Recombinant Shewanella sediminis ATP synthase subunit b (atpF)

What is the structure and function of ATP synthase subunit b (atpF) in Shewanella sediminis?

ATP synthase subunit b (atpF) in S. sediminis is a 156-amino acid membrane protein component of the F-type ATP synthase complex. It functions as part of the F0 sector that spans the bacterial membrane. According to the amino acid sequence data, the protein begins with "MNINATLLGQTVAFIIFVWFCMKFVWPPLMNA..." and contains both hydrophobic and hydrophilic regions .

The atpF protein serves as a critical structural stator element within the ATP synthase complex, connecting the membrane-embedded F0 sector to the catalytic F1 sector. This structural role is essential for proper function of the rotary mechanism of ATP synthase. The protein is encoded by the atpF gene (locus tag Ssed_4490) in the S. sediminis genome .

Within the ATP synthase complex, the b subunit prevents rotation of the catalytic portions of the enzyme while allowing the central rotor to turn, thereby enabling the conversion of proton motive force (PMF) into chemical energy in the form of ATP. The hydrophobic N-terminal region anchors the protein in the membrane, while the hydrophilic C-terminal region interacts with other components of the F1 sector.

Unlike in some well-studied bacteria, the ATP synthase in Shewanella species may play a more complex role in cellular energetics, particularly during anaerobic growth conditions where substrate-level phosphorylation appears to be more important for ATP generation than oxidative phosphorylation .

How does S. sediminis ATP synthase differ from other bacterial ATP synthases?

The ATP synthase of S. sediminis shows several distinctive features compared to well-characterized homologs from mesophilic bacteria like E. coli. These differences reflect adaptations to its psychrophilic, marine lifestyle.

Most notably, as a psychrophilic bacterium isolated from marine sediment, S. sediminis ATP synthase likely exhibits cold-adaptive features in its protein structure. While direct comparative structural data is limited in the available research, several inferences can be made based on studies of other cold-adapted proteins and Shewanella species :

• Amino acid composition: Psychrophilic proteins typically contain decreased levels of proline and arginine residues with increased levels of glycine, providing greater structural flexibility at low temperatures. The precise amino acid composition of S. sediminis atpF suggests adaptations for function in cold environments .

• Membrane environment: S. sediminis produces eicosapentaenoic acid (EPA), an unsaturated fatty acid that increases membrane fluidity at low temperatures. This modified lipid environment likely influences ATP synthase function and stability .

• Salt requirements: S. sediminis belongs to the Na+-requiring group of Shewanella species, suggesting its ATP synthase may have adaptations for functioning in marine environments with higher salt concentrations .

• Energy metabolism context: Unlike in E. coli, studies of Shewanella species suggest that under anaerobic conditions, substrate-level phosphorylation rather than oxidative phosphorylation via ATP synthase may be the primary source of ATP. This indicates potential differences in the regulation and function of ATP synthase within cellular metabolism .

These adaptations ensure optimal enzymatic performance in the cold, high-pressure, marine environment from which S. sediminis was isolated.

What research approaches have been used to characterize the ATP synthase complex in Shewanella species?

Research on ATP synthase in Shewanella species has employed multiple complementary approaches:

• Genomic analysis: Whole genome sequencing has enabled identification and annotation of ATP synthase components across Shewanella species. Comparative genomics has revealed conservation patterns and potential adaptations to different environments .

• Transcriptomic studies: RNA-seq analysis has been used to examine expression of ATP synthase genes under different environmental conditions, including varying pressure, temperature, and oxygen availability .

• Metabolic modeling: Genome-scale metabolic models of Shewanella species have been developed to predict and analyze the role of ATP synthase in cellular energetics. These models integrate reaction stoichiometry, gene-protein-reaction associations, and experimental data to simulate metabolism under various conditions .

• Mutant analysis: Creation and characterization of deletion mutants for various components of energy metabolism pathways have helped elucidate the role of ATP synthase. For example, studies in S. oneidensis MR-1 examined strains lacking genes for acetate kinase (ΔackA) and phosphotransacetylase (Δpta) to understand the relationship between substrate-level phosphorylation and oxidative phosphorylation .

• Protein characterization: Recombinant expression and purification of ATP synthase components, including the atpF subunit, have enabled detailed biochemical and structural studies .

• Flux analysis: Metabolic flux analysis using isotopically labeled substrates has helped trace carbon flow through central metabolism and determine the relative contributions of different ATP-generating pathways .

These multidisciplinary approaches have collectively contributed to our understanding of how the ATP synthase complex functions within the unique metabolic network of Shewanella species.

What are the optimal conditions for expressing recombinant S. sediminis ATP synthase subunits?

Successful expression of recombinant S. sediminis ATP synthase subunits requires optimization of several parameters to ensure high yield and proper folding of these membrane-associated proteins. Based on available product information and general practices for expressing recombinant proteins from psychrophilic organisms, the following methodological approaches are recommended:

Expression system:

• E. coli BL21(DE3) or C41(DE3)/C43(DE3) strains that are optimized for membrane protein expression

• Cold-inducible promoter systems to mimic native low-temperature expression conditions

• Codon optimization for E. coli, particularly if rare codons are present in the S. sediminis sequence

Culture conditions:

• Growth at lower temperatures (16-20°C) during induction phase

• Use of rich media such as Terrific Broth supplemented with appropriate antibiotics

• Low IPTG concentrations (0.1-0.5 mM) for induction to prevent formation of inclusion bodies

• Extended expression times (16-24 hours) at reduced temperatures

For the ATP synthase beta subunit (atpD), expression in E. coli has been successfully demonstrated, as evidenced by the availability of the recombinant protein with high purity (>85% by SDS-PAGE) . For atpF specifically, similar conditions likely apply, though membrane association may require additional optimization.

After expression, the recombinant protein should be stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage, with working aliquots maintained at 4°C for up to one week to avoid repeated freeze-thaw cycles .

What purification strategies are most effective for recombinant ATP synthase subunits?

Purification of recombinant ATP synthase subunits from S. sediminis requires strategies optimized for membrane-associated proteins. Based on established protocols and product information, the following multi-step approach is recommended:

• Membrane fraction isolation:

  • Cell lysis via sonication or French press in a buffer containing protease inhibitors

  • Low-speed centrifugation to remove cell debris

  • Ultracentrifugation to collect membrane fractions

  • Selective solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or CHAPS

• Affinity chromatography:

  • Utilize affinity tags (His-tag is commonly used, though specific tag types may vary as noted in product descriptions)

  • Equilibrate columns with detergent-containing buffers to maintain protein solubility

  • Include gradual imidazole gradients for elution to maximize purity

• Ion exchange chromatography:

  • Secondary purification step based on the theoretical isoelectric point of the protein

  • Removal of remaining contaminants based on charge differences

• Size exclusion chromatography:

  • Final polishing step to separate aggregates and ensure homogeneity

  • Analysis of oligomeric state in solution

For the ATP synthase beta subunit (atpD), purification protocols have achieved >85% purity as determined by SDS-PAGE . Similar approaches can be applied to atpF, with modifications to account for its membrane-association properties.

Quality control measures should include SDS-PAGE analysis, Western blotting for identity confirmation, and mass spectrometry for sequence verification. The final product should be stored in a Tris-based buffer with 50% glycerol at appropriate temperatures with consideration for avoiding repeated freeze-thaw cycles .

How can researchers verify the structural integrity and functionality of purified recombinant atpF?

Verifying both structural integrity and functional activity of recombinant S. sediminis atpF requires a comprehensive approach combining biophysical, biochemical, and functional assays:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy to evaluate secondary structure content and proper folding

• Thermal shift assays to determine stability and melting temperature

• Limited proteolysis to probe for properly folded domains resistant to digestion

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess oligomeric state

Biochemical characterization:

• Western blotting with specific antibodies to confirm identity

• Mass spectrometry for accurate mass determination and peptide mapping

• N-terminal sequencing to verify proper processing

• Dynamic light scattering to evaluate homogeneity and detect aggregation

Functional verification:

• Binding assays with other ATP synthase subunits (particularly the F1 components) to verify proper interaction interfaces

• Reconstitution experiments combining recombinant subunits to build partial or complete ATP synthase complexes

• ATP hydrolysis assays with reconstituted complexes

• Proton pumping assays using pH-sensitive fluorescent dyes in proteoliposomes containing reconstituted complexes

For temperature adaptation studies, these assays should be performed across a range of temperatures (4-37°C) to evaluate cold adaptation features. Additionally, for pressure adaptation studies, high-pressure biophysical techniques can be employed to examine structural stability under pressure conditions mimicking the natural habitat of S. sediminis.

Successful verification would demonstrate that the recombinant atpF maintains native-like structure, can interact properly with partner proteins, and contributes to the functional activity of the ATP synthase complex.

How does ATP synthase function differ under aerobic versus anaerobic conditions in Shewanella species?

The function of ATP synthase in Shewanella species shows remarkable differences between aerobic and anaerobic conditions, revealing a metabolic flexibility that distinguishes these bacteria from many other model organisms:

Under aerobic conditions:

• ATP synthase primarily functions in its canonical role, synthesizing ATP using the proton motive force generated by the respiratory chain

• The electron transport chain pumps protons across the membrane during NADH and succinate oxidation

• Oxygen serves as the terminal electron acceptor with high redox potential, enabling efficient energy conservation

Under anaerobic conditions:

• Studies in S. oneidensis MR-1 suggest a paradigm shift in energy metabolism

• Substrate-level phosphorylation through enzymes like acetate kinase (AckA) becomes the primary source of ATP rather than oxidative phosphorylation via ATP synthase

• ATP synthase may have minor contributions to ATP production or, surprisingly, might function in reverse as an ATP-driven proton pump to generate proton motive force

• Various terminal electron acceptors (nitrate, fumarate, metals, etc.) with lower redox potentials are utilized, affecting the energetics of the electron transport chain

Flux analysis in S. oneidensis revealed that in wild-type cells under anaerobic conditions, acetate kinase (AckA) was primarily responsible for ATP production through substrate-level phosphorylation, while ATP synthase played a minor role in ATP generation . When the ackA gene was deleted, significant metabolic reorganization occurred, affecting the roles of numerous enzymes including ATP synthase.

Interestingly, the genome-scale metabolic model of Shewanella demonstrated that, under some anaerobic conditions, ATP synthase might actually consume ATP to generate proton motive force, which is then used for other cellular processes including the reduction of external electron acceptors . This reverse function represents a fundamental difference from the typical operation in model organisms like E. coli under similar conditions.

What role does ATP synthase play in S. sediminis' adaptation to cold temperatures and high pressure?

ATP synthase plays multiple critical roles in S. sediminis' adaptation to its psychrophilic and piezotolerant (pressure-tolerant) lifestyle:

Cold temperature adaptations:

• The ATP synthase complex in S. sediminis likely contains structural modifications that maintain catalytic efficiency at low temperatures

• These may include increased flexibility in catalytic domains, reduced hydrophobic interactions in the core structure, and optimized energy coupling at lower temperatures

• EPA (eicosapentaenoic acid) production in S. sediminis, confirmed by fatty acid profiling, modifies membrane fluidity and likely influences ATP synthase function at low temperatures

• The ATP synthase must maintain efficient energy conservation despite the reduced kinetic energy available at low temperatures

High pressure adaptations:

• Studies of Shewanella species from deep-sea environments indicate that high pressure affects intracellular energy production pathways

• S. sediminis ATP synthase likely has structural features that maintain proper function under elevated pressure conditions

• These may include reduced void volumes, pressure-resistant protein-protein interfaces, and optimized rotary mechanism that functions effectively under compression

• Under high pressure, Shewanella species tend to shift from aerobic respiration to anaerobic respiration, affecting the role of ATP synthase

Metabolic integration:

• ATP synthase function is integrated with broader metabolic adaptations in S. sediminis

• Transcriptomic analysis of the related psychrophilic Shewanella strain YLB-09 revealed that high-pressure conditions cause widespread downregulation of genes involved in the TCA cycle, pyruvate metabolism, and oxidative phosphorylation, while genes in glycolysis/gluconeogenesis were generally upregulated

• This suggests a pressure-induced metabolic shift that likely affects ATP synthase function and regulation

These adaptations collectively enable S. sediminis to maintain energy homeostasis in its cold, high-pressure marine environment, demonstrating sophisticated evolutionary tuning of the ATP synthase complex to support life under challenging conditions.

How does ATP synthase contribute to S. sediminis' ability to perform environmental adaptations?

ATP synthase contributes fundamentally to S. sediminis' environmental adaptations through several interconnected mechanisms:

Respiratory flexibility:

• S. sediminis, like other Shewanella species, can utilize a broad range of electron acceptors including oxygen, nitrate, metals, and organohalides

• ATP synthase works in concert with this flexible respiratory system, helping to optimize energy conservation regardless of available electron acceptors

• During tetrachloroethene reduction to trichloroethene (a capability confirmed in S. sediminis), the enzyme shows Michaelis-Menten kinetics, indicating a well-regulated process that requires proper energy management via ATP synthase

Integration with cellular redox state:

• The Arc two-component system in Shewanella species senses redox conditions and regulates metabolism accordingly

• While ArcS (sensor kinase) differs significantly from its E. coli counterpart, the system maintains functional conservation in regulating the response to changing oxygen levels

• ATP synthase activity is likely modulated by this regulatory network to maintain appropriate energy balance under different redox conditions

Nutrient acquisition and utilization:

• S. sediminis can grow on several carbon sources, including N-acetyl-d-glucosamine, Tween 40, Tween 80, acetate, succinate, butyrate, and serine

• The BtuB protein in Shewanella species, including S. sediminis, is involved in cobalamin (vitamin B12) transport

• ATP synthase provides the energy required for active transport systems and biosynthetic pathways necessary for utilizing these diverse nutrients

Membrane homeostasis:

• S. sediminis produces eicosapentaenoic acid (EPA), which influences membrane fluidity

• ATP synthase must function within this specialized membrane environment

• The EPA synthesis gene cluster is conserved across Shewanella genomes, though only some species (including S. sediminis) produce significant amounts of EPA

These interconnected systems highlight how ATP synthase serves as a central hub in the adaptive network of S. sediminis, supporting its remarkable environmental versatility and specialized niche adaptation.

What techniques are most effective for studying ATP synthase activity under simulated deep-sea conditions?

Studying ATP synthase activity under simulated deep-sea conditions requires specialized techniques that can maintain cold temperatures and high pressure while enabling precise measurements. The following methodological approaches are particularly effective:

High-pressure bioreactor systems:

• Custom-designed pressure vessels that allow real-time monitoring of bacterial cultures under high pressure (up to 50 MPa to simulate deep-sea conditions)

• Temperature-controlled systems operating at 4-15°C to match the psychrophilic nature of S. sediminis

• Sampling ports that maintain pressure during sample collection for biochemical analysis

• Integration with optical sensors for non-invasive monitoring of growth and metabolic activity

Biochemical assays under pressure:

• High-pressure stopped-flow spectrophotometry for enzyme kinetics studies

• Pressure-resistant cuvettes with ATP synthesis/hydrolysis coupled enzyme assays

• Fluorescent probes for measuring membrane potential and pH gradients in pressurized systems

• Luciferase-based ATP detection optimized for low-temperature conditions

Structural biology under pressure:

• High-pressure NMR to study protein dynamics under deep-sea conditions

• Small-angle X-ray scattering (SAXS) with pressure cells to analyze protein conformational changes

• Pressure-adapted circular dichroism spectroscopy for secondary structure analysis

Genetic and molecular biology approaches:

• Expression of recombinant ATP synthase components under pressure-mimicking conditions

• Site-directed mutagenesis to identify pressure-sensitive residues

• Complementation studies using ATP synthase variants in pressure-sensitive mutants

A particularly valuable approach would be to compare the activity profiles of ATP synthase from S. sediminis (pressure-tolerant) with homologs from surface-dwelling relatives across a range of pressure and temperature conditions. This comparative analysis would highlight specific adaptations that enable function in the deep-sea environment.

For S. eurypsychrophilus YLB-09, a related deep-sea Shewanella strain, research has confirmed pressure-tolerant characteristics with capability to grow under high pressure (50 MPa) and at low temperatures (4°C) . Similar methodologies can be applied to study S. sediminis ATP synthase under these extreme conditions.

How does the function of S. sediminis ATP synthase relate to its ability to degrade environmental contaminants?

The relationship between S. sediminis ATP synthase function and its remarkable ability to degrade environmental contaminants involves sophisticated energy coupling and metabolic integration:

Connection to reductive dehalogenation:

• S. sediminis is notably capable of degrading hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and reductively dechlorinating tetrachloroethene (PCE) to trichloroethene (TCE)

• Cell extracts of S. sediminis catalyze PCE dechlorination with a specific activity of approximately 1 nmol TCE min−1 (mg protein)−1

• This process follows Michaelis-Menten kinetics, indicating enzyme-mediated transformation that requires energy investment

• ATP synthase supplies the energy needed to maintain cellular functions during these energy-intensive biodegradation processes

Redox balancing during anaerobic respiration:

• Reductive dehalogenation serves as a terminal electron-accepting process during anaerobic respiration

• ATP synthase function during anaerobic growth in Shewanella species shows unique characteristics, with potential reverse function to generate proton motive force

• This PMF may drive electron transfer processes required for reductive dehalogenation

Genomic evidence for metabolic integration:

• The genome of S. sediminis contains five putative reductive dehalogenase (Rdh) genes

• These dehalogenases are part of the complex electron transport network that ultimately affects ATP synthase function

• The presence of genes like napAB (for nitrate reduction), hydAB (for hydrogen production), and genes for sulfur compound reduction indicates diverse respiratory capabilities that all connect to energy conservation via ATP synthase

Environmental adaptation synergy:

• ATP synthase adaptations for cold temperatures and marine conditions (including Na+ requirement) likely optimize energy conservation during biodegradation

• The psychrophilic nature of S. sediminis provides advantages for bioremediation in cold environments, with ATP synthase supporting metabolism under these conditions

Understanding this relationship has practical applications for environmental biotechnology, potentially enabling optimization of bioremediation processes using S. sediminis or engineered derivatives targeting specific contaminants.

What insights can comparative genomics provide about ATP synthase evolution in Shewanella species?

Comparative genomics offers powerful insights into ATP synthase evolution across Shewanella species, revealing adaptation patterns related to diverse environments:

Conservation and variation patterns:

• Genome-level phylogenetic analysis of Shewanella species (including 19 Shewanella taxa and 3 outgroup species) provides a robust evolutionary framework for examining ATP synthase evolution

• ATP synthase genes show strong conservation of core functional domains across Shewanella species, reflecting the essential nature of this complex

• Variations in sequence appear to correlate with environmental parameters such as temperature, pressure, and salinity

• These analyses help distinguish between ancestral traits and derived adaptations in the ATP synthase complex

Environmental adaptation signatures:

• Cold-adapted Shewanella species (including S. sediminis HAW-EB3, S. loihica PV-4, and S. frigidimarina NCIMB 400) share specific genomic features not found in mesophilic counterparts

• The LIV-I branched-chain amino acid ABC transporter, important for regulating branched-chain fatty acid synthesis, was identified only in cold-adapted Shewanella species, suggesting a role in cold adaptation

• Similar comparative approaches can reveal adaptations in ATP synthase genes specific to cold-adapted species

Horizontal gene transfer assessment:

• Dinucleotide bias analysis of gene clusters in Shewanella can identify potential horizontal gene transfer events

• Analysis of the LIV-I gene cluster showed high dinucleotide bias in all four Shewanella species containing it, suggesting acquisition through horizontal gene transfer

• Similar analyses of ATP synthase genes could reveal whether any components have been acquired horizontally, potentially bringing adaptive advantages

Functional divergence analysis:

• Genome-scale metabolic models of Shewanella species provide functional context for ATP synthase evolution

• These models reveal how ATP synthase integrates with species-specific metabolic networks

• Comparison of substrate-level phosphorylation versus oxidative phosphorylation across species illuminates evolutionary shifts in energy conservation strategies

Reconstruction of ancestral states:

• Phylogenomic approaches enable reconstruction of ancestral ATP synthase sequences

• These reconstructions help track the evolutionary trajectory of adaptations to different environments

• Understanding when and how adaptations arose provides insight into the selective pressures shaping ATP synthase evolution

Through these comparative genomic approaches, researchers can reconstruct the evolutionary history of ATP synthase in Shewanella species and identify specific adaptations that enable functioning across diverse environments from the deep sea to freshwater systems.