Recombinant Syntrophobacter fumaroxidans ATP synthase subunit c (atpE)

What is Syntrophobacter fumaroxidans and why is it significant in microbiological research?

Syntrophobacter fumaroxidans strain MPOB is the best-studied species of the genus Syntrophobacter. This bacterium is particularly significant in microbiological research due to its anaerobic syntrophic lifestyle and its crucial role in the conversion of propionate to acetate, hydrogen, and carbon dioxide during organic matter degradation. These metabolic products serve as substrates for other microorganisms in anaerobic environments .

S. fumaroxidans exhibits remarkable metabolic versatility. It can ferment fumarate to carbon dioxide and succinate in pure culture, and also functions as a sulfate reducer with propionate as an electron donor. This bacterium belongs to the family Syntrophobacteraceae within the order Syntrophobacterales, which contains sulfate-reducing species capable of syntrophy . The availability of its complete genome sequence has made it an important model organism for studying anaerobic syntrophic metabolism and interspecies electron transfer.

What is the functional role of ATP synthase subunit c (atpE) in S. fumaroxidans metabolism?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F₁F₀-ATP synthase complex in S. fumaroxidans. This protein functions as part of the membrane-embedded F₀ portion of the ATP synthase, forming a ring structure that facilitates proton translocation across the membrane. This proton movement drives ATP synthesis through oxidative phosphorylation.

In S. fumaroxidans, ATP synthase is particularly important given the energy constraints of syntrophic propionate metabolism. The bacterium operates close to thermodynamic limits when growing syntrophically, making efficient energy conservation essential. The ATP synthase complex harnesses the transmembrane proton gradient, which is crucial for the endergonic oxidation of succinate to fumarate during propionate metabolism via the methylmalonyl-CoA pathway . This reaction is highly endergonic since the midpoint potential of succinate (+30 mV) is much more positive than menaquinone (-80 mV), requiring a transmembrane proton gradient to function .

How does the genomic context of atpE differ between S. fumaroxidans and other syntrophic bacteria?

The atpE gene in S. fumaroxidans exists within the ATP synthase operon, which contains other genes encoding various subunits of the F₁F₀-ATP synthase complex. While the search results do not provide specific information about the genomic context of atpE in S. fumaroxidans, genomic analyses have revealed that S. fumaroxidans possesses unique adaptations for its syntrophic lifestyle.

What are the optimal conditions for culturing S. fumaroxidans for atpE expression studies?

For optimal cultivation of S. fumaroxidans for atpE expression studies, strict anaerobic conditions are essential. Research protocols indicate that S. fumaroxidans MPOB (DSM10017) should be grown at 35°C in serum bottles with bicarbonate-buffered medium under a N₂/CO₂ (80:20, v/v) headspace .

For pure culture studies focused on gene expression, the bacterium can be grown on propionate (20 mM) plus fumarate (60 mM). These substrates can be added from 1 M sterile anoxic stock solutions. Adaptation to growth conditions is achieved through at least five subsequent transfers (10% v/v) to fresh media containing the respective electron donors and acceptors .

For studies examining atpE expression in syntrophic conditions, S. fumaroxidans can be co-cultured with hydrogen/formate-utilizing partners such as Methanospirillum hungatei or Geobacter sulfurreducens. When establishing such co-cultures, careful adaptation through multiple transfers is necessary to ensure stable syntrophic growth. Growth can be monitored by analyzing substrate depletion, product formation, and increases in volatile suspended solids (VSS) .

What methodological approaches are effective for isolating and characterizing recombinant S. fumaroxidans atpE?

For isolation and characterization of recombinant S. fumaroxidans atpE, a multi-step methodological approach is recommended:

• Genetic cloning strategy: The atpE gene can be amplified from genomic DNA using PCR with primers designed based on the genome sequence. The amplified gene should be cloned into an appropriate expression vector containing a suitable tag (such as His-tag) for purification.

• Expression system selection: Given the anaerobic nature of S. fumaroxidans, heterologous expression in E. coli may require codon optimization. Selection of an expression system that can accommodate membrane proteins is crucial, as atpE encodes a highly hydrophobic membrane protein.

• Protein purification: A two-phase purification approach is recommended:

  • Initial isolation of membrane fractions through differential centrifugation

  • Solubilization with appropriate detergents (e.g., n-dodecyl-β-D-maltoside)

  • Affinity chromatography using the attached tag

  • Size exclusion chromatography for final purification

• Functional characterization: Assembly of the purified atpE into proteoliposomes can allow for functional studies, including proton translocation assays and ATP synthesis measurements.

• Structural analysis: Techniques such as circular dichroism can provide information about secondary structure, while advanced methods like cryo-electron microscopy might reveal detailed structural information of the assembled c-ring.

How can researchers effectively measure ATP synthase activity in S. fumaroxidans?

Measuring ATP synthase activity in S. fumaroxidans requires specialized techniques that account for the anaerobic nature of this organism and the complex energetics of syntrophic metabolism:

• Membrane vesicle preparation: Researchers should prepare inverted membrane vesicles from S. fumaroxidans cells grown under different conditions (e.g., propionate plus fumarate, propionate plus sulfate, or syntrophic growth).

• ATP synthesis assays: ATP synthesis can be measured by energizing the membrane vesicles with an artificial pH gradient or membrane potential and providing ADP and Pi. ATP formation can be quantified using the luciferase assay.

• ATP hydrolysis assays: The reverse reaction can be measured by monitoring inorganic phosphate release from ATP hydrolysis, using colorimetric methods such as the malachite green assay.

• Inhibitor studies: Using specific inhibitors of ATP synthase (such as oligomycin or DCCD) can confirm the specificity of the measured activity. DCCD specifically binds to the c subunit, making it particularly relevant for atpE studies.

• Correlation with energy conservation: In S. fumaroxidans, ATP synthase activity should be correlated with succinate dehydrogenase activity, as the endergonic oxidation of succinate to fumarate is coupled to energy conservation and requires a transmembrane proton gradient .

How does atpE expression change during syntrophic versus non-syntrophic growth of S. fumaroxidans?

The expression of atpE and other energy metabolism genes in S. fumaroxidans varies significantly between syntrophic and non-syntrophic growth conditions. Proteomic analyses reveal differential protein abundance patterns that reflect the metabolic adjustments required for different growth modes:

• Syntrophic growth conditions: During syntrophic growth with partners like methanogens or Geobacter, S. fumaroxidans must optimize energy conservation mechanisms due to the minimal energy available from propionate oxidation. Under these conditions, components of ATP synthase may show higher expression to maximize ATP yield from the limited energy available.

• Non-syntrophic growth with alternative electron acceptors: When growing with sulfate or fumarate as electron acceptors, S. fumaroxidans shows distinct protein expression patterns. For example, proteomic analyses have shown that the membrane-bound succinate dehydrogenase SdhABC complex (Sfum_1998–2000) shows significantly lower abundance in syntrophic co-cultures compared to pure cultures . This suggests that the energy conservation system, including ATP synthase components, may be differentially regulated.

• Growth substrate effects: The expression of energy metabolism genes, including atpE, likely varies not only with the presence of syntrophic partners but also with the specific growth substrates. When growing on propionate versus alternative substrates, different electron transport chains and energy conservation mechanisms may be employed.

Comparative proteomic studies of S. fumaroxidans grown under different conditions provide insights into these regulatory patterns, though specific data on atpE expression is limited in the available literature.

What is the relationship between atpE function and interspecies electron transfer in syntrophic cultures?

The function of ATP synthase, including its subunit c (atpE), is intricately connected to interspecies electron transfer (IET) in syntrophic cultures involving S. fumaroxidans. This relationship operates through several mechanisms:

• Energy conservation during hydrogen/formate production: In syntrophic cultures, S. fumaroxidans produces hydrogen and formate as electron carriers for interspecies electron transfer. The production of these electron carriers is energetically unfavorable and requires energy input, which is linked to the proton motive force utilized by ATP synthase .

• Reverse electron transport: The oxidation of succinate to fumarate during propionate metabolism is highly endergonic and requires reverse electron transport. This process depends on the transmembrane proton gradient that is also used by ATP synthase, creating a direct link between ATP synthesis/hydrolysis and electron transfer capabilities .

• Different IET mechanisms: In co-cultures of S. fumaroxidans with different partners, various IET mechanisms may operate:

  • With methanogens like M. hungatei, hydrogen and formate serve as primary electron carriers

  • With Geobacter sulfurreducens, there is evidence for both hydrogen/formate interspecies transfer (HFIT) and possibly direct interspecies electron transfer (DIET)

How can structural studies of atpE contribute to understanding energy conservation in S. fumaroxidans?

Structural studies of the ATP synthase subunit c (atpE) from S. fumaroxidans can provide crucial insights into energy conservation mechanisms in this metabolically unique organism:

• c-ring stoichiometry: Determining the number of c subunits in the complete ring structure is critical for understanding the H⁺/ATP ratio and therefore the bioenergetic efficiency of ATP synthesis. Different organisms have different numbers of c subunits per ring, which affects their bioenergetic efficiency.

• Proton-binding site adaptations: Structural analysis can reveal specific adaptations in the proton-binding site of atpE that might optimize function under the energy-limited conditions of syntrophic growth.

• Membrane integration: Understanding how the c-ring integrates into the membrane of S. fumaroxidans could provide insights into how the ATP synthase functions in concert with other membrane complexes involved in reverse electron transport, such as the succinate dehydrogenase SdhABC complex .

• Interaction with other ATP synthase subunits: Structural studies can elucidate interactions between atpE and other subunits of the ATP synthase complex, particularly those unique to S. fumaroxidans or other syntrophic bacteria.

• Comparative structural biology: Comparing the structure of S. fumaroxidans atpE with homologs from other bacteria can highlight adaptations specific to the syntrophic lifestyle and the challenging energetics of propionate metabolism.

These structural insights could ultimately help explain how S. fumaroxidans manages to conserve sufficient energy during syntrophic growth despite operating near thermodynamic limits.

What approaches can resolve contradictory data in S. fumaroxidans atpE functional studies?

Researchers working with S. fumaroxidans atpE may encounter contradictory data due to the complex nature of energy metabolism in this organism. The following methodological approaches can help resolve such contradictions:

• Standardization of growth conditions: Ensure strict consistency in anaerobic cultivation conditions, as subtle variations can significantly affect energy metabolism. S. fumaroxidans should be grown at 35°C in bicarbonate-buffered medium under N₂/CO₂ (80:20, v/v) headspace with carefully controlled substrate concentrations .

• Multi-omics integration: Combine proteomic data with transcriptomics and metabolomics to obtain a more complete picture. For example, proteomic analyses of S. fumaroxidans in syntrophic co-cultures have revealed significant differences in protein abundance compared to pure cultures , which can be correlated with functional measurements.

• Time-course experiments: Collect data at multiple time points during growth to capture dynamic changes in gene expression and enzyme activity, as energy metabolism may shift during different growth phases.

• Genetic manipulation verification: When using recombinant systems, verify that any tags or modifications do not disrupt the native function of atpE by comparing with wild-type controls whenever possible.

• Context-dependent analysis: Interpret data in the context of the specific growth condition being tested. For example, energy conservation mechanisms in S. fumaroxidans differ significantly between growth with fumarate, sulfate, or syntrophic partners .

• Statistical validation: Apply rigorous statistical analyses to distinguish significant differences from experimental noise, especially in proteomic studies where fold changes may be subtle but biologically meaningful.

How can researchers effectively compare ATP synthase activity across different growth conditions?

To effectively compare ATP synthase activity across different growth conditions in S. fumaroxidans, researchers should implement the following methodological strategies:

• Standardized biochemical assays: Develop and consistently apply standardized assays for both ATP synthesis and hydrolysis activities that can be used across all growth conditions. These assays should be optimized for the specific biochemical properties of S. fumaroxidans ATP synthase.

• Normalization methods: Carefully select appropriate normalization methods, such as:

  • Per unit of total protein

  • Per cell dry weight

  • Relative to marker enzyme activities

  • Per copy number of ATP synthase complexes (quantified by targeted proteomics)

• Isolation of consistent membrane fractions: Ensure that membrane fractions are isolated using identical protocols across all conditions to avoid preparation artifacts.

• Control for membrane energization state: Since ATP synthase activity depends on the proton motive force, control for or measure the energization state of the membranes in different preparations.

• Parallel omics analyses: Accompany activity measurements with proteomic analyses to correlate functional changes with protein abundance changes. For example, studies have shown significant differences in the abundance of energy metabolism proteins like the SdhABC complex under different growth conditions .

• In situ activity probes: Where possible, develop methods to measure ATP synthase activity in living cells under different growth conditions, which may provide more physiologically relevant data than in vitro assays.

By systematically applying these approaches, researchers can generate reliable comparative data on ATP synthase activity across the diverse metabolic modes of S. fumaroxidans.

What bioinformatic tools are most effective for analyzing atpE sequence conservation across syntrophic bacteria?

For analyzing atpE sequence conservation across syntrophic bacteria like S. fumaroxidans, the following bioinformatic approaches and tools are particularly effective:

• Multiple sequence alignment tools:

  • MUSCLE or MAFFT for initial alignment of atpE sequences

  • T-Coffee for refinement of alignments, especially for transmembrane regions

  • Clustal Omega for handling large numbers of sequences

• Phylogenetic analysis software:

  • IQ-TREE for maximum likelihood phylogenetic tree construction

  • MrBayes for Bayesian phylogenetic analysis

  • TreeTime for time-calibrated phylogenetic trees to understand evolutionary trajectories

• Comparative genomics platforms:

  • IMG/M (Integrated Microbial Genomes & Microbiomes) for genomic context analysis

  • KEGG for metabolic pathway comparisons

  • OrthoMCL for identification of orthologous proteins across multiple genomes

• Structural prediction tools:

  • AlphaFold2 for predicting protein structures from sequences

  • ConSurf for mapping conservation onto structural models

  • TMHMM or TOPCONS for transmembrane topology prediction

• Specialized analyses:

  • Selection pressure analysis using PAML or HyPhy to identify sites under positive selection

  • Coevolution analysis using tools like EVcouplings to identify co-evolving residues

  • Synteny analysis using tools like SynMap to examine conservation of genomic context

• Visualization tools:

  • Jalview for visualizing multiple sequence alignments and conservation

  • PyMOL or UCSF Chimera for mapping sequence conservation onto structural models

  • Interactive Tree Of Life (iTOL) for visualizing phylogenetic trees with metadata

These tools can help researchers identify conserved functional residues, evolutionary adaptations specific to syntrophic lifestyles, and potential structure-function relationships in ATP synthase subunit c across diverse syntrophic bacteria including S. fumaroxidans.

How might CRISPR-Cas9 techniques advance functional studies of S. fumaroxidans atpE?

CRISPR-Cas9 gene editing technologies offer promising approaches for advancing functional studies of atpE in S. fumaroxidans, despite the challenges of working with anaerobic syntrophic bacteria:

• Site-directed mutagenesis: CRISPR-Cas9 could enable precise modification of specific residues in the atpE gene to investigate structure-function relationships. Researchers could target:

  • Proton-binding residues to alter H⁺/ATP ratios

  • Interface residues that mediate c-ring assembly

  • Residues involved in interactions with other ATP synthase subunits

• Promoter modifications: Engineering the native promoter of atpE could allow for controlled expression studies, helping to understand how expression levels affect syntrophic growth capabilities and energy conservation efficiency.

• Reporter gene fusions: CRISPR-Cas9 could facilitate the creation of fluorescent or luminescent reporter fusions to monitor atpE expression in real-time under different growth conditions.

• Complementation studies: For essential genes like atpE, conditional knockdown approaches combined with expression of variant forms could help elucidate functional requirements.

• Adaptation for anaerobes: Development of anaerobic-compatible CRISPR-Cas9 protocols for S. fumaroxidans would represent a significant methodological advancement for the broader field of syntrophic microbiology.

• Genomic integration of affinity tags: Precise integration of affinity tags at the genomic level would allow purification of native ATP synthase complexes without overexpression artifacts.

These applications could provide unprecedented insights into how ATP synthase functions within the energy conservation system of S. fumaroxidans, particularly in the context of its syntrophic lifestyle where energy conservation is critical .

What emerging technologies show promise for investigating atpE role in interspecies electron transfer?

Several emerging technologies show particular promise for investigating the role of ATP synthase (including atpE) in interspecies electron transfer in S. fumaroxidans:

• Bioelectrochemical systems: These systems allow for the measurement and manipulation of electron transfer processes in real-time. By combining electrochemical measurements with proteomic analysis, researchers can establish correlations between ATP synthase activity and electron transfer rates.

• Single-cell techniques:

  • Fluorescence lifetime imaging microscopy (FLIM) to measure membrane potential at the single-cell level

  • Single-cell proteomics to examine cell-to-cell variation in atpE expression

  • Nanoelectrodes for measuring electron transfer at specific cell-to-cell interfaces

• Advanced microscopy:

  • Super-resolution microscopy to visualize the spatial organization of ATP synthase relative to electron transfer complexes

  • Correlative light and electron microscopy (CLEM) to connect functional data with ultrastructural information

  • Cryo-electron tomography to visualize ATP synthase in situ within the native membrane environment

• Real-time metabolic measurements:

  • Isotope probing combined with mass spectrometry to track metabolic fluxes

  • Biosensors for ATP, proton gradients, or redox states to monitor energy conservation in real-time

• Synthetic biology approaches:

  • Engineering artificial syntrophic systems with defined components

  • Creating chimeric ATP synthases with components from different organisms

These technologies could help resolve the complex relationship between ATP synthesis, proton gradients, and interspecies electron transfer in syntrophic cultures. Recent proteomic studies of S. fumaroxidans in co-culture with G. sulfurreducens have already suggested that different interspecies electron transfer mechanisms may operate simultaneously , and these emerging technologies could provide more detailed insights.

How might structural modifications of atpE be used to engineer more efficient syntrophic relationships?

Strategic structural modifications of ATP synthase subunit c (atpE) could potentially be used to engineer more efficient syntrophic relationships involving S. fumaroxidans through several approaches:

• Optimizing c-ring stoichiometry: Modifying the number of c subunits in the ring could alter the H⁺/ATP ratio, potentially allowing for ATP synthesis at lower proton motive force. This could be achieved through mutations at the c-c interface that favor assemblies with fewer subunits.

• Enhancing proton affinity: Targeted modifications of the proton-binding site could increase the affinity for protons, potentially allowing ATP synthesis to occur at lower proton gradients. This would be particularly beneficial in syntrophic conditions where energy is limited.

• Improving thermal stability: Introducing mutations that enhance the thermal stability of the c-ring could maintain ATP synthase function under stress conditions or allow operations at higher temperatures, potentially increasing reaction rates.

• Interfaces with electron transport components: Engineering the interaction surfaces between ATP synthase and electron transport components could improve the coupling efficiency between electron transfer and proton translocation.

• Synthetic consortia design: Modified atpE variants could be incorporated into synthetic microbial consortia designed for specific applications such as:

  • Enhanced biogas production from propionate-rich waste streams

  • Improved bioremediation capabilities

  • Optimized bioelectrochemical systems

Proteomic studies have shown that energy metabolism proteins in S. fumaroxidans show differential abundance in syntrophic versus pure cultures , indicating that ATP synthase function is adapted to different growth modes. Rational engineering of atpE could potentially enhance these adaptations for improved syntrophic efficiency.