygen conditions.

Researchers can use the recombinant protein to study:

• Adaptation of energy metabolism in bacterial symbionts

• Comparative bioenergetics between free-living and symbiotic bacteria

• The molecular basis for energy exchange in host-symbiont relationships

• Evolution of bioenergetic systems in long-term symbioses

Such studies might involve reconstitution experiments where recombinant atpE is incorporated into artificial membrane systems to assess its functional properties compared to ATP synthase components from other bacteria.

What purification methods are optimal for recombinant atpE?

The purification of recombinant Verminephrobacter eiseniae ATP synthase subunit c (atpE) requires specialized approaches due to its highly hydrophobic nature as a membrane protein. Based on standard practices for similar proteins, the following methodological approach is recommended:

• Expression optimization: When expressing in E. coli systems, use specialized strains designed for membrane protein expression (e.g., C41(DE3) or C43(DE3)).

• Solubilization: Effective extraction from membranes requires appropriate detergents. For ATP synthase subunit c, detergents such as n-dodecyl β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are often effective.

• Purification steps:

  • Affinity chromatography using appropriate tags (His-tag is common)

  • Size exclusion chromatography to separate protein-detergent complexes

  • Ion exchange chromatography for final polishing

• Quality assessment: SDS-PAGE analysis should confirm purity of ≥85%, consistent with commercially available standards .

• Storage: The purified protein should be stored in Tris-based buffer with 50% glycerol optimized for stability, typically at -20°C for short-term or -80°C for long-term storage .

What experimental approaches can elucidate the structure-function relationship of atpE?

Understanding the structure-function relationship of Verminephrobacter eiseniae ATP synthase subunit c requires multiple complementary experimental approaches:

Structural Analysis:

• X-ray crystallography or Cryo-EM: To determine the three-dimensional structure of the protein either individually or as part of the ATP synthase complex.

• NMR spectroscopy: Particularly useful for analyzing the dynamics of specific regions within the protein.

• Molecular dynamics simulations: To explore conformational changes during function.

Functional Analysis:

• Reconstitution into liposomes: Incorporating purified atpE into artificial membrane systems to measure proton translocation.

• Site-directed mutagenesis: Systematic modification of key residues to assess their role in function.

• ATP synthesis/hydrolysis assays: To measure the functional consequences of structural manipulations.

Interaction Studies:

• Chemical cross-linking: To identify interactions with other ATP synthase subunits.

• Native gel electrophoresis: To analyze complex formation.

• Lipid binding assays: To characterize interactions with specific membrane lipids.

These approaches can be particularly informative when comparing atpE from Verminephrobacter eiseniae with homologs from related free-living bacteria to identify adaptations related to the symbiotic lifestyle.

How can recombinant DNA technology be optimized for atpE expression?

Optimizing recombinant DNA technology for Verminephrobacter eiseniae atpE expression involves several key considerations:

Vector Design:

• Select appropriate vectors with promoters that allow controlled expression, as membrane proteins can be toxic when overexpressed.

• Include fusion tags that facilitate both purification and solubility (e.g., SUMO, MBP).

• Consider codon optimization based on the host expression system to enhance translation efficiency.

Cloning Strategy:

• The standard approach involves restriction enzyme digestion and ligation , but gateway cloning or Gibson assembly may offer advantages for membrane proteins.

• When designing primers, ensure the full expression region (amino acids 1-89) is included .

Expression Optimization:

• Test multiple expression systems (E. coli, yeast, baculovirus, mammalian cells) as each may yield different results for membrane proteins .

• For E. coli expression, evaluate different strains, media compositions, induction conditions, and growth temperatures.

• Consider expression with and without fusion partners to identify the approach yielding the highest functional protein.

Expression Verification:

• Use Western blotting with antibodies against the fusion tag or the protein itself.

• Assess membrane localization using cell fractionation procedures.

• Perform functional assays to confirm that the recombinant protein retains native properties.

How can atpE be used to study the evolutionary dynamics of the Verminephrobacter-earthworm symbiosis?

The ATP synthase subunit c (atpE) offers a valuable molecular marker for studying the evolutionary dynamics of the Verminephrobacter-earthworm symbiosis due to several key characteristics:

• Evolutionary Conservation: As part of the essential energy metabolism machinery, atpE is under selective pressure, making sequence changes potentially informative about adaptation.

• Methodological Approach:

  • Comparative sequence analysis of atpE across Verminephrobacter strains from different earthworm species can reveal patterns of coevolution.

  • Ratio of synonymous to non-synonymous substitutions can indicate selective pressures.

  • Phylogenetic analysis using atpE sequences can complement 16S rRNA-based phylogenies to refine our understanding of symbiont evolution.

• Research Questions:

  • Has atpE undergone adaptive evolution specific to different earthworm hosts?

  • Does the biparental transmission of Verminephrobacter leave signatures in atpE sequence variation?

  • How does atpE evolution compare between Verminephrobacter and free-living relatives?

This approach is particularly powerful when integrated with whole-genome analyses to understand how symbiosis shapes the evolution of core metabolic functions.

What insights can comparative analysis of ATP synthase components provide about energy metabolism in symbiotic bacteria?

Comparative analysis of ATP synthase components, including atpE, from Verminephrobacter eiseniae and other bacteria can provide significant insights into symbiotic energy metabolism:

Research Approach:

• Compare sequences and structures of ATP synthase components from:

  • Verminephrobacter eiseniae

  • Free-living relatives (e.g., Acidovorax species)

  • Other bacterial symbionts with different degrees of genome reduction

• Analyze adaptations in relation to the ecological niche:

  • Adaptations for low oxygen conditions preferred by Verminephrobacter

  • Modifications related to the chemical environment of earthworm nephridia

• Functional studies:

  • Compare ATP synthesis efficiency under varying conditions

  • Assess proton conductance properties of the F0 sector

Expected Insights:

• Understanding how energy metabolism is optimized in symbiotic relationships

• Identifying adaptations that reflect the metabolic integration between host and symbiont

• Revealing how biparental transmission may influence the evolution of bioenergetic systems compared to strictly vertical transmission

This research direction connects the molecular structure of atpE to broader questions about metabolic coevolution in symbiotic systems.