Recombinant Syntrophomonas wolfei subsp. wolfei ATP synthase subunit b (atpF)

What is the genomic organization of the ATP synthase gene cluster in Syntrophomonas wolfei?

The ATP synthase complex in Syntrophomonas wolfei is encoded by a single gene cluster in the genome, specifically identified as Swol_2381-2388 . This organized genetic architecture is typical of bacterial ATP synthases but with specific adaptations in S. wolfei that may relate to its syntrophic lifestyle. When designing experiments to study atpF specifically, researchers should consider the genomic context and potential co-regulation with other ATP synthase subunits. Sequence analysis and comparative genomics approaches can help identify regulatory elements and promoter regions that control expression of this operon.

How many subunits comprise the ATP synthase complex in S. wolfei?

Proteomic analyses have identified at least six subunits of the S. wolfei ATP synthase complex: α, β, γ, δ, b, and c . Three of these subunits were detected under all growth conditions tested, suggesting constitutive expression of core components . The b subunit (atpF) is particularly important as it forms part of the peripheral stalk connecting the F₁ and F₀ portions of the ATP synthase complex. When planning recombinant expression studies, consider that proper folding and function of the b subunit may depend on interactions with other ATP synthase components.

What methods are most effective for detecting ATP synthase subunits in S. wolfei?

Based on published research, the following methods have proven effective:

• Blue Native Gel Electrophoresis: This technique successfully resolves membrane protein complexes from S. wolfei, allowing identification of ATP synthase subunits while maintaining native protein interactions .

• Mass Spectrometry-Based Proteomics (LC-MS/MS): This approach has been used to characterize protein profiles in S. wolfei and can identify ATP synthase subunits in complex samples .

• 2D Gel Electrophoresis: This method separates proteins by both isoelectric point and molecular weight, helping to identify ATP synthase subunits at specific molecular weights and pI values .

• Activity Staining: While not specific to ATP synthase, activity staining has been successfully used to identify enzymatic activity in membrane protein complexes from S. wolfei .

How do growth conditions affect the expression of ATP synthase in S. wolfei?

Growth conditions significantly influence ATP synthase expression in S. wolfei:

The ATP synthase complex appears to play a role in energy conservation during syntrophic growth, particularly under conditions requiring reverse electron transfer . When designing experiments with recombinant atpF, replicating these physiological conditions may be important for proper functional assessment.

How does the expression of ATP synthase subunit b (atpF) differ between syntrophic and axenic growth conditions?

The expression of ATP synthase subunits, including subunit b, shows significant variation between syntrophic and axenic growth conditions. Proteomic analysis reveals that certain ATP synthase components are more abundant in membranes of butyrate-grown S. wolfei cells in co-culture with methanogens compared to axenically grown cells on crotonate .

For experimental design, consider:

• Using quantitative proteomics to measure specific changes in atpF abundance

• Performing qRT-PCR to analyze gene expression levels across different growth conditions

• Investigating post-translational modifications that may affect protein function in different metabolic states

The differential expression suggests that ATP synthase may have specialized functions during syntrophic metabolism, potentially related to energy conservation mechanisms required for the thermodynamically unfavorable oxidation of butyrate .

What protocols are most effective for the recombinant expression of S. wolfei ATP synthase subunit b?

While the search results don't provide specific protocols for recombinant expression of S. wolfei atpF, general methodological approaches for membrane proteins from anaerobic bacteria can be adapted:

What role does ATP synthase play in reverse electron transfer during syntrophic butyrate degradation?

ATP synthase appears to be integral to the energy conservation mechanisms required for syntrophic butyrate degradation. The process involves:

• Proton/Sodium Gradient Utilization: The ATP synthase complex in S. wolfei is described as "proton- or sodium-driven" , suggesting it can utilize ion gradients for ATP synthesis.

• Energy Conservation: During syntrophic butyrate oxidation, which is thermodynamically unfavorable, reverse electron transfer requires energy input. The ATP synthase likely works in reverse (ATP hydrolysis) to generate the required proton gradient .

• Co-localization with Electron Transfer Components: Proteomic analyses show that ATP synthase subunits are present in membrane fractions along with hydrogenases and FeS oxidoreductases that are essential for reverse electron transfer .

• Differential Expression: ATP synthase components show altered expression patterns during syntrophic growth on butyrate compared to axenic growth on crotonate, highlighting their specialized role in this metabolic mode .

This functional role should be considered when designing experiments with recombinant atpF, as the protein may have adapted to function optimally under conditions requiring tight coupling with electron transfer components.

How does the ATP synthase of S. wolfei compare to homologous complexes in other syntrophic bacteria?

While the search results don't provide direct comparative information, several observations about S. wolfei ATP synthase suggest unique adaptations:

• The ATP synthase in S. wolfei is encoded by a single gene cluster (Swol_2381-2388) , which is consistent with other bacterial F-type ATP synthases but may contain syntrophy-specific regulatory elements.

• The complex is described as "proton- or sodium-driven" , suggesting flexibility in ion coupling that might be adaptive for syntrophic lifestyle.

• The observation of differential expression under different growth conditions indicates specialized regulatory mechanisms that may differ from non-syntrophic bacteria .

Research approaches to explore these comparisons should include:

• Comparative genomics across syntrophic bacteria species

• Phylogenetic analysis of ATP synthase subunits

• Structural modeling to identify unique features

• Functional studies under varying ion gradients to determine specificity

What are the critical factors affecting the functionality of recombinant S. wolfei ATP synthase subunit b in experimental systems?

Several critical factors likely influence the functionality of recombinant atpF:

• Membrane Environment: The lipid composition of the expression host may differ significantly from S. wolfei, potentially affecting proper folding and function. Consider lipid supplementation or reconstitution in liposomes mimicking the native membrane composition.

• Protein-Protein Interactions: The b subunit forms part of the peripheral stalk and interacts with other ATP synthase components. Co-expression with partner proteins (particularly the δ and α subunits) may be necessary for proper folding and function.

• Post-Translational Modifications: S. wolfei proteins show evidence of acylation modifications related to metabolic intermediates . These modifications might be essential for function but difficult to replicate in heterologous expression systems.

• Redox Environment: As an anaerobe, S. wolfei proteins may be sensitive to oxidation. Expression and purification under anaerobic conditions might preserve critical structural features.

• Energy Coupling Mechanisms: The ATP synthase of S. wolfei appears adapted to function in reverse electron transfer conditions . Functional assays should consider this specialized role rather than standard ATP synthase activity measurements.

How can structural biology approaches be used to understand the unique adaptations of S. wolfei ATP synthase to syntrophic lifestyle?

Structural biology approaches offer powerful tools to investigate the specialized features of S. wolfei ATP synthase:

These approaches would help identify structural adaptations that enable ATP synthase to function efficiently in the energy-limited conditions of syntrophic metabolism.

What methodologies are most appropriate for investigating the interaction between ATP synthase and other membrane complexes during syntrophic metabolism?

Investigating protein-protein interactions in membrane systems requires specialized approaches:

• Blue Native PAGE coupled with second-dimension SDS-PAGE: This approach has already proven effective in S. wolfei studies and can identify stable protein complexes and their components.

• Proximity Labeling Techniques: Methods such as BioID or APEX2 could be adapted to identify proteins that transiently interact with ATP synthase subunits in living cells.

• Co-immunoprecipitation with Crosslinking: Chemical crosslinking can capture transient interactions before extraction from the membrane, followed by immunoprecipitation and mass spectrometry.

• Fluorescence Resonance Energy Transfer (FRET): If genetic manipulation of S. wolfei becomes feasible, FRET could be used to study interactions between ATP synthase and other membrane complexes in living cells.

• Super-Resolution Microscopy: Techniques like STORM or PALM could map the spatial distribution of ATP synthase relative to other membrane complexes involved in syntrophic metabolism.

What implications does the ATP synthase have for metabolic engineering of syntrophic consortia for bioenergy applications?

Understanding the ATP synthase of S. wolfei has significant implications for metabolic engineering:

• Energy Conservation Enhancement: Optimizing ATP synthase function could potentially improve energy conservation during syntrophic metabolism, enhancing the efficiency of bioenergy production from fatty acids.

• Syntrophic Partner Compatibility: The adaptations of S. wolfei ATP synthase for interspecies electron transfer suggest that engineered consortia should consider compatible energy coupling mechanisms between partners.

• Reverse Engineering for Synthetic Biology: The mechanisms by which S. wolfei couples ATP synthesis/hydrolysis to reverse electron transfer could inspire novel synthetic biology designs for energy-efficient biocatalysis.

• Biomarkers for Process Monitoring: Expression levels of ATP synthase subunits could serve as biomarkers for monitoring the metabolic state of syntrophic consortia in bioreactors.

• Thermodynamic Bottleneck Resolution: Understanding how ATP synthase helps overcome thermodynamic barriers in syntrophic metabolism could inform strategies to overcome similar bottlenecks in engineered pathways.