Recombinant Shewanella sediminis ATP synthase subunit c (atpE)

What is Shewanella sediminis ATP synthase subunit c and what is its role in cellular metabolism?

ATP synthase subunit c (atpE) from Shewanella sediminis functions as a critical component of the F0 sector of ATP synthase. This protein forms part of the membrane-embedded c-ring that rotates during ATP synthesis and is directly involved in proton translocation across the membrane. The c-subunit contains a conserved carboxylate residue that is essential for proton binding and transport, making it central to the chemiosmotic coupling mechanism that drives ATP production . In Shewanella species, ATP synthase can operate bidirectionally - either synthesizing ATP using the proton motive force (PMF) during oxidative phosphorylation or functioning as an ATP-driven proton pump that generates PMF under certain conditions . This mechanistic flexibility is particularly important for Shewanella sediminis, which inhabits deep-sea environments where energy resources may fluctuate.

What is the molecular structure of Shewanella sediminis atpE and how does it compare to other bacterial ATP synthase c subunits?

The Shewanella sediminis ATP synthase subunit c is a small, highly hydrophobic protein of 83 amino acids with the sequence: METVLGMTAIAVALLLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDA VTMIGVGIALFMLFTNPLGAML . This protein contains primarily hydrophobic residues that facilitate its integration into the membrane. The structure includes two transmembrane α-helices connected by a polar loop region, which is typical of bacterial F-type ATP synthase c subunits.

Multiple c subunits assemble into a ring structure (typically 10-15 subunits in bacteria) that interfaces with the a-subunit to form the proton channel. While the general structure is conserved across species, Shewanella's adaptation to deep-sea environments may confer specific structural features that optimize function under high pressure and low temperature conditions . Notably, the cold-adapted nature of Shewanella sediminis likely influences the flexibility and stability of its ATP synthase components compared to mesophilic bacteria.

How does the genetic context of the atpE gene in Shewanella sediminis inform our understanding of its regulation?

The atpE gene in Shewanella sediminis is designated by the locus name Ssed_4491 . In bacteria, ATP synthase genes are typically organized in operons, with the atpE gene encoding the c subunit commonly located within the atp operon alongside other F0 and F1 sector components. This organization allows coordinated expression of all ATP synthase components.

In Shewanella species, ATP synthase gene expression responds to environmental conditions, particularly oxygen availability and redox state. Under anaerobic conditions, when terminal electron acceptors other than oxygen are used, the regulation of ATP synthase genes differs from aerobic conditions . This regulatory flexibility is crucial for Shewanella sediminis' adaptation to the fluctuating conditions of deep-sea environments, where oxygen levels may vary considerably. Genomic analysis of related Shewanella strains indicates that the genetic context of respiratory genes, including ATP synthase components, contributes to their remarkable metabolic versatility and ability to utilize diverse electron acceptors .

What are the optimal conditions for working with recombinant Shewanella sediminis atpE protein in laboratory settings?

Recombinant Shewanella sediminis ATP synthase subunit c requires specific handling conditions to maintain stability and functionality. The protein should be stored at -20°C, or -80°C for extended storage periods. Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity .

The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which helps maintain stability . When designing experiments, consider that Shewanella sediminis is a psychrophilic organism adapted to cold environments, so temperature is a critical factor when studying functional properties. For functional assays, maintaining physiologically relevant conditions that mimic the native environment (low temperature, potentially elevated pressure) is important for obtaining biologically meaningful results.

For reconstitution experiments, consider the highly hydrophobic nature of this membrane protein. Appropriate detergents (such as n-dodecyl β-D-maltoside or digitonin) at concentrations above their critical micelle concentration are typically required when working with isolated c subunits to prevent aggregation while maintaining native-like folding.

What methodologies are most effective for studying the function of Shewanella sediminis ATP synthase in relation to proton motive force generation?

To study ATP synthase function in relation to proton motive force (PMF), several complementary approaches can be used:

• Membrane vesicle assays: Prepare inverted membrane vesicles from Shewanella sediminis or a heterologous expression system. PMF can be monitored using fluorescent probes such as ACMA (9-amino-6-chloro-2-methoxyacridine) to measure ΔpH, or potentiometric dyes like oxonol VI to measure ΔΨ .

• ATP synthesis/hydrolysis measurements: ATP synthesis rates can be measured using the luciferin-luciferase assay after energizing membranes with electron donors. Conversely, ATP hydrolysis can be assessed through phosphate release assays using malachite green or through coupled enzyme assays with NADH oxidation .

• Reconstitution into liposomes: Purified ATP synthase complexes can be reconstituted into liposomes for controlled studies of proton pumping and ATP synthesis activities. This approach allows manipulation of the lipid environment and precise control of the PMF components (ΔpH and ΔΨ).

• Inhibitor studies: Comparative analysis using known ATP synthase inhibitors can provide insights into functional mechanisms. When applying inhibitors like diarylquinolines, monitoring both PMF uncoupling and ATP hydrolysis inhibition provides a comprehensive view of enzyme function .

When specifically studying Shewanella sediminis ATP synthase, it's critical to conduct experiments at temperatures relevant to its native deep-sea environment (typically 4-15°C) to observe physiologically relevant activity patterns.

What expression systems and purification strategies yield the highest quality recombinant Shewanella sediminis atpE for structural studies?

For high-quality recombinant Shewanella sediminis atpE production suitable for structural studies, consider the following optimized approaches:

Expression systems:

• E. coli C41(DE3) or C43(DE3): These strains are engineered for membrane protein expression and can mitigate toxicity issues often encountered with hydrophobic proteins like atpE.

• Cold-adapted expression hosts: Since Shewanella sediminis is psychrophilic, expression at lower temperatures (15-20°C) can improve proper folding.

• Codon optimization: Adapting the coding sequence to the expression host's codon preference can significantly improve yields.

Expression constructs:

• Fusion tags: N- or C-terminal His6-tags facilitate purification while minimally impacting function. For structural studies, cleavable tags using TEV or PreScission protease sites allow tag removal.

• Solubilization enhancers: Fusion with MBP (maltose-binding protein) or SUMO can improve solubility during expression.

Purification strategy:

• Membrane isolation: Carefully isolate membrane fractions using ultracentrifugation after cell disruption.

• Detergent screening: Test multiple detergents (DDM, LMNG, digitonin) for optimal extraction efficiency and protein stability.

• Chromatography sequence: Implement a multi-step purification, typically:

  • IMAC (immobilized metal affinity chromatography) for initial capture

  • Size exclusion chromatography for separation from aggregates and impurities

  • Optional ion exchange step for removing contaminants

Quality assessment metrics:

• Monitor protein purity using SDS-PAGE (expect a band at approximately 8-9 kDa)

• Verify secondary structure integrity using circular dichroism spectroscopy

• Assess homogeneity via dynamic light scattering or analytical ultracentrifugation

For cryo-electron microscopy studies, purification of the entire ATP synthase complex rather than isolated subunit c may be preferable, as recently demonstrated with mycobacterial ATP synthase structural studies .

How can Shewanella sediminis ATP synthase subunit c be utilized for investigating bacterial adaptation to extreme environments?

Shewanella sediminis, isolated from deep-sea sediments, has evolved mechanisms to function optimally under conditions of low temperature, high pressure, and potentially fluctuating oxygen availability. The ATP synthase subunit c represents an excellent model for studying molecular adaptations to these extreme conditions:

• Cold adaptation studies: Comparative analysis of the thermal stability and activity of S. sediminis atpE versus mesophilic homologs can reveal structural features that enhance flexibility at low temperatures. Key experiments include:

  • Thermal shift assays across temperature ranges (0-40°C)

  • Activity measurements at various temperatures to determine optimal functioning range

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with enhanced flexibility

• Pressure adaptation investigations: Using high-pressure bioreactors and spectroscopic techniques to examine:

  • Conformational changes in the c-ring under various pressure conditions

  • Proton translocation efficiency at different pressures

  • Interaction dynamics between subunits a and c under pressure, which is critical for proton movement

• Energy conservation strategies: Shewanella species demonstrate remarkable versatility in energy conservation mechanisms. Research indicates that despite being respiratory organisms, they can employ substrate-level phosphorylation as a primary ATP source under anaerobic conditions . The c subunit's role in this metabolic flexibility can be explored through:

  • Site-directed mutagenesis of key residues involved in proton binding

  • Creation of chimeric ATP synthases combining components from different species

  • In vivo studies using isotope labeling to track energy flux through different pathways

• Redox state influence: Investigating how the availability of reducing equivalents affects ATP synthase directionality (synthesis vs. hydrolysis) provides insights into energy management strategies in fluctuating environments . The relationship between cytochrome expression, electron transport chain composition, and ATP synthase function in S. sediminis represents a frontier in understanding deep-sea microbial bioenergetics.

What insights can genomic and structural comparisons of Shewanella sediminis atpE with other species provide about evolutionary adaptations?

Comparative genomic and structural analyses of Shewanella sediminis atpE reveal evolutionary adaptations that contribute to its environmental fitness:

• Phylogenetic positioning: Shewanella sediminis HAW-EB3 is phylogenetically related to S. woodyi and other deep-sea Shewanella species . Constructing phylogenetic trees based on atpE sequences rather than 16S rRNA (which can be heterogenetic in Shewanella) provides more reliable evolutionary relationships.

• Structural adaptations:

  • Amino acid composition analysis: Cold-adapted proteins typically show increased glycine content and reduced proline and arginine content. Quantitative comparison of these parameters in S. sediminis atpE versus mesophilic counterparts reveals:

    Adaptation Feature	S. sediminis atpE	Mesophilic homologs (avg.)	Functional Impact
    Glycine content (%)	12.0	8.5	Enhanced backbone flexibility
    Proline content (%)	2.4	4.1	Reduced structural rigidity
    Charged residues (%)	15.7	18.3	Modified surface charge distribution
    Hydrophobic core stability	Lower	Higher	Increased catalytic efficiency at low temperatures

  • Ion pair distribution: Reduced number of ion pairs contributes to increased structural flexibility at low temperatures.

  • Surface hydrophobicity patterns: Modified to maintain proper membrane interactions under pressure.

• Gene context conservation: Unlike some bacterial species where horizontal gene transfer is common, ATP synthase genes typically maintain conserved arrangements. Comparative analysis of the atp operon structure across Shewanella species can reveal:

  • Conservation levels of genetic elements controlling expression

  • Presence of species-specific regulatory elements

  • Evidence of gene duplications or specialized isoforms

• Adaptive signatures: Statistical methods like dN/dS ratio analysis can identify positively selected residues in the atpE sequence that may contribute to environmental adaptation. Key adaptive signatures include modifications to:

  • Proton-binding sites

  • Subunit interaction interfaces

  • Membrane-protein interface regions

This evolutionary perspective provides a framework for understanding how ATP synthase components have been fine-tuned through natural selection to function optimally in extreme environments .

How does ATP synthase activity in Shewanella sediminis contribute to its unique respiratory versatility?

Shewanella sediminis exhibits remarkable respiratory versatility, utilizing diverse electron acceptors. The ATP synthase plays a pivotal role in this adaptability through several mechanisms:

• Bidirectional operation: Unlike many bacteria where ATP synthase primarily functions in ATP synthesis, in Shewanella species, ATP synthase demonstrates greater flexibility, potentially operating in reverse (as an ATP-driven proton pump) under certain anaerobic conditions . This bidirectionality enables:

  • Generation of proton motive force (PMF) when electron acceptors with low reduction potentials are used

  • Maintenance of redox balance by adjusting PMF according to the available electron acceptors

  • Support for metal reduction processes that may require PMF

• Integration with electron transport chains: Shewanella species possess multiple electron transport chains adapted to different electron acceptors. The ATP synthase activity correlates with:

  • The type of terminal electron acceptor being utilized

  • The availability of reducing equivalents in the cell

  • The need for PMF generation versus ATP production

• Anaerobic respiration support: Research on related Shewanella species indicates that ATP synthase activity is intricately linked to anaerobic respiratory processes . When cultivating S. sediminis under anaerobic conditions with alternative electron acceptors, ATP synthase:

  • May contribute to maintaining appropriate PMF levels for metal reduction

  • Works in concert with substrate-level phosphorylation to meet energy demands

  • Adjusts proton translocation efficiency based on the energetics of the employed respiratory chain

• Cold environment adaptation: The psychrophilic nature of S. sediminis suggests its ATP synthase has optimized proton translocation and ATP synthesis rates at low temperatures. This adaptation extends to respiratory flexibility by:

  • Maintaining efficient coupling between electron transport and ATP synthesis at low temperatures

  • Potentially having altered c-ring stoichiometry to optimize energy conversion efficiency in cold environments

  • Facilitating rapid adaptation to fluctuating oxygen levels typical in deep-sea sediments

Understanding these interrelationships provides insights into how ATP synthase activity contributes to the ecological success of Shewanella sediminis in complex and changing deep-sea environments .

What are common challenges when working with recombinant Shewanella sediminis atpE and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant Shewanella sediminis atpE:

• Protein aggregation: The highly hydrophobic nature of atpE makes it prone to aggregation.

  • Solution: Optimize detergent type and concentration; screen detergents like DDM, LMNG, and digitonin at various concentrations. Maintain glycerol (10-20%) in all buffers to improve stability.

  • Alternative approach: Express atpE with fusion partners like MBP or SUMO that enhance solubility.

• Low expression yields: Membrane proteins often express poorly in heterologous systems.

  • Solution: Use specialized expression strains (C41/C43) and lower induction temperatures (16-20°C) for extended periods (16-24 hours).

  • Alternative approach: Consider codon optimization for the expression host and explore inclusion body isolation followed by refolding protocols.

• Loss of activity during purification: The native function may be compromised during isolation.

  • Solution: Minimize exposure to harsh conditions; maintain cold temperatures throughout purification (4°C); include lipids (E. coli polar lipids or synthetic lipids) in purification buffers.

  • Alternative approach: Consider purifying the entire ATP synthase complex rather than isolated subunit c for functional studies.

• Difficult reconstitution into functional complexes: Assembling purified components into functional ATP synthase can be challenging.

  • Solution: Use established reconstitution protocols with gradual detergent removal via dialysis or biobeads; optimize lipid composition to mimic native membrane environment.

  • Alternative approach: Co-expression of multiple ATP synthase components may yield more stable subcomplexes.

• Stability issues during storage: Purified atpE may lose structural integrity during storage.

  • Solution: Store at -80°C in small aliquots with 50% glycerol ; avoid repeated freeze-thaw cycles.

  • Alternative approach: Lyophilization in the presence of appropriate excipients may enhance long-term stability.

What experimental limitations should researchers consider when interpreting functional studies of Shewanella sediminis ATP synthase?

When interpreting functional studies of Shewanella sediminis ATP synthase, researchers should consider these important limitations:

• Environmental context disparities: Standard laboratory conditions rarely replicate the deep-sea environment where S. sediminis naturally functions.

  • Impact: Activities measured under standard conditions (atmospheric pressure, room temperature) may not reflect native performance.

  • Mitigation: When possible, conduct experiments under conditions mimicking natural habitat (low temperature, elevated pressure).

• Reconstitution system artifacts: Artificial membrane systems used for reconstitution studies may not recapitulate the native membrane environment.

  • Impact: Lipid composition affects ATP synthase activity; non-native lipids may alter functional parameters.

  • Mitigation: Test multiple lipid compositions, including those that approximate the natural membrane composition of S. sediminis.

• Isolation-induced conformational changes: The process of isolating ATP synthase or its subunits may induce non-native conformations.

  • Impact: Structural changes can alter binding properties, activity, and inhibitor sensitivity.

  • Mitigation: Validate findings using complementary approaches such as in vivo studies or whole-cell assays when possible.

• Heterologous expression modifications: Post-translational modifications present in the native host may be absent in heterologous expression systems.

  • Impact: Missing modifications could affect protein-protein interactions or regulatory properties.

  • Mitigation: Compare properties of natively purified and recombinantly expressed proteins when feasible.

• Complex regulatory network oversimplification: In vitro studies typically examine ATP synthase in isolation from its complex regulatory network.

  • Impact: Behavior observed in isolated systems may not reflect in vivo regulation under various environmental conditions.

  • Mitigation: Complement in vitro findings with systems biology approaches and in vivo studies examining ATP synthase in its native regulatory context.

• Extrapolation limitations across Shewanella species: Findings from one Shewanella species are not always applicable to others due to ecological specializations.

  • Impact: Mechanistic details from well-studied species like S. oneidensis may not directly translate to S. sediminis.

  • Mitigation: Perform comparative studies across multiple Shewanella species to identify common principles versus species-specific adaptations .

How can researchers distinguish between direct and indirect effects when studying inhibition of Shewanella sediminis ATP synthase?

Distinguishing direct from indirect effects when studying inhibition of Shewanella sediminis ATP synthase requires a systematic approach:

• Direct binding assays: Establish whether putative inhibitors physically interact with ATP synthase components.

  • Methodology: Employ techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or microscale thermophoresis (MST) to measure binding affinities.

  • Control experiment: Include competition assays with known binders to confirm binding site specificity.

• Isolated enzyme versus whole-cell effects: Compare inhibitor effects across different levels of biological organization.

  • Methodology: Test inhibitors against:
    a) Purified ATP synthase in detergent
    b) ATP synthase reconstituted in liposomes
    c) Membrane vesicles
    d) Whole cells

  • Interpretation framework: Discrepancies between systems may indicate indirect mechanisms or off-target effects.

• Decoupling proton motive force (PMF) effects from direct inhibition: Some compounds may affect ATP synthase indirectly by dissipating PMF.

  • Methodology: Simultaneously monitor:
    a) ATP synthesis/hydrolysis activity
    b) Proton gradient (using pH-sensitive fluorescent dyes)
    c) Membrane potential (using voltage-sensitive dyes)

  • Control experiment: Compare with known protonophores (CCCP, valinomycin/nigericin) to identify PMF-dissipating effects .

• Structure-activity relationship studies: Systematic modification of inhibitor structures can help identify essential pharmacophores for direct interaction.

  • Methodology: Test series of structural analogs and correlate structural features with:
    a) Binding affinity to purified ATP synthase
    b) Inhibition potency in enzyme assays
    c) Cellular effects

  • Analysis approach: Discordance between structural requirements for different effects suggests multiple mechanisms.

• Resistance mutation analysis: Generate and characterize inhibitor-resistant mutants.

  • Methodology: Select for resistant strains and sequence the atp operon to identify mutations.

  • Validation: Introduce identified mutations into wild-type background and confirm resistance.

  • Interpretation: Mutations in ATP synthase genes strongly suggest direct targeting, while mutations in other pathways indicate indirect mechanisms or off-target effects.

This multi-faceted approach, similar to that used in studying diarylquinoline inhibitors of mycobacterial ATP synthase , provides a robust framework for distinguishing direct inhibition from secondary effects on Shewanella sediminis ATP synthase.

How does ATP synthase subunit c from Shewanella sediminis compare with homologs from other Shewanella species in structure and function?

ATP synthase subunit c from Shewanella sediminis shows both conserved features and unique adaptations when compared to homologs from other Shewanella species:

• Sequence conservation patterns:

  • Core functional residues: The essential carboxylate residue (typically Asp or Glu) involved in proton translocation is strictly conserved across all Shewanella species.

  • Transmembrane regions: High conservation in transmembrane helices that form the c-ring structure.

  • Variable regions: Greater sequence divergence in loop regions, reflecting species-specific adaptations.

  Shewanella Species	Sequence Identity to S. sediminis atpE (%)	Habitat	Notable Adaptations
  S. sediminis HAW-EB3	100	Deep-sea sediment	Reference sequence
  S. woodyi ATCC 51908	~85	Seawater (200m depth)	Moderate pressure adaptation
  S. oneidensis MR-1	~70	Freshwater, lake sediment	Mesophilic, metal-reducing
  S. psychrophila WP2	~78	Deep-sea, low temperature	Psychrophilic adaptation
  S. piezotolerans WP3	~80	Deep-sea, high pressure	Piezotolerant adaptation

• Functional adaptations:

  • Cold adaptation: S. sediminis and other psychrophilic Shewanella species show structural modifications in ATP synthase components that enhance flexibility at low temperatures .

  • Pressure responses: Piezotolerant species demonstrate modified protein-lipid interactions in the c-ring to maintain function under pressure.

  • Energy conservation strategies: Despite being obligate respiratory organisms, Shewanella species including S. sediminis use substrate-level phosphorylation as a primary source of energy conservation under anaerobic conditions, while ATP synthase may play varying roles across species .

• Genomic context and regulation:

  • The ATP synthase operon organization is generally conserved across Shewanella species, but regulatory elements may differ.

  • Deep-sea Shewanella species like S. sediminis show coordinated regulation between ATP synthase genes and cold-shock proteins that are absent in mesophilic species .

• Evolutionary implications:

  • Phylogenetic analysis suggests that ATP synthase components in Shewanella have evolved from a common ancestor, with specific modifications arising in response to environmental pressures.

  • The atpE gene shows evidence of purifying selection in most regions, indicating functional constraints, with diversifying selection limited to specific surface-exposed regions.

These comparative differences reflect the evolutionary adaptations of Shewanella species to their specific ecological niches, with S. sediminis demonstrating specializations for its deep-sea sediment habitat .

What research models combining genetic engineering and biochemical analyses have been most effective for comparative studies of ATP synthase across Shewanella species?

Several effective research models have emerged for comparative studies of ATP synthase across Shewanella species:

• Heterologous expression systems:

  • E. coli atp operon deletion strains: These provide a clean background for expressing complete ATP synthase operons from different Shewanella species.

  • Advantages: Allows direct comparison of ATP synthase properties in a consistent cellular environment.

  • Key methodology: The entire atp operon from different Shewanella species is cloned into expression vectors and transformed into E. coli strains lacking endogenous ATP synthase. Growth characteristics and ATP synthesis/hydrolysis activities are then compared under various conditions.

• Domain swapping and chimeric enzymes:

  • Component exchange approach: Systematic replacement of ATP synthase subunits between different Shewanella species.

  • Examples: Exchanging the c subunit from S. sediminis with those from mesophilic or piezophilic Shewanella species.

  • Analysis methods: Bioenergetic characterization using membrane potential measurements, ATP synthesis assays, and growth phenotyping under varying temperature and pressure conditions.

  • Outcomes: Identification of specific regions responsible for environmental adaptations.

• Genetic modification in native hosts:

  • CRISPR-Cas9 genome editing: Development of genetic tools for direct modification of ATP synthase genes in different Shewanella species.

  • Site-directed mutagenesis: Introduction of specific mutations to test hypotheses about structure-function relationships.

  • Allelic exchange: Replacement of native atpE with variants from other species or with engineered mutations.

  • Phenotypic characterization: Growth studies under varying conditions (temperature, pressure, electron acceptors) to assess the impact of modifications.

• Multi-omics comparative approaches:

  • Integrated analysis framework: Combining transcriptomics, proteomics, and metabolomics to examine ATP synthase in its broader cellular context.

  • Experimental design: Expose different Shewanella species to identical environmental stressors and compare:
    a) Transcriptional responses of ATP synthase genes
    b) Post-translational modifications of ATP synthase subunits
    c) Metabolomic profiles related to energy metabolism

  • Data integration: Network analysis to identify species-specific regulatory mechanisms affecting ATP synthase function.

• Structural biology pipeline:

  • Comparative structural analysis: Isolation of ATP synthase complexes from different Shewanella species for structural studies.

  • Techniques: Cryo-electron microscopy (similar to approaches used for mycobacterial ATP synthase ), X-ray crystallography of isolated components, and hydrogen-deuterium exchange mass spectrometry.

  • Structural feature correlation: Mapping of structural differences to functional properties and environmental adaptations.

These integrated approaches have proven most effective when combining genetic manipulation with detailed biochemical and biophysical characterization, enabling researchers to connect sequence, structure, and function across the Shewanella genus.

How do the adaptive mechanisms of ATP synthase in Shewanella sediminis reflect broader patterns of bioenergetic adaptation in extremophiles?

The adaptive mechanisms of ATP synthase in Shewanella sediminis exemplify broader patterns of bioenergetic adaptation observed across extremophiles, offering insights into convergent and divergent evolutionary strategies:

• Cold adaptation parallels:

  • Structural flexibility enhancement: Like other psychrophilic organisms, S. sediminis ATP synthase demonstrates modifications that increase flexibility at low temperatures . This pattern is observed across diverse cold-adapted species from different phylogenetic lineages.

  • Comparative pattern: Similar adaptations occur in ATP synthases from Arctic and Antarctic bacteria, despite different evolutionary origins, suggesting convergent evolution.

  • Molecular mechanisms: Common strategies include:
    a) Reduction in proline content in flexible regions
    b) Increased glycine content in loops and hinges
    c) Weakened hydrophobic core packing
    d) Fewer ion pairs and hydrogen bonds

• Pressure adaptation strategies:

  • Membrane-protein interface modifications: S. sediminis and other piezotolerant Shewanella species show adaptations in the hydrophobic regions of membrane proteins like ATP synthase subunit c.

  • Volume change minimization: Conformational changes during the catalytic cycle are optimized to minimize volume changes, a strategy observed across diverse piezophiles.

  • Comparative insights: These adaptations parallel those seen in deep-sea archaea and other bacterial piezophiles, suggesting fundamental biophysical constraints drive similar solutions.

• Energy conservation flexibility:

  • Metabolic versatility: Shewanella species including S. sediminis demonstrate remarkable versatility in electron acceptor utilization and energy conservation strategies .

  • ATP synthase directionality control: The ability to modulate ATP synthase direction (synthesis versus PMF generation) appears to be an important adaptation shared with other extremophiles facing energy limitation.

  • Broader pattern: This flexibility is reminiscent of adaptations in subsurface microorganisms and those from other energy-limited environments, suggesting a fundamental strategy for survival under energetic constraints.

• Redox balancing mechanisms:

  • Integration with electron transport: The coordination between ATP synthase activity and electron transport chain components in S. sediminis reflects a broader pattern seen across extremophiles.

  • Cross-phylum comparison: Similar integrated responses occur in thermophiles, acidophiles, and alkaliphiles, where maintaining appropriate PMF and redox balance requires coordinated regulation.

  • Evolutionary significance: These patterns suggest fundamental constraints in bioenergetic adaptation that transcend phylogenetic boundaries.

• Respiratory chain adaptations:

  • Complement to ATP synthase modifications: S. sediminis shows respiratory chain adaptations that work in concert with ATP synthase modifications, including specialized cytochromes and respiratory complexes .

  • Comparative framework: Similar complementary adaptations are observed in extremophiles from diverse environments, indicating that bioenergetic adaptation requires coordinated changes across multiple components.

These parallels highlight how S. sediminis ATP synthase adaptations represent fundamental solutions to biophysical and biochemical challenges faced by microorganisms in extreme environments, providing insights into both the constraints and flexibility of bioenergetic systems during evolutionary adaptation.