Recombinant Pelobacter propionicus ATP synthase subunit c 1 (atpE1)

What distinguishes Pelobacter propionicus from other anaerobic bacteria in terms of energy metabolism?

Pelobacter propionicus is a strictly anaerobic, Gram-negative bacterium belonging to the Deltaproteobacteria class. While closely related to Geobacter species, P. propionicus exhibits distinct metabolic pathways. Unlike Geobacter, which utilizes outer-surface c-type cytochromes for Fe(III) reduction, P. propionicus contains very low amounts of b-type cytochrome (approximately 46 nmol·g protein⁻¹) . P. propionicus primarily ferments ethanol to propionate and acetate, employing a randomizing pathway for propionate formation as demonstrated through ¹³C-NMR experiments . The bacterium's energy conservation occurs primarily through substrate-level phosphorylation, with ATP generated during the acetate kinase reaction .

How does the ATP synthase c subunit contribute to energy conservation in anaerobic bacteria?

The ATP synthase subunit c forms the critical c-ring in the membrane-embedded F₀ portion of ATP synthase. In P. propionicus, this structure likely functions similarly to other bacterial ATP synthases, creating a proton channel that couples proton translocation across the membrane to ATP synthesis. Given P. propionicus's fermentative metabolism, the ATP synthase likely plays a role in maintaining ion gradients rather than being the primary ATP generation mechanism. The protein's exact stoichiometry and structure would determine the H⁺/ATP ratio, which influences the bacterium's bioenergetic efficiency in energy-limited environments.

What genomic evidence exists for ATP synthase function in Pelobacter species?

While specific data on P. propionicus ATP synthase genes is limited in the provided search results, related Pelobacter species show genomic evidence of energy conservation mechanisms. In P. carbinolicus, genome-wide expression studies have identified upregulation of genes involved in energy metabolism during different growth conditions . The presence of genes encoding complete pathways for substrate oxidation, coupled with genes for cytochrome c biogenesis organized in two operons (particularly Pcar_1953 to Pcar_1954, which showed 8- to 10-fold upregulation during Fe(III) reduction), suggests sophisticated energy conservation systems .

What expression systems are most appropriate for producing functional recombinant ATP synthase subunit c 1 from P. propionicus?

For recombinant expression of hydrophobic membrane proteins like ATP synthase subunit c 1, specialized bacterial expression systems are recommended. Based on approaches used for similar proteins, E. coli C41(DE3) or C43(DE3) strains designed for membrane protein expression would be optimal. Expression should be conducted at lower temperatures (16-20°C) with reduced inducer concentrations to prevent protein aggregation. For construct design, incorporating a cleavable N-terminal fusion partner (such as MBP or SUMO) can improve solubility while maintaining native protein folding after tag removal.

What are the methodological considerations for functional characterization of P. propionicus ATP synthase components?

Functional characterization requires:

• Membrane Preparation Protocol:

  • Culture cells in anaerobic conditions similar to those used for P. propionicus growth

  • Harvest cells in mid-logarithmic phase by centrifugation (5,000×g, 15 min, 4°C)

  • Resuspend in buffer containing 50 mM HEPES-KOH pH 7.5, 250 mM sucrose, 2 mM MgCl₂

  • Disrupt cells using French press (20,000 psi) or sonication

  • Remove unbroken cells (5,000×g, 10 min, 4°C)

  • Isolate membranes by ultracentrifugation (100,000×g, 1 h, 4°C)

• Activity Assays:

  • ATP synthesis activity: luciferin/luciferase-based luminescence assay

  • ATP hydrolysis: coupled enzyme assay with pyruvate kinase and lactate dehydrogenase

  • Proton translocation: ACMA fluorescence quenching assay

This methodology can be adapted from techniques established for analyzing ATP synthases from other anaerobic bacteria.

How can researchers overcome challenges in purifying functional ATP synthase subunit c 1?

Purification of ATP synthase subunit c 1 requires careful consideration of its hydrophobic nature. A multi-step protocol is recommended:

• Solubilization:

  • Solubilize membranes using mild detergents (DDM or LMNG at 1% w/v)

  • Incubate with gentle agitation for 1 hour at 4°C

  • Remove insoluble material by ultracentrifugation (100,000×g, 30 min, 4°C)

• Purification Steps:

  • Immobilized metal affinity chromatography (if His-tagged)

  • Ion exchange chromatography: Resource Q column with gradient elution

  • Size exclusion chromatography: Superdex 200 column

• Protein Stabilization:

  • Maintain purified protein in buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, and 0.02% DDM

  • For functional reconstitution, incorporate into nanodiscs or liposomes using E. coli polar lipids

This approach minimizes aggregation while preserving the native structure necessary for functional studies.

How does the electron transport system of P. propionicus differ from that of Geobacter species, and what implications does this have for ATP synthase function?

Despite phylogenetic proximity to Geobacter species, P. propionicus employs fundamentally different electron transport mechanisms. While Geobacter species utilize numerous c-type cytochromes for extracellular electron transfer to Fe(III), P. propionicus lacks these components . P. propionicus contains minimal amounts of b-type cytochrome (~46 nmol·g protein⁻¹) , suggesting a limited conventional electron transport chain.

This difference implies that the ATP synthase in P. propionicus likely operates under distinct bioenergetic conditions compared to Geobacter species. Rather than being driven by a robust respiratory electron transport chain, the P. propionicus ATP synthase likely functions in concert with fermentative metabolism, potentially:

• Operating in reverse (ATP hydrolysis) to maintain membrane potential

• Utilizing smaller ion gradients established during fermentation

• Having evolved structural adaptations for efficient function at lower proton motive force

These differences would be reflected in the c subunit's structure and the c-ring stoichiometry, which directly influence the H⁺/ATP ratio.

What can be inferred about P. propionicus ATP synthase based on the organism's fermentation pathways?

P. propionicus ferments ethanol to propionate and acetate through a distinct pathway that includes:

This fermentation pathway generates ATP through substrate-level phosphorylation, primarily via the acetate kinase reaction . The presence of a pyruvate:ferredoxin oxidoreductase (pyruvate synthase) suggests electron transfer to ferredoxin during metabolism .

Given this metabolic framework, the ATP synthase in P. propionicus likely:

• Does not serve as the primary ATP generation mechanism

• May function in maintaining ion homeostasis

• Could potentially operate bidirectionally depending on cellular energetic state

• May have evolved specialized adaptations for function in an organism with limited electron transport components

How might analysis of the atpE1 gene from P. propionicus inform our understanding of ATP synthase evolution in anaerobic bacteria?

Comparative analysis of the P. propionicus atpE1 gene could reveal important evolutionary adaptations to anaerobic lifestyles. Key research approaches should include:

• Phylogenetic Analysis: Constructing phylogenetic trees of atpE genes from diverse anaerobic bacteria to determine evolutionary relationships and potential horizontal gene transfer events.

• Sequence Motif Identification: Analyzing conserved residues critical for:

  • Proton binding and translocation

  • Subunit-subunit interactions within the c-ring

  • Interactions with other F₀ components

• Structural Prediction: Homology modeling to identify structural adaptations that might reflect:

  • Adaptation to specific membrane lipid composition

  • Optimization for function at different proton motive force values

  • Altered c-ring stoichiometry affecting bioenergetic efficiency

• Genomic Context Analysis: Examining the organization of ATP synthase genes to identify potential regulatory elements or operonic arrangements unique to P. propionicus.

This systematic approach could reveal how ATP synthases have evolved specialized features in bacteria adapted to energy-limited anaerobic environments.

What approaches can be used to determine the c-ring stoichiometry of P. propionicus ATP synthase, and why is this parameter biochemically significant?

The c-ring stoichiometry (number of c subunits per ring) directly determines the H⁺/ATP ratio and thus the bioenergetic efficiency of ATP synthesis. For P. propionicus, which survives in energy-limited environments, this parameter is particularly crucial.

Research Methodology:

• Atomic Force Microscopy (AFM):

  • Isolate native membranes or reconstituted proteoliposomes

  • Image c-rings at high resolution to count individual subunits

  • Compare ring diameters with known reference proteins

• Mass Determination:

  • Native mass spectrometry of purified c-rings

  • Analytical ultracentrifugation to determine molecular mass

  • Size exclusion chromatography coupled with multi-angle light scattering

• Cryo-Electron Microscopy:

  • Single-particle analysis of purified ATP synthase

  • Sub-tomogram averaging of membrane-embedded complexes

  • Direct visualization and counting of c subunits

Biochemical Significance:
The c-ring stoichiometry determines the theoretical minimum proton motive force required for ATP synthesis. For P. propionicus, which contains limited cytochromes and relies primarily on fermentation , a lower c-ring stoichiometry (and thus H⁺/ATP ratio) would be advantageous, enabling ATP synthesis with smaller ion gradients.

How might the ATP synthase of P. propionicus be involved in its unique iron reduction mechanism?

Although P. propionicus is related to Geobacter species, it appears to employ a fundamentally different mechanism for Fe(III) reduction. While Geobacter species use direct electron transfer via outer-surface c-type cytochromes, evidence from related Pelobacter species suggests an indirect reduction mechanism, possibly involving sulfide production .

The ATP synthase might contribute to this process through:

• Energetic Support: Maintaining membrane potential required for iron reduction processes

• Ion Homeostasis: Regulating intracellular pH during Fe(III) reduction, which can produce protons

• Reverse Operation: Under certain conditions, ATP synthase might operate in reverse, hydrolyzing ATP to pump protons and maintain membrane potential essential for electron transport processes

Experimental approaches to elucidate this relationship could include:

• ATP synthase inhibition studies during Fe(III) reduction

• Membrane potential measurements in wild-type vs. ATP synthase-deficient mutants

• Isotope labeling experiments to track proton flux during Fe(III) reduction

What post-translational modifications might occur in P. propionicus ATP synthase subunit c 1, and how would they affect enzyme function?

Post-translational modifications (PTMs) of ATP synthase subunits can significantly impact assembly, stability, and function. For the c subunit specifically, several PTMs have been identified in other organisms that might also occur in P. propionicus:

Research methodology to investigate these modifications should include:

• Identification Protocol:

  • Purify native ATP synthase complex from P. propionicus membranes

  • Separate subunits by 2D gel electrophoresis

  • Perform in-gel digestion with multiple proteases

  • Analyze peptides by LC-MS/MS with ETD and CID fragmentation

  • Compare spectra to theoretical unmodified peptides

• Functional Analysis:

  • Site-directed mutagenesis of modified residues

  • Comparison of ATP synthesis/hydrolysis activities

  • Membrane incorporation efficiency measurement

  • Proton translocation assays

These investigations would provide insights into how P. propionicus might regulate ATP synthase function through post-translational mechanisms, potentially revealing adaptations specific to its anaerobic lifestyle.

What experimental approaches can determine the regulation of ATP synthase gene expression in P. propionicus under different growth conditions?

Understanding ATP synthase gene regulation is crucial for elucidating how P. propionicus adapts its energy metabolism to different environmental conditions. A comprehensive experimental approach should include:

• Transcriptomic Analysis:

  • RNA-Seq or microarray analysis of P. propionicus grown under different conditions:

    • Different carbon sources (ethanol vs. other alcohols)

    • Presence/absence of electron acceptors

    • Different growth phases

  • qRT-PCR validation of differential expression using primers designed similarly to those described for P. carbinolicus

  • Identification of co-regulated genes to establish regulons

• Promoter Analysis:

  • 5' RACE to map transcription start sites

  • Reporter gene fusions (e.g., lacZ) to quantify promoter activity

  • DNA footprinting to identify transcription factor binding sites

  • Gel shift assays to confirm protein-DNA interactions

• Proteomics:

  • Quantitative proteomics to correlate transcript levels with protein abundance

  • Pulse-chase experiments to determine protein turnover rates

  • Membrane proteome analysis to examine ATP synthase assembly

Similar approaches have revealed differential gene expression patterns in the related organism P. carbinolicus, where microarray and qRT-PCR analyses identified genes upregulated during Fe(III) reduction versus fermentative growth .

How might interspecies electron transfer influence the expression and function of ATP synthase in P. propionicus?

Interspecies electron transfer is an important ecological process in anaerobic communities. For P. propionicus, which contains limited cytochromes , interactions with other microorganisms might influence its energy metabolism and ATP synthase function.

Research approaches should include:

• Co-culture Experiments:

  • Establish defined co-cultures of P. propionicus with:

    • Hydrogenotrophic methanogens

    • Other iron-reducing bacteria

    • Sulfate-reducing bacteria

  • Monitor growth parameters, metabolite production, and gene expression

• Transcriptomic/Proteomic Analysis:

  • Compare ATP synthase gene expression in pure culture vs. co-culture

  • Identify other differentially expressed genes that might interact with ATP synthase

  • Quantify ATP synthase protein levels and post-translational modifications

• Bioenergetic Measurements:

  • Membrane potential determination using fluorescent probes

  • Intracellular ATP concentration measurement

  • Proton motive force calculation under different co-culture conditions

Understanding these interactions could reveal how P. propionicus adapts its ATP synthase function to optimize energy conservation in complex microbial communities.

What molecular techniques can be employed to create site-directed mutations in the atpE1 gene to study structure-function relationships?

Site-directed mutagenesis of the atpE1 gene enables detailed structure-function analysis of the ATP synthase c subunit. For P. propionicus, which lacks established genetic systems, several approaches could be employed:

• Homologous Recombination Strategy:

  • Construct allelic exchange vector containing:

    • Mutated atpE1 gene flanked by ~1 kb homologous regions

    • Antibiotic resistance marker for selection

    • Counter-selectable marker (e.g., sacB) for identifying double crossovers

  • Introduce vector by electroporation or conjugation

  • Select recombinants on appropriate media

  • Confirm mutations by sequencing

• CRISPR-Cas9 Approach:

  • Design guide RNA targeting atpE1

  • Provide repair template containing desired mutation

  • Introduce components via conjugation

  • Screen transformants for successful editing

• Heterologous Expression System:

  • Express wild-type and mutant versions in:

    • ATP synthase-deficient E. coli strain

    • Complementation system in another anaerobe

  • Compare functional parameters including:

    • ATP synthesis/hydrolysis rates

    • Proton translocation efficiency

    • Complex assembly and stability

Key residues to target would include the essential glutamate involved in proton translocation, residues at subunit interfaces, and positions potentially involved in c-ring assembly or interaction with other ATP synthase components.