Recombinant Geobacter bemidjiensis ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c in Geobacter bemidjiensis?

ATP synthase subunit c (atpE) in Geobacter bemidjiensis functions as an essential component of the F-type ATP synthase complex, specifically within the membrane-embedded F0 sector. The protein consists of 91 amino acids with the sequence: MEFFTMCVLAAGIGMALGTLGTGIGQGLAVKSAVEGTSRNPGASGKILTTMMIGLAMIESLAIYALVVCLIILFANPYKDIALELAKSVAK . Structurally, multiple copies of this subunit assemble into a cylindrical c-ring oligomer embedded in the membrane. This c-ring works in conjunction with subunit a to facilitate proton translocation across the membrane, which drives the conformational changes necessary for ATP synthesis in the F1 sector. Like in other organisms, the c-subunit in G. bemidjiensis plays a critical role in coupling the proton motive force to ATP production, making it central to cellular energy generation during growth and metabolism .

How does the atpE gene of G. bemidjiensis compare to homologous genes in other bacterial species?

The atpE gene (locus tag Gbem_3932) of Geobacter bemidjiensis encodes the ATP synthase subunit c protein, which shares structural and functional similarities with homologs in other bacteria . Unlike in eukaryotes where multiple isoforms with different targeting peptides exist, G. bemidjiensis has a single atpE gene coding for the c subunit . Comparative genomic analyses reveal that while the core functional regions of ATP synthase subunit c are generally conserved across bacterial species, G. bemidjiensis exhibits specific adaptations potentially related to its subsurface lifestyle and unique bioenergetic requirements .

When compared to other Geobacter species like G. sulfurreducens and G. metallireducens, the atpE gene shows sequence conservation reflective of their phylogenetic relationship, while maintaining species-specific characteristics. These differences may contribute to the varying metabolic capabilities observed among Geobacter species, including G. bemidjiensis' enhanced capacity for fumarate disproportionation due to its possession of different dicarboxylic acid transporters and two oxaloacetate decarboxylases .

What are the optimal storage and handling conditions for recombinant G. bemidjiensis ATP synthase subunit c protein?

For optimal preservation of recombinant G. bemidjiensis ATP synthase subunit c, store the protein at -20°C for regular use, or at -80°C for extended storage periods . The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for stability. When working with the protein, prepare small working aliquots and store them at 4°C for no longer than one week to maintain activity .

Avoid repeated freeze-thaw cycles as they can significantly compromise protein integrity and function . When planning experiments, consider the following methodology to maximize protein stability:

• Thaw aliquots rapidly at room temperature or in a 37°C water bath

• Keep on ice once thawed

• Centrifuge briefly before opening tubes to collect all liquid

• Use appropriate buffer conditions for downstream applications, maintaining pH and ionic strength compatible with protein stability

• Consider adding protease inhibitors if working with cell or tissue extracts

For long-term studies, monitoring protein integrity through techniques such as SDS-PAGE or circular dichroism spectroscopy is recommended to ensure experimental reproducibility.

What methods are recommended for expression and purification of recombinant G. bemidjiensis ATP synthase subunit c?

For efficient expression and purification of recombinant G. bemidjiensis ATP synthase subunit c, a methodical approach utilizing heterologous expression systems is recommended. The preferred methodology encompasses:

Expression System Selection:

• E. coli BL21(DE3) or similar strains are typically used for expression of membrane proteins

• Consider using specialized strains for membrane proteins like C41(DE3) or C43(DE3)

• Expression vectors containing T7 or similar strong promoters with inducible control

Expression Protocol:

• Transform expression plasmid into host cells

• Culture cells in rich media (e.g., LB) until mid-log phase (OD600 ~0.6-0.8)

• Induce with IPTG at reduced temperature (16-25°C) to enhance proper folding

• Continue expression for 4-16 hours depending on protein stability

Purification Strategy:

• Cell disruption by sonication or French press in buffer containing detergents suitable for membrane proteins (e.g., DDM, LDAO)

• Initial purification step: Immobilized metal affinity chromatography (IMAC) if His-tagged

• Secondary purification: Size exclusion chromatography to obtain homogeneous protein

• Quality assessment by SDS-PAGE and mass spectrometry to confirm identity

The final product should be stored in a Tris-based buffer with 50% glycerol or lyophilized for long-term storage . During purification, maintaining conditions that prevent protein aggregation is crucial for obtaining functional protein suitable for downstream structural and biochemical studies.

How can recombinant G. bemidjiensis ATP synthase subunit c be used to study bioenergetics in subsurface microorganisms?

Recombinant G. bemidjiensis ATP synthase subunit c serves as an excellent model for investigating bioenergetic processes in subsurface microorganisms. G. bemidjiensis, as a member of the subsurface clade 1 of Geobacter species, has evolved specialized energy conservation mechanisms for Fe(III)-reducing environments . Research methodologies utilizing this recombinant protein include:

Comparative Structural Studies:

• Reconstitution of the c-subunit into liposomes or nanodiscs to study proton translocation

• Site-directed mutagenesis of key residues to determine their role in proton binding and transport

• Cross-linking studies to investigate subunit interactions within the ATP synthase complex

Bioenergetic Function Analysis:

• In vitro ATP synthesis assays using reconstituted proteoliposomes

• Measurement of proton transport using pH-sensitive fluorescent dyes

• Analysis of c-ring stoichiometry and its impact on the H⁺/ATP ratio

These approaches provide insights into how G. bemidjiensis has adapted its energy conservation mechanisms to thrive in subsurface environments with limited electron acceptors. The abundance of ATP synthase subunits detected during biostimulation experiments highlights the importance of this complex in energy generation during periods of rapid growth . Studying these adaptations contributes to our understanding of microbial survival strategies in extreme environments and may inform bioremediation approaches utilizing Geobacter species.

What techniques can be used to investigate the interaction between ATP synthase subunit c and other components of the ATP synthase complex in G. bemidjiensis?

Investigating the interactions between ATP synthase subunit c and other components of the ATP synthase complex in G. bemidjiensis requires sophisticated protein-protein interaction techniques. Several methodological approaches are particularly effective:

Structural Biology Approaches:

• Cryo-electron microscopy of the intact ATP synthase complex

• X-ray crystallography of subcomplexes containing the c-ring

• NMR spectroscopy for dynamic studies of isolated subunits

Biochemical Interaction Analysis:

• Chemical cross-linking combined with mass spectrometry (XL-MS) to map interaction sites

• Co-immunoprecipitation assays using antibodies against different subunits

• Blue native PAGE to analyze intact complex assembly

• Surface plasmon resonance to measure binding kinetics between subunits

Genetic and In Vivo Studies:

• Site-directed mutagenesis of interaction interfaces followed by functional assays

• FRET-based approaches using fluorescently labeled subunits

• Bacterial two-hybrid systems to screen for interactions

These techniques can reveal how subunit c interacts with subunit a during proton pumping and how the c-ring couples with the central stalk components to drive ATP synthesis in the F1 sector. The insights gained from such studies would be particularly valuable given G. bemidjiensis' adaptation to subsurface environments and its different dicarboxylic acid metabolism compared to other Geobacter species, which might be reflected in unique aspects of its ATP synthase structure and function.

How does G. bemidjiensis ATP synthase subunit c differ from eukaryotic homologs in structure and regulation?

G. bemidjiensis ATP synthase subunit c exhibits several fundamental differences from its eukaryotic counterparts in both structure and regulation:

Genetic Organization:

• G. bemidjiensis has a single atpE gene encoding the c-subunit (locus tag Gbem_3932)

• Mammals possess three distinct isoforms of F1F0-ATP synthase subunit c, which differ primarily in their targeting peptides while maintaining identical mature sequences

• In yeast, subunit c (also called subunit 9) is encoded by mitochondrial DNA, whereas in G. bemidjiensis and other bacteria, it is encoded in the nuclear genome

Structural Differences:

• The bacterial c-subunit forms a cylindrical c10 oligomer in most cases, though the exact stoichiometry may vary

• Eukaryotic mitochondrial c-rings typically contain 8-10 subunits depending on the species

• The G. bemidjiensis c-subunit lacks the N-terminal mitochondrial targeting peptides found in eukaryotic homologs

Regulatory Mechanisms:

• Eukaryotic ATP synthase is regulated by inhibitory proteins (IF1) and natural inhibitors like oligomycin

• G. bemidjiensis lacks these eukaryotic regulatory mechanisms but likely employs alternative regulatory strategies adapted to its subsurface lifestyle

• In eukaryotes, the different targeting peptides of c-subunit isoforms play crucial roles beyond import, including maintenance of respiratory chain function , whereas G. bemidjiensis utilizes different mechanisms for respiratory regulation

These differences reflect the divergent evolutionary paths and environmental adaptations of bacterial and eukaryotic ATP synthases, with G. bemidjiensis specifically adapted to function optimally in subsurface environments where Fe(III) reduction is the primary electron-accepting process .

What evolutionary insights can be gained from studying ATP synthase subunit c across different Geobacter species?

Studying ATP synthase subunit c across different Geobacter species provides valuable evolutionary insights into adaptation and specialization within this genus. Comparative analysis reveals:

Phylogenetic Relationships:

• G. bemidjiensis belongs to the subsurface clade 1 of Geobacter species

• Sequence analysis of ATP synthase subunits can help resolve evolutionary relationships between G. bemidjiensis, G. sulfurreducens, G. metallireducens, G. uraniireducens, and other members of the genus

Adaptive Evolution:

• Variations in ATP synthase c-subunit sequences across Geobacter species may reflect adaptations to different environmental niches

• G. bemidjiensis shows specific adaptations for subsurface environments where Fe(III) reduction predominates

• These adaptations may include modifications in the c-subunit to optimize energy conservation under specific redox conditions

Functional Divergence:

• G. bemidjiensis possesses unique metabolic capabilities including carbon dioxide fixation and growth on glucose

• Its enhanced abilities to respire, detoxify, and avoid oxygen may be reflected in adaptations in the ATP synthase complex

• The differential expression of ATP synthase subunits observed during biostimulation experiments suggests environmental regulation of energy metabolism

Methodologically, researchers can:

• Perform multiple sequence alignments of c-subunits across Geobacter species

• Calculate selection pressures on specific residues using dN/dS analysis

• Conduct ancestral sequence reconstruction to trace evolutionary changes

• Correlate sequence variations with ecological niches and metabolic capabilities

Such analyses contribute to our understanding of how this ancient molecular machine has been fine-tuned in different Geobacter species to optimize energy conservation in diverse environmental conditions.

How does ATP synthase activity in G. bemidjiensis contribute to its role in environmental bioremediation processes?

ATP synthase activity in G. bemidjiensis plays a crucial role in powering the metabolic processes that make this organism valuable for bioremediation applications. The following methodological framework explains this connection:

Energy Generation for Bioremediation:

• During biostimulation experiments, abundant peptides matching ATP synthase subunits have been detected, indicating high energy demands during periods of rapid growth

• This energy production fuels the electron transfer processes required for reduction of metals and contaminants

• The ATP generated supports cellular growth and maintenance in contaminated subsurface environments

Metabolic Integration:

• ATP synthase works in concert with the tricarboxylic acid cycle to generate energy from acetate, which is often used as an electron donor in bioremediation settings

• This energy powers the extensive electron transport chain of G. bemidjiensis, which culminates in the reduction of Fe(III) or other electron acceptors

• G. bemidjiensis' unique ability to fix carbon dioxide and grow on glucose expands its metabolic versatility in contaminated environments

Adaptation to Contaminated Environments:

• The enhanced ability of G. bemidjiensis to respire, detoxify, and avoid oxygen is critical for function in subsurface environments

• These adaptations allow continued ATP production even under challenging conditions

The predominance of G. bemidjiensis (60-70%) in biostimulation samples demonstrates its competitive advantage in such environments , which is supported by efficient energy conservation mechanisms including ATP synthase. Understanding the regulation and activity of ATP synthase in this organism can help optimize bioremediation strategies by ensuring adequate energy supply for contaminant transformation and microbial growth.

What research techniques can be used to study the expression and activity of ATP synthase in G. bemidjiensis during environmental bioremediation?

Investigating ATP synthase expression and activity in G. bemidjiensis during environmental bioremediation requires a multifaceted approach combining molecular, biochemical, and field techniques:

Field Monitoring Techniques:

• Metatranscriptomics: Extraction of total RNA from environmental samples to measure in situ expression of ATP synthase genes

• Metaproteomics: Analysis of protein extracts from environmental samples to detect and quantify ATP synthase subunits, as demonstrated by the detection of abundant ATP synthase peptides during biostimulation

• Stable Isotope Probing: Tracking carbon flow through ATP synthase-dependent metabolic pathways

Laboratory Methods for Expression Analysis:

• Quantitative RT-PCR: Measuring transcript levels of atpE and other ATP synthase genes under varying environmental conditions

• Reporter Gene Constructs: Creating fusion proteins with fluorescent or luminescent reporters to visualize expression patterns

• Western Blotting: Using antibodies against ATP synthase subunits to quantify protein levels

Activity Measurement Approaches:

• ATP Production Assays: Direct measurement of ATP synthesis rates in cell extracts or whole cells

• Membrane Potential Measurements: Using fluorescent dyes to monitor the proton motive force that drives ATP synthase

• Oxygen Consumption Rates: Measuring respiratory activity which is coupled to ATP synthesis

Integrative Approaches:

• Multi-omics Integration: Combining transcriptomic, proteomic, and metabolomic data to create a comprehensive view of energy metabolism during bioremediation

• Biomarker Development: Identifying ATP synthase expression patterns that correlate with effective bioremediation activity

These techniques can help researchers understand how G. bemidjiensis regulates its energy metabolism during different phases of bioremediation, particularly during the documented predominance of this species in subsurface environments where Fe(III) reduction is the primary electron-accepting process . The abundance of ATP synthase subunits observed during biostimulation suggests their importance as potential biomarkers for active remediation processes .

What are the common challenges in working with recombinant G. bemidjiensis ATP synthase subunit c and how can they be addressed?

Working with recombinant G. bemidjiensis ATP synthase subunit c presents several technical challenges due to its hydrophobic nature and membrane association. Common problems and methodological solutions include:

Expression and Solubility Issues:

• Challenge: Low expression levels and inclusion body formation
  Solution: Optimize expression conditions by lowering induction temperature (16-20°C), reducing inducer concentration, and using specialized bacterial strains like C41(DE3) designed for membrane protein expression

• Challenge: Poor solubility due to hydrophobic nature
  Solution: Use appropriate detergents (DDM, LDAO, or Fos-choline) for extraction; consider fusion tags that enhance solubility such as MBP or SUMO

Purification Difficulties:

• Challenge: Co-purification of contaminant proteins
  Solution: Implement multi-step purification strategy combining affinity chromatography with ion exchange and size exclusion steps

• Challenge: Protein aggregation during concentration
  Solution: Add glycerol (10-20%) to buffers, maintain low protein concentration during handling, and consider using amphipols or nanodiscs for final preparation

Functional Characterization Obstacles:

• Challenge: Distinguishing activity of recombinant protein from contaminating ATPases
  Solution: Include specific inhibitors of other ATPases in assay buffers; create negative controls using site-directed mutagenesis of catalytic residues

• Challenge: Reconstituting functional protein into liposomes
  Solution: Optimize lipid composition to mimic G. bemidjiensis membrane environment; carefully control protein-to-lipid ratios; use gentle detergent removal methods

Storage and Stability:

• Challenge: Activity loss during storage
  Solution: Store at -20°C or -80°C in buffer containing 50% glycerol; avoid repeated freeze-thaw cycles ; consider flash-freezing small aliquots in liquid nitrogen

By systematically addressing these challenges, researchers can successfully work with this technically demanding but scientifically valuable protein to advance understanding of bioenergetics in subsurface microorganisms.

How can researchers troubleshoot inconsistent results when studying the structure-function relationship of G. bemidjiensis ATP synthase subunit c?

When investigating structure-function relationships of G. bemidjiensis ATP synthase subunit c, inconsistent results often arise from several methodological and experimental variables. The following troubleshooting framework addresses these challenges:

Protein Quality Assessment:

• Issue: Heterogeneous protein preparations
  Solution: Implement rigorous quality control using analytical SEC, dynamic light scattering, and mass spectrometry to confirm protein homogeneity and integrity

• Issue: Post-translational modifications affecting function
  Solution: Characterize protein using mass spectrometry to identify any modifications; compare to expected sequence (MEFFTMCVLAAGIGMALGTLGTGIGQGLAVKSAVEGTSRNPGASGKILTTMMIGLAMIESLAIYALVVCLIILFANPYKDIALELAKSVAK)

Experimental Condition Optimization:

• Issue: Variable activity in different buffer conditions
  Solution: Systematically test buffer components, pH ranges, and ionic strengths; document optimal conditions thoroughly for reproducibility

• Issue: Temperature sensitivity affecting structural measurements
  Solution: Maintain strict temperature control during experiments; perform measurements at multiple temperatures to establish stability ranges

Structural Analysis Refinement:

• Issue: Conflicting structural data from different techniques
  Solution: Use complementary approaches (X-ray crystallography, NMR, cryo-EM) and reconcile results through computational modeling

• Issue: Oligomeric state variability
  Solution: Employ native mass spectrometry and analytical ultracentrifugation to definitively establish oligomeric state under experimental conditions

Control Experiments and Validation:

• Issue: Lack of appropriate controls
  Solution: Include well-characterized homologs from related organisms (e.g., other Geobacter species) as comparative controls

• Issue: Inability to validate functional effects of structural changes
  Solution: Develop robust functional assays specifically optimized for G. bemidjiensis ATP synthase; consider whole-cell complementation studies

Data Integration and Analysis:

• Issue: Difficulty correlating structural and functional data
  Solution: Develop integrated data analysis workflows; use structure-based computational predictions to guide experimental design

• Issue: Unexpected results compared to related species
  Solution: Consider G. bemidjiensis' unique ecological niche and adaptations ; examine literature on subsurface microorganisms for context

By implementing this comprehensive troubleshooting approach, researchers can improve consistency and reliability when investigating the complex structure-function relationships of this important bioenergetic protein.