Recombinant Geobacter sulfurreducens ATP synthase subunit c (atpE)

What is the amino acid sequence and structural characteristics of Geobacter sulfurreducens atpE?

The ATP synthase subunit c (atpE) from Geobacter species is a membrane protein consisting of 91 amino acids with the sequence: MEFFTMCVLAAGIGMALGTLGTGIGQGLAVKSAVEGTSRNPGASGKILTTMMIGLAMIES LAIYALVVCLIILFANPYKDIALELAKSVAK . This hydrophobic protein is part of the F₀ sector of the F₀F₁ ATP synthase complex, embedded in the membrane where it forms an oligomeric ring structure. The protein contains two transmembrane α-helices connected by a polar loop, with the conserved carboxyl residue essential for proton translocation. Structural analysis indicates that atpE's hydrophobic nature allows it to function within the membrane environment while facilitating proton movement across the membrane during ATP synthesis .

How does the Geobacter atpE differ from ATP synthase subunit c in other bacterial species?

The ATP synthase subunit c in Geobacter shows distinct sequence characteristics compared to other bacterial species, particularly those from non-iron reducing bacteria. While maintaining the core functional domains, alignment studies show that Geobacter atpE shares approximately 88-91% sequence similarity with mycobacterial species like M. tuberculosis , but differs significantly in specific residues that may relate to its function in electron transfer chains.

Unlike many other bacteria, Geobacter species have adapted their ATP synthase components to function efficiently under anaerobic conditions where Fe(III) serves as the terminal electron acceptor rather than oxygen . The sequence variations in key residues likely reflect adaptations to the unique electron transport mechanisms employed by Geobacter in its subsurface, metal-reducing environment. These differences make Geobacter atpE particularly interesting for comparative studies of energy conservation mechanisms across diverse bacterial metabolic strategies.

What are the optimal conditions for heterologous expression of recombinant Geobacter sulfurreducens atpE protein?

For optimal heterologous expression of recombinant G. sulfurreducens atpE, E. coli-based expression systems have proven most effective . The protein should be expressed with an N-terminal His-tag to facilitate purification while minimizing interference with the protein's structure and function.

The recommended expression protocol involves:

• Cloning the full-length atpE gene (273 bp) into a pET-based vector with an N-terminal His-tag

• Transforming into an E. coli strain optimized for membrane protein expression (e.g., C43(DE3))

• Culturing at 30°C until OD₆₀₀ reaches 0.6-0.8

• Inducing with 0.5 mM IPTG

• Continuing expression at reduced temperature (18°C) for 16-18 hours to minimize inclusion body formation

This approach yields functional protein that can be extracted in detergent micelles while maintaining native conformation. Notably, expression at higher temperatures results in reduced yield and functionality due to the hydrophobic nature of the protein .

What purification strategy yields the highest purity and retention of function for recombinant Geobacter atpE?

Purification of recombinant Geobacter atpE requires a strategic approach to maintain protein integrity while achieving high purity. Based on established protocols, the most effective purification strategy involves:

• Cell lysis under gentle conditions (French press or sonication with cooling intervals)

• Membrane fraction isolation through differential centrifugation

• Solubilization of membrane proteins using mild detergents (0.5-1% n-dodecyl β-D-maltoside)

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography as a polishing step

This process typically yields protein with >90% purity as determined by SDS-PAGE . For functional studies, it's critical to maintain the protein in appropriate detergent concentrations above the critical micelle concentration throughout purification. Reconstitution into liposomes may be necessary for functional assays, as this membrane protein requires a lipid environment to maintain its native conformation and activity.

Storage recommendations include aliquoting in buffer containing 6% trehalose at pH 8.0 and storing at -80°C to maintain stability . Repeated freeze-thaw cycles should be avoided as they significantly reduce protein activity.

How can researchers accurately measure ATP synthase activity of recombinant Geobacter atpE in vitro?

To accurately measure ATP synthase activity of recombinant Geobacter atpE in vitro, researchers should employ inverted membrane vesicle assays that mimic the natural environment of the protein. This methodology has been successfully used to compare ATP production between wild-type and mutant ATP synthase complexes .

The recommended protocol involves:

• Reconstitution of purified recombinant atpE into proteoliposomes with other necessary F₀F₁ ATP synthase subunits

• Creation of inverted membrane vesicles through sonication or extrusion

• Establishment of a proton gradient using NADH or an artificial electron donor

• Measurement of ATP synthesis using a luciferase-based ATP detection assay

Control experiments should include measurements with known ATP synthase inhibitors and with vesicles lacking the reconstituted protein. The assay can be adapted to measure ATP hydrolysis activity by monitoring the release of inorganic phosphate or through a coupled enzyme assay. Researchers should note that the anaerobic lifestyle of Geobacter may necessitate performing these assays under oxygen-limited conditions to obtain physiologically relevant results.

What experimental approaches can reveal the role of atpE in the electron transport chain of Geobacter sulfurreducens?

To investigate the role of atpE in Geobacter's electron transport chain, researchers should employ a multi-faceted approach combining genetic manipulation with biophysical measurements:

• Gene deletion/complementation studies: Create atpE knockout strains and complemented strains to observe phenotypic changes in electron transfer capabilities .

• Membrane potential measurements: Use fluorescent probes (e.g., DiOC₂) to quantify membrane potential changes in wild-type versus atpE-modified strains.

• Respiration rate analysis: Measure electron transfer to various acceptors (Fe(III), electrodes) in strains with modified atpE expression levels .

• ATP/ADP ratio monitoring: Quantify energy conservation efficiency using LC-MS/MS to determine how atpE modifications affect energy capture.

• Transcriptomic profiling: Analyze changes in expression of other electron transport components when atpE function is altered .

Research has demonstrated that manipulating ATP synthase activity in G. sulfurreducens affects respiratory rates and electron transfer efficiency. For example, creating an ATP demand by overexpressing the F₁ portion of the ATP synthase complex resulted in significantly increased respiration rates accompanied by upregulation of TCA cycle enzymes and electron transport components . This suggests that atpE and the ATP synthase complex play a key regulatory role in balancing energy conservation with electron transfer rates in Geobacter species.

How does the structure and function of Geobacter atpE compare to homologous proteins in other bacteria used in genetic engineering?

Geobacter sulfurreducens atpE shares fundamental structural features with homologous ATP synthase c-subunits from other bacteria, but displays distinct adaptations that reflect its specialized role in anaerobic respiration. Comparative analysis reveals:

These differences manifest in altered sensitivity to inhibitors and environmental conditions. For example, while mutations in the atpE gene of M. tuberculosis at positions 28 (Asp), 61 (Glu), 63 (Ala), and 66 (Ile) confer resistance to bedaquiline , similar positions in Geobacter atpE may affect its function in iron-reducing conditions.

When engineering ATP synthase components for biotechnological applications, these species-specific adaptations must be considered to optimize function in the target environment. The unique properties of Geobacter atpE make it particularly suitable for applications requiring electron transfer to extracellular acceptors, such as microbial fuel cells or bioremediation systems .

What insights can be gained from studying mutations in the atpE gene across different bacterial species?

Studying mutations in the atpE gene across different bacterial species provides valuable insights into both fundamental biology and applied biotechnology. Comparative mutation analysis reveals:

• Conservation of critical functional domains: Despite evolutionary divergence, certain regions of atpE show high conservation across species, indicating essential functional roles. For example, the proton-binding glutamate residue is preserved across diverse bacteria.

• Species-specific adaptation mechanisms: In M. tuberculosis, mutations at positions 28, 61, 63, and 66 in atpE confer resistance to bedaquiline , while in S. aureus, mutations in atpE result in altered ATP production and membrane properties .

• Metabolic reprogramming responses: In Geobacter, manipulating ATP synthase function leads to compensatory changes in electron transport chain components and central carbon metabolism, demonstrating the interconnected nature of these systems .

For researchers, these insights enable rational design of experiments targeting specific atpE functions. For instance, by comparing the effects of homologous mutations across species, one can predict how specific amino acid substitutions might affect Geobacter's unique extracellular electron transfer capabilities. This knowledge also informs potential genetic engineering strategies to enhance desirable traits such as higher rates of Fe(III) reduction or improved performance in microbial fuel cells .

How can atpE modifications enhance Geobacter sulfurreducens performance in microbial fuel cells?

Modifying atpE in Geobacter sulfurreducens offers significant potential for enhancing microbial fuel cell (MFC) performance through strategic manipulation of energy conservation and electron transfer processes. Research has demonstrated that increasing cellular ATP demand by engineering the ATP synthase complex leads to higher respiration rates and enhanced electron transfer capabilities .

Specific engineering approaches include:

• Controlled expression of F₁ ATP synthase components: Overexpressing the hydrolytic F₁ portion creates an ATP drain that stimulates respiratory activity. In engineered strains, this approach decreased cellular ATP content by more than 50% while significantly increasing respiration rates .

• Point mutations in proton-conducting residues: Strategic mutations can modify the proton/ATP ratio, redirecting more electrons to external acceptors rather than ATP synthesis.

• Promoter engineering: Developing inducible systems to dynamically control atpE expression allows researchers to optimize the balance between growth and electricity production.

The enhanced performance results from metabolic restructuring, where cells compensate for ATP depletion by increasing TCA cycle activity and upregulating electron transport chain components . This leads to greater electron flux to the anode in MFCs. Implementation requires careful balance, as excessive ATP drainage reduces growth and biomass accumulation, potentially limiting long-term electricity production. Optimal engineering creates a "sweet spot" where energy conservation is sufficiently maintained for cell viability while maximizing electron transfer to the anode.

What are the methodological considerations when engineering Geobacter atpE for enhanced bioremediation of metal-contaminated environments?

Engineering Geobacter atpE for enhanced bioremediation requires careful methodological considerations to balance increased metal reduction rates with bacterial survival in contaminated environments. Research strategies should address:

• Modification approach selection:

  • CRISPR-Cas9 genome editing offers precise modifications to native atpE

  • Plasmid-based overexpression provides tunable control but requires selection pressure

  • Chromosomal integration ensures stability but may have lower expression levels

• Physiological balance optimization:

  • Partial ATP synthase inhibition increases respiration rates but must maintain sufficient energy for stress response mechanisms

  • Monitoring growth rates and metal reduction activity under relevant field conditions is essential

• Environmental resilience enhancement:

  • Co-expression of metal resistance genes alongside atpE modifications

  • Testing engineered strains under fluctuating conditions (pH, temperature, competing organisms)

• Field deployment considerations:

  • Bioaugmentation strategies (pre-grown biomass vs. in situ stimulation)

  • Biocontainment features to prevent horizontal gene transfer

  • Monitoring protocols to track persistence and activity of engineered strains

How can researchers effectively employ adaptive laboratory evolution (ALE) to study atpE function in Geobacter species?

Adaptive Laboratory Evolution (ALE) offers a powerful approach to study atpE function in Geobacter by allowing natural selection to reveal adaptations under specific selective pressures. A methodological framework for effective ALE studies includes:

• Experimental design considerations:

  • Selection of appropriate growth medium and electron acceptors to target specific metabolic pathways

  • Determination of optimal transfer intervals (shorter intervals favor faster growing mutants)

  • Implementation of parallel evolutionary lines to distinguish random from adaptive mutations

  • Selection of appropriate control strains for comparative analysis

• Specific ALE strategies for studying atpE:

  • Employing ATP synthase inhibitors at sub-lethal concentrations to select for compensatory mutations

  • Gradually reducing available carbon sources to force more efficient energy conservation

  • Using non-traditional electron acceptors to select for altered electron transport coupling

  • Transferring cultures in media containing formate as the electron donor and carbon source with Fe(III) citrate as the electron acceptor to explore chemolithoautotrophic capabilities

• Post-evolution analysis:

  • Whole-genome sequencing of evolved strains to identify mutations

  • Transcriptomic profiling to understand system-wide adaptations

  • Proteomics to detect changes in protein abundance and post-translational modifications

  • Functional characterization of atpE and ATP synthase activity in evolved strains

  • Construction of defined mutants to verify causality of identified mutations

ALE has successfully revealed hidden metabolic capabilities in Geobacter, including the potential for chemolithoautotrophic growth using the roTCA cycle . When applied specifically to study atpE function, ALE can provide insights into the protein's role in energy conservation and its adaptability to different environmental conditions that may not be evident through direct genetic manipulation alone.

What advanced imaging techniques are most suitable for studying the localization and dynamics of atpE in Geobacter membranes?

Advanced imaging techniques provide crucial insights into the localization, organization, and dynamics of atpE within the complex membrane architecture of Geobacter sulfurreducens. Researchers should consider these methodological approaches:

• Super-resolution microscopy techniques:

  • PALM (Photoactivated Localization Microscopy) using photoactivatable fluorescent protein fusions to atpE

  • STORM (Stochastic Optical Reconstruction Microscopy) using appropriate antibodies against atpE

  • These techniques overcome the diffraction limit, achieving resolution down to ~20 nm to visualize ATP synthase distribution patterns

• Cryo-electron microscopy applications:

  • Single-particle cryo-EM to determine the structure of the ATP synthase complex containing atpE

  • Cryo-electron tomography to visualize ATP synthase in situ within intact bacterial membranes

  • Sub-tomogram averaging to improve resolution of the ATP synthase complex

• Fluorescence-based dynamic techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure lateral mobility of atpE

  • FRET (Förster Resonance Energy Transfer) to study interactions between atpE and other membrane proteins

  • Single-molecule tracking to follow individual ATP synthase complexes

• Sample preparation considerations:

  • Expression of fluorescent protein fusions at near-native levels to avoid artifacts

  • Careful membrane isolation procedures to maintain native protein arrangements

  • Development of specific antibodies against Geobacter atpE for immunolabeling

These techniques should be applied with awareness of Geobacter's unique membrane characteristics, including the presence of c-type cytochromes and multicopper proteins that contribute to extracellular electron transfer . Understanding the spatial organization of ATP synthase complexes relative to these electron transport components may reveal functional coupling mechanisms between energy conservation and respiration. The dynamic redistribution of ATP synthase complexes under different growth conditions (varying electron acceptors or donors) could provide insights into how Geobacter adapts its energy conservation strategy to environmental changes.

What strategies can address protein aggregation issues when working with recombinant Geobacter atpE?

Recombinant Geobacter atpE, being a highly hydrophobic membrane protein, frequently presents aggregation challenges during expression and purification. Researchers can implement these methodological solutions:

• Expression optimization:

  • Reduce expression temperature to 18-20°C during induction

  • Use slower induction with lower IPTG concentrations (0.1-0.3 mM)

  • Select specialized E. coli strains (C41/C43(DE3)) designed for membrane protein expression

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Extraction and solubilization:

  • Test a panel of detergents for optimal solubilization (LDAO, DDM, UDM)

  • Implement a detergent screening approach:

  Detergent	Concentration	Solubilization Efficiency	Functional Retention
  DDM	1%	High	Good
  LDAO	0.5%	Moderate	Excellent
  UDM	1%	High	Moderate
  Digitonin	1%	Low	Very good

• Purification considerations:

  • Include glycerol (10-15%) in all buffers to stabilize protein

  • Maintain detergent above critical micelle concentration throughout purification

  • Consider adding lipids (E. coli polar lipid extract) during purification

  • Implement on-column refolding protocols during affinity purification

• Storage optimization:

  • Add 6% trehalose to storage buffer as recommended

  • Flash-freeze small aliquots to avoid freeze-thaw cycles

  • Consider storing as membrane fractions rather than purified protein for some applications

Researchers should validate protein quality using analytical size exclusion chromatography before functional studies. For particularly challenging constructs, fusion partners such as GFP can be employed as folding indicators, allowing fluorescence-based monitoring of proper membrane insertion during expression.

How can researchers overcome challenges in measuring electron transfer activities in atpE-modified Geobacter strains?

Measuring electron transfer activities in atpE-modified Geobacter strains presents several methodological challenges that researchers can address with these approaches:

• Chronoamperometric measurements in microbial electrochemical systems:

  • Use three-electrode configuration with working, counter, and reference electrodes

  • Maintain strictly anaerobic conditions using continuous N₂ purging

  • Standardize biomass loading through OD₆₀₀ measurements or protein quantification

  • Compare current density (μA/cm²) rather than absolute current to normalize for electrode surface area

  • Implement technical replicates (minimum n=3) to account for biological variability

• Fe(III) reduction assays:

  • Use ferrozine assay for Fe(II) quantification with appropriate controls for abiotic reduction

  • Account for potential precipitation of Fe minerals through parallel acid-extractable iron measurements

  • Consider kinetic measurements at multiple timepoints rather than endpoint assays

  • Normalize reduction rates to cell protein content or cell number

• Respiratory activity measurements:

  • Adapt oxygen electrode methods for anaerobic respiration using alternative electron acceptors

  • Implement redox indicator dyes (e.g., DCPIP) coupled to spectrophotometric detection

  • Consider flow cytometry with redox-sensitive fluorescent dyes for single-cell analysis

• Controls and validation:

  • Include wild-type G. sulfurreducens as positive control in all experiments

  • Use metabolically inactive cells (heat-killed) as negative controls

  • Verify genetic modifications through sequencing before phenotypic testing

  • Complement genetic deletions to confirm phenotype causality

  • Measure ATP/ADP ratios to confirm the energetic impact of atpE modifications

When interpreting results, researchers should consider that alterations in atpE may have pleiotropic effects on cellular physiology. Changes in electron transfer rates may result from direct effects on energy conservation or indirect effects through altered expression of electron transport components such as c-type cytochromes and multicopper proteins . Therefore, comprehensive analysis should include transcriptomic or proteomic profiling alongside functional measurements to establish mechanistic understanding.