Recombinant Geobacter uraniireducens ATP synthase subunit c (atpE)

What is the ATP synthase subunit c in Geobacter uraniireducens and why is it significant for research?

ATP synthase subunit c in G. uraniireducens is a component of the Fo domain of ATP synthase, forming the membrane-embedded c-ring responsible for proton translocation. This protein is significant because G. uraniireducens possesses both F-type and V-type ATPase complexes, which is uncommon among Geobacter species. Only G. uraniireducens and Geobacter sp. M18 have been identified to contain both ATPase complexes . Analysis of subunit c from both complexes shows that neither appears to have Na⁺-binding sites, suggesting both ATPase complexes are proton-dependent . This characteristic is crucial for understanding the energy metabolism of G. uraniireducens in uranium-contaminated environments where it plays a significant role in bioremediation.

How does the ATP synthase structure in G. uraniireducens compare to other Geobacter species?

The ATP synthase in G. uraniireducens is unique among most Geobacter species in that it possesses both F-type and V-type ATPase complexes. This dual ATPase system is only shared with Geobacter sp. M18 among the Geobacter genus . Most other Geobacter species, including the well-studied G. sulfurreducens and G. metallireducens, contain only the F-type ATPase complex that is common in most bacteria .

The genomic comparison reveals functional differences that may relate to the unique environmental adaptations of G. uraniireducens:

The presence of both ATP synthase types may contribute to G. uraniireducens' ability to thrive in uranium-contaminated subsurface environments and its effectiveness in uranium bioremediation .

What are the recommended expression systems for recombinant production of G. uraniireducens ATP synthase subunit c?

For recombinant production of G. uraniireducens ATP synthase subunit c, an E. coli expression system is typically recommended, similar to the approach used for other membrane proteins. The methodology should be adapted from successful approaches used for other ATP synthase subunit c proteins .

The expression protocol typically includes:

• Gene synthesis or PCR amplification of the atpE gene from G. uraniireducens genomic DNA

• Cloning into an expression vector with an appropriate promoter (T7 or tac promoters are commonly used)

• Transformation into an E. coli strain optimized for membrane protein expression (C41(DE3), C43(DE3), or BL21(DE3) with additional plasmids like pLysS)

• Induction with IPTG at lower temperatures (16-25°C) to facilitate proper folding

• Membrane fraction isolation followed by detergent solubilization

The hydrophobic nature of subunit c requires careful optimization of detergents for extraction and purification. Typical detergents include n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG), or digitonin depending on downstream applications .

What purification challenges are specific to recombinant G. uraniireducens ATP synthase subunit c and how can they be addressed?

Purification of recombinant G. uraniireducens ATP synthase subunit c presents several challenges inherent to highly hydrophobic membrane proteins:

• Aggregation during extraction: This can be addressed by careful optimization of detergent concentration and buffer composition. A stepwise screening of detergents at various concentrations is recommended, starting with milder detergents like DDM (0.5-2%) and increasing to stronger ones only if necessary.

• Maintaining native conformation: The subunit c often exists as an oligomeric ring in native conditions. To preserve this structure, purification should be performed at 4°C with stabilizing agents like glycerol (10-20%) and appropriate ionic strength (150-300 mM NaCl).

• Protein yield optimization: Based on experiences with other ATP synthase subunit c proteins, expression can be optimized by:

  • Using chemical chaperones like glycerol or trehalose in the growth medium

  • Co-expression with specific chaperones

  • Employing fusion tags that enhance solubility (MBP or SUMO)

  • Using controlled expression systems with lower inducer concentrations

• Verification of functionality: Unlike soluble proteins, demonstrating that the purified recombinant subunit c remains functional requires specialized assays:

  • Reconstitution into liposomes followed by proton translocation assays

  • Circular dichroism to verify secondary structure integrity

  • Blue native PAGE to assess oligomerization state

The purification protocol should incorporate affinity chromatography (if a tag is used), followed by size exclusion chromatography to separate the properly folded protein from aggregates and other impurities.

How does ATP synthase subunit c contribute to the uranium reduction capabilities of G. uraniireducens?

The ATP synthase subunit c in G. uraniireducens indirectly contributes to uranium reduction capabilities through its central role in energy metabolism. This relationship operates through several interconnected mechanisms:

• Energy conservation for uranium reduction: The proton-translocating function of ATP synthase is crucial for generating ATP during respiratory processes. G. uraniireducens requires this energy for the expression and maintenance of extracellular electron transfer components necessary for uranium reduction .

• Maintenance of proton gradient: The c-ring of ATP synthase facilitates proton translocation across the membrane, maintaining the proton motive force. This process is essential for several electron transport proteins involved in uranium reduction, particularly c-type cytochromes .

• Support for biofilm formation: G. uraniireducens transitions between planktonic and biofilm physiologies during active uranium reduction, and ATP generated via ATP synthase is required for the energy-intensive process of biofilm formation . Multilayer biofilms have been demonstrated to reduce and tolerate substantially more uranium than planktonic cells .

• Metabolic adaptation to uranium-contaminated environments: The dual ATP synthase system (both F-type and V-type) in G. uraniireducens may provide metabolic flexibility that contributes to its success in uranium-contaminated environments, allowing for adaptation to varying redox conditions and energy demands .

Research indicates that G. uraniireducens responds to changes in environmental conditions by altering expression of metabolic genes, including those related to energy conservation, which would encompass ATP synthase components .

Can recombinant ATP synthase subunit c be used to enhance uranium bioremediation technologies?

Recombinant ATP synthase subunit c from G. uraniireducens has potential applications in enhancing uranium bioremediation technologies, though this represents an advanced area of research. The methodological approach would include:

• Engineered Geobacter strains: Overexpression or modification of ATP synthase subunit c could potentially enhance energy conservation, allowing for more efficient uranium reduction. This approach would require:

  • Site-directed mutagenesis to optimize proton translocation efficiency

  • Controlled expression systems to balance energy conservation with cellular stress

  • Assessment of uranium reduction rates in laboratory and field conditions

• Biofilm enhancement strategies: Since biofilms show superior uranium reduction capabilities , recombinant approaches that optimize ATP synthase function might promote biofilm formation. Research indicates that G. uraniireducens forms biofilms during uranium reduction, and enhancing ATP production could support this process .

• Creation of biohybrid materials: Recombinant ATP synthase subunit c could be incorporated into artificial membrane systems to create biohybrid materials that generate proton gradients to power uranium reduction by attached cytochromes.

• Field application considerations: Implementation would require:

  • Assessment of stability and activity under relevant environmental conditions

  • Integration with existing bioremediation approaches

  • Monitoring of metabolic activity using biomarkers like citrate synthase

This application remains theoretical and would require extensive laboratory validation before field implementation. Researchers should be aware that uranium reduction by Geobacter species involves multiple components beyond ATP synthase, particularly conductive pili and various c-type cytochromes that directly interact with uranium .

What are the best approaches to study the proton translocation mechanism of recombinant G. uraniireducens ATP synthase subunit c?

Studying the proton translocation mechanism of recombinant G. uraniireducens ATP synthase subunit c requires specialized techniques that address its membrane-embedded nature. Recommended methodological approaches include:

• Liposome reconstitution assays: Purified recombinant subunit c can be reconstituted into liposomes containing pH-sensitive fluorescent dyes (such as ACMA or pyranine). Changes in internal pH upon addition of ionophores or establishment of ion gradients can provide direct evidence of proton translocation activity.

• Site-directed mutagenesis: Create targeted mutations of key residues predicted to be involved in proton binding and translocation. Comparative functional analysis between wild-type and mutant proteins can identify essential residues. Based on analyses of other ATP synthases, focus should be on conserved polar residues within transmembrane helices.

• Structural studies:

  • Cryo-electron microscopy of the reconstituted c-ring

  • NMR spectroscopy for dynamics studies (particularly solid-state NMR)

  • X-ray crystallography if the protein can be crystallized

• Electrophysiological measurements: Incorporation of the c-ring into planar lipid bilayers allows direct measurement of proton conductance using voltage-clamp techniques.

• Computational approaches: Molecular dynamics simulations can model proton movement through the c-ring and identify structural determinants of specificity.

Analysis should focus on comparing the proton translocation mechanisms between the F-type and V-type ATP synthases in G. uraniireducens, as this dual system is relatively rare in Geobacter species . The lack of Na⁺-binding sites in both ATPase complexes suggests proton-specificity that may be advantageous in the uranium-contaminated subsurface environments where G. uraniireducens thrives .

How can researchers assess the impact of uranium exposure on ATP synthase structure and function in G. uraniireducens?

Assessing the impact of uranium exposure on ATP synthase structure and function in G. uraniireducens requires a multi-faceted experimental approach:

• In vivo studies:

  • Culture G. uraniireducens at varying uranium concentrations

  • Monitor growth rates, ATP production levels, and membrane potential

  • Measure expression levels of ATP synthase genes using qRT-PCR

  • Assess protein abundance through western blotting with specific antibodies

  • Analyze changes in membrane composition that might affect ATP synthase function

• Recombinant protein studies:

  • Expose purified recombinant ATP synthase components to uranium at various concentrations

  • Measure ATPase activity using standard enzymatic assays before and after uranium exposure

  • Analyze structural changes using circular dichroism, fluorescence spectroscopy, or structural methods

  • Assess oligomerization state changes using native PAGE or analytical ultracentrifugation

• Structural analysis of uranium interaction:

  • Use X-ray absorption spectroscopy to identify uranium binding sites on ATP synthase

  • Employ isothermal titration calorimetry to determine binding constants

  • Perform competitive binding studies with other metal ions

• Functional impact assessment:

  • Measure proton translocation efficiency in reconstituted systems with and without uranium

  • Assess ATP synthesis rates in cell extracts or reconstituted systems

  • Monitor membrane integrity and potential during uranium exposure

• Comparative analysis with uranium-sensitive bacteria:

  • Compare effects with species lacking the dual ATP synthase system

  • Identify protective mechanisms specific to G. uraniireducens

This research would be particularly valuable given that G. uraniireducens has been observed to maintain functionality in uranium-contaminated environments and can reduce uranium(VI) to uranium(IV), suggesting adaptations that protect essential cellular machinery including ATP synthase .

How does the ATP synthase subunit c of G. uraniireducens differ from that of other Geobacter species, and what are the functional implications?

The ATP synthase subunit c of G. uraniireducens differs from most other Geobacter species in several significant aspects:

• Dual ATP synthase systems: G. uraniireducens possesses both F-type and V-type ATP synthase complexes, a feature shared only with Geobacter sp. M18 among the Geobacter genus . Most other Geobacter species, including G. sulfurreducens and G. metallireducens, possess only the F-type ATPase common to most bacteria.

• Sequence and structural variations: While specific sequence data for G. uraniireducens ATP synthase subunit c is not provided in the search results, comparative genomic analyses of Geobacter species reveal substantial genetic divergence in energy metabolism components despite conservation of core functions . These variations likely extend to the ATP synthase subunit c.

• Proton specificity: Analysis indicates that both ATP synthase complexes in G. uraniireducens lack Na⁺-binding sites, making them exclusively proton-dependent . This characteristic influences their function in environments with varying pH and ion concentrations.

The functional implications of these differences include:

• Metabolic flexibility: The dual ATP synthase system likely provides G. uraniireducens with greater metabolic flexibility, potentially allowing it to thrive under varying environmental conditions in uranium-contaminated subsurface environments .

• Energy conservation efficiency: The presence of two different ATP synthase types may allow for optimization of energy conservation under different growth conditions, contributing to the organism's success in challenging environments.

• Adaptation to contaminated environments: These differences may contribute to G. uraniireducens' effectiveness in uranium bioremediation by supporting its metabolism in contaminated settings where other bacteria may struggle .

Comparative analysis suggests that these adaptations in G. uraniireducens are part of its specialized metabolism for environments with metal contaminants, particularly uranium.

What advanced techniques can be used to study the evolutionary relationships between ATP synthase subunit c across Geobacter species and their adaptation to different environments?

Advanced techniques for studying evolutionary relationships and environmental adaptations of ATP synthase subunit c across Geobacter species include:

• Comparative genomics and phylogenetic analysis:

  • Whole-genome sequencing of multiple Geobacter strains from diverse environments

  • Construction of phylogenetic trees based on ATP synthase genes

  • Analysis of selection pressure using dN/dS ratios to identify positively selected residues

  • Ancestral sequence reconstruction to trace evolutionary changes

• Environmental transcriptomics and proteomics:

  • RNA-Seq analysis of Geobacter species under varying environmental conditions

  • Quantitative proteomics to measure ATP synthase abundance across species

  • Correlation of expression patterns with environmental parameters

  • Transcript abundance measurements similar to those used for monitoring gene expression in subsurface environments

• Structural biology approaches:

  • Comparative structural analysis of ATP synthase c-rings from different Geobacter species

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Cryo-EM structures of ATP synthase complexes from multiple species

  • Molecular dynamics simulations to compare functional dynamics

• Functional genomics:

  • CRISPR-Cas9 gene editing to create chimeric ATP synthase components

  • Heterologous expression systems to test functionality across species boundaries

  • Site-directed mutagenesis to test the importance of species-specific residues

  • Growth complementation assays under varying environmental conditions

• Environmental adaptation studies:

  • Isolation of Geobacter strains from environments with varying metal concentrations

  • Experimental evolution studies under controlled selective pressures

  • Comparison of ATP synthase performance metrics across environmental gradients

  • Analysis of horizontal gene transfer events affecting ATP synthase genes

These methodological approaches can reveal how ATP synthase subunit c has evolved in Geobacter species in response to their diverse environments, particularly in metal-contaminated subsurface settings where G. uraniireducens demonstrates specialized adaptation for uranium reduction .

How can single-molecule techniques be applied to study the rotation mechanics of recombinant G. uraniireducens ATP synthase subunit c ring?

Single-molecule techniques offer powerful approaches to directly observe the rotation mechanics of the ATP synthase c-ring, providing insights that bulk measurements cannot capture. Methodological approaches include:

• Single-molecule FRET (smFRET):

  • Label specific residues on the c-ring and adjacent subunits with FRET pairs

  • Monitor conformational changes during proton translocation and rotation in real-time

  • Observe step-wise motion corresponding to individual proton binding events

  • Calculate rotation speeds and dwell times under varying conditions

• Gold nanorod tracking:

  • Attach gold nanorods to the c-ring in reconstituted systems

  • Use dark-field microscopy to track rotational motion with high temporal resolution

  • Correlate rotation with proton motive force under varying conditions

  • Measure torque generation through viscous drag calculations

• Magnetic bead rotation assays:

  • Immobilize the ATP synthase complex on a surface

  • Attach magnetic beads to the c-ring

  • Apply magnetic fields to either drive rotation or measure resistance to rotation

  • Calculate the mechanical work performed during ATP synthesis or hydrolysis

• High-speed atomic force microscopy (HS-AFM):

  • Directly visualize structural changes in the c-ring during rotation

  • Monitor interactions with other subunits in real-time

  • Observe natural fluctuations in the absence of ATP or proton gradients

  • Compare dynamics between F-type and V-type ATP synthases from G. uraniireducens

• Nanodiscs and lipid bilayer systems:

  • Reconstitute the ATP synthase complex in nanodiscs with controlled lipid composition

  • Create artificial proton gradients using photosensitive proton pumps

  • Measure rotation events triggered by light-activated proton translocation

  • Test the effects of uranium exposure on rotation mechanics

These techniques can reveal unique features of G. uraniireducens ATP synthase function that may contribute to its specialized role in uranium-contaminated environments. The dual ATP synthase system in G. uraniireducens presents a unique opportunity to compare the rotation mechanics of F-type and V-type complexes within the same organism, potentially revealing adaptations specific to metal-reducing bacteria.

What are the challenges and strategies for integrating structural data with functional assays when studying G. uraniireducens ATP synthase subunit c in uranium reduction pathways?

Integrating structural data with functional assays for G. uraniireducens ATP synthase subunit c presents several challenges and requires sophisticated methodological strategies:

• Challenges in structural-functional integration:

  • Membrane protein structures often represent static states, while function is dynamic

  • Environmental conditions during structural determination may differ from physiological conditions

  • Uranium interaction may induce conformational changes not captured in native structures

  • Connecting ATP synthase function to downstream uranium reduction involves multiple cellular components

• Methodological strategies for integration:

  a. Time-resolved structural approaches:

  • Time-resolved cryo-EM to capture intermediates during the catalytic cycle

  • Time-resolved X-ray free-electron laser (XFEL) crystallography

  • EPR spectroscopy with site-directed spin labeling to monitor conformational changes

  • Nuclear magnetic resonance (NMR) to detect dynamic regions and binding events

  b. Structure-guided functional assays:

  • Design mutants based on structural data to test mechanistic hypotheses

  • Create chimeric proteins exchanging structural elements between the F-type and V-type complexes

  • Use computational predictions to identify uranium binding sites for experimental validation

  • Develop structure-based biosensors for uranium using engineered ATP synthase components

  c. Systems biology approaches:

  • Integrate ATP synthase structural data with metabolic flux analysis

  • Correlate structural features with transcriptomic and proteomic data

  • Develop kinetic models that incorporate structural constraints

  • Map energy conservation pathways connecting ATP synthesis to uranium reduction

  d. Advanced imaging techniques:

  • Correlative light and electron microscopy to localize ATP synthase in intact cells

  • Super-resolution microscopy to visualize ATP synthase distribution relative to uranium reduction sites

  • Cryo-electron tomography of intact cells to visualize ATP synthase in native environment

  • Live-cell imaging with fluorescent probes to track ATP production and uranium reduction simultaneously

• Implementation strategy:

  • Begin with structural determination of both F-type and V-type ATP synthase c-rings

  • Develop functional assays calibrated to physiological conditions

  • Create a computational model integrating structural constraints with functional parameters

  • Validate predictions through targeted mutations and chimeric constructs

  • Scale to whole-cell models connecting ATP synthesis to uranium reduction