Recombinant Shewanella amazonensis ATP synthase subunit c (atpE)

What is Shewanella amazonensis and why is its ATP synthase subunit c (atpE) of research interest?

Shewanella amazonensis is a Gram-negative, facultatively anaerobic, motile, polarly flagellated, rod-shaped bacterium that was isolated from shallow-water marine sediments in the Amazon River delta. It belongs to the Gammaproteobacteria class and is exceptionally active in the anaerobic reduction of iron, manganese, and sulfur compounds . This metal-reducing capability makes it important for bioremediation applications involving contaminated metals and radioactive wastes .

The ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase, which is responsible for ATP production in the cell. This protein is of particular research interest because:

• It functions as part of the membrane-embedded F0 sector of ATP synthase

• It plays a crucial role in proton translocation across the membrane

• Its structure and function are conserved across many species, making it useful for comparative studies

• Understanding this protein can provide insights into bioenergetics and membrane biology

What are the key structural and biochemical characteristics of recombinant S. amazonensis atpE protein?

The recombinant S. amazonensis ATP synthase subunit c (atpE) is characterized by the following features:

• Protein length: Full length (1-83 amino acids)

• Amino acid sequence: METILGFTAIAVALLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDA VTMIGVGIALYMLFTNPLGAML

• UniProt ID: A1SBU5

• Synonyms: ATP synthase F0 sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, Lipid-binding protein

• Typical recombinant form: His-tagged at the N-terminus, expressed in E. coli

The protein is predominantly hydrophobic, consistent with its role as a membrane-embedded component. Its structure includes transmembrane domains that form the c-ring of the ATP synthase complex, which is essential for the rotary mechanism of ATP synthesis.

How should recombinant S. amazonensis atpE protein be stored and handled in a laboratory setting?

For optimal stability and activity of recombinant S. amazonensis atpE protein, the following storage and handling protocols are recommended:

• Storage conditions:

  • Store at -20°C/-80°C upon receipt

  • For extended storage, conserve at -20°C or -80°C

  • Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Reconstitution procedure:

  • Briefly centrifuge the vial prior to opening to bring contents to the bottom

  • Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) and aliquot for long-term storage

  • The default final concentration of glycerol is typically 50%

• Storage buffer composition:

  • Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • Alternative: Tris-based buffer with 50% glycerol

These handling procedures help maintain protein stability and functional integrity for experimental use.

How does the structure and function of S. amazonensis atpE compare to ATP synthase c subunits from other bacterial species?

The ATP synthase subunit c (atpE) from S. amazonensis, while sharing fundamental structural features with other bacterial ATP synthase c subunits, displays certain distinctive characteristics:

• Sequence comparison:

  • The 83-amino acid sequence of S. amazonensis atpE (METILGFTAIAVALLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDA VTMIGVGIALYMLFTNPLGAML) contains the conserved acidic residue (aspartate or glutamate) essential for proton translocation

  • Phylogenetic analysis reveals greatest similarity to Shewanella putrefaciens, though DNA-DNA hybridization shows low similarity values (24.6-42.7%), confirming its species-level distinction

• Functional implications:

  • The membrane-spanning regions contain predominantly hydrophobic residues, characteristic of proteins that form the c-ring structure

  • The conserved acidic residue plays a crucial role in the proton translocation mechanism during ATP synthesis

  • The protein's adaptation to S. amazonensis's optimal growth conditions (35°C, 1-3% NaCl, pH 7-8) suggests potential structural adaptations for function in these specific environmental conditions

• Evolutionary significance:

  • Comparative analysis with other Shewanella species provides insights into the evolution of ATP synthase in bacteria adapted to different environmental niches

  • The gyrB sequence analysis supports its taxonomic position as a distinct species within the Shewanella genus

This comparative structural and functional analysis is valuable for understanding the adaptation of ATP synthase to different ecological niches and physiological conditions.

What methodological approaches are most effective for studying the interaction of S. amazonensis atpE with other ATP synthase subunits?

Investigating the interactions between S. amazonensis atpE and other ATP synthase subunits requires sophisticated methodological approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation (Co-IP) using His-tagged atpE as bait protein

  • Cross-linking studies combined with mass spectrometry to identify interaction sites

  • Surface plasmon resonance (SPR) to measure binding kinetics between atpE and other subunits

  • Biolayer interferometry for real-time analysis of protein-protein interactions

• Structural analysis techniques:

  • Cryo-electron microscopy (cryo-EM) to visualize the entire ATP synthase complex with atpE in its native conformation

  • X-ray crystallography of reconstituted subcomplexes containing atpE

  • Nuclear magnetic resonance (NMR) spectroscopy for analyzing the dynamics of specific interaction domains

  • Molecular dynamics simulations to predict conformational changes during subunit interactions

• Functional assays:

  • Reconstitution of atpE with other ATP synthase subunits in liposomes to measure ATP synthesis activity

  • Site-directed mutagenesis of key residues followed by functional assays to identify critical interaction points

  • Proton translocation assays using pH-sensitive fluorescent probes to assess functional coupling between atpE and other subunits

These methodological approaches can provide comprehensive insights into how atpE interacts with other ATP synthase components to enable the enzyme's function in energy transduction.

How does sodium chloride stress affect the expression and function of ATP synthase components in S. amazonensis?

S. amazonensis demonstrates a complex response to sodium chloride stress that affects ATP synthase expression and function:

• Temporal expression profile:

  • During NaCl stress, S. amazonensis SB2B shows an orchestrated sequence of events involving increased signal transduction associated with motility and restricted growth

  • Following a metabolic shift to branched chain amino acid degradation, motility and cellular replication proteins return to pre-perturbed levels

  • Unlike other organisms, S. amazonensis does not change its membrane fatty acid composition during NaCl stress, as fatty acid degradation pathways are not expressed

• ATP synthase regulation:

  • The ATP synthase complex, including atpE, likely undergoes expression changes during salt stress that align with the energetic demands of the stress response

  • The absence of membrane fatty acid composition changes suggests that ATP synthase must maintain functionality within the existing membrane environment

  • The metabolic shift to branched chain amino acid degradation may provide alternative energy sources when ATP synthesis is affected by salt stress

• Experimental evidence:

  • Proteomics analysis has revealed that S. amazonensis responds to salt stress through pulse expression of proteases and nucleases immediately following NaCl exposure

  • The expression of protease/chaperone ClpA (Sama2056) decreases until growth resumes between 60-90 minutes after stress application

  • These changes suggest a coordinated response affecting energy metabolism and protein turnover, which would impact ATP synthase function

Understanding these stress response mechanisms provides valuable insights into how S. amazonensis maintains energy homeostasis under challenging environmental conditions.

What are the optimal expression systems and conditions for producing high-yield, functional recombinant S. amazonensis atpE?

Optimizing the expression of recombinant S. amazonensis atpE requires careful consideration of expression systems and conditions:

• Expression systems comparison:

• Optimization parameters:

  • Temperature: Lower temperatures (18-25°C) often reduce inclusion body formation for membrane proteins

  • Induction: Low IPTG concentration (0.1-0.5 mM) and induction at mid-log phase (OD600 0.6-0.8)

  • Media composition: Enriched media (2XYT, TB) supplemented with glucose (0.5-1%)

  • Additives: Addition of membrane-stabilizing compounds (glycerol 5-10%, specific detergents)

  • Harvest timing: 4-6 hours post-induction for E. coli systems to balance yield and quality

• Recommended protocol:

  • Transform expression vector containing His-tagged atpE into E. coli C41(DE3)

  • Grow cultures in TB media supplemented with appropriate antibiotics at 37°C to OD600 of 0.7

  • Reduce temperature to 20°C and induce with 0.2 mM IPTG

  • Continue expression for 16-18 hours

  • Harvest cells and process immediately or store at -80°C

  • For membrane protein extraction, use specialized detergents suitable for ATP synthase components

These optimized conditions help ensure high yield and functional integrity of the recombinant protein for downstream applications.

What purification strategies are most effective for obtaining high-purity S. amazonensis atpE suitable for structural and functional studies?

Purifying recombinant S. amazonensis atpE presents unique challenges due to its hydrophobic nature and membrane association. The following purification strategies have proven effective:

• Initial extraction approaches:

• Chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC): Utilizing the His-tag for initial capture (Ni-NTA resin with imidazole gradient elution)

  • Size exclusion chromatography (SEC): Separation based on molecular size to remove aggregates and impurities

  • Ion exchange chromatography (IEX): Optional polishing step for removing remaining impurities

• Optimized purification protocol:

  • Cell lysis: Sonication or high-pressure homogenization in buffer containing 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% appropriate detergent (e.g., n-dodecyl-β-D-maltoside)

  • IMAC purification: Load clarified lysate on Ni-NTA column, wash with increasing imidazole concentrations (10 mM, 30 mM), elute with 250 mM imidazole

  • Buffer exchange: Dialysis or desalting to remove imidazole and adjust detergent concentration

  • SEC purification: Superdex 200 column in buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and appropriate detergent below CMC

  • Quality assessment: SDS-PAGE, Western blot, and mass spectrometry to confirm purity and integrity

• Special considerations:

  • Detergent selection is critical; screen multiple options (DDM, LDAO, CHAPS)

  • Include protease inhibitors throughout the purification process

  • Maintain low temperature (4°C) during all steps

  • Consider using amphipols or nanodiscs for stabilization after purification

Following this strategy typically yields protein with >90% purity as determined by SDS-PAGE , suitable for structural and functional studies.

How can researchers effectively analyze the structure-function relationship of recombinant S. amazonensis atpE?

Investigating the structure-function relationship of S. amazonensis atpE requires a multi-faceted approach combining structural analysis, functional assays, and computational methods:

• Structural characterization methods:

• Functional analysis approaches:

  • Reconstitution in liposomes: Incorporate purified atpE with other ATP synthase subunits to measure ATP synthesis activity

  • Proton translocation assays: Using pH-sensitive fluorescent dyes to monitor proton movement across membranes

  • Site-directed mutagenesis: Systematic mutation of key residues followed by functional assays to identify critical sites

  • Cross-linking studies: Identify interaction partners and conformational changes during the catalytic cycle

• Computational analysis:

  • Molecular dynamics simulations to predict protein behavior in membrane environments

  • Homology modeling based on related structures from other bacterial species

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for proton transfer events

  • Evolutionary analysis to identify conserved functional residues across species

• Integrative approach:

  • Correlate structural features with functional outcomes

  • Map mutations that affect function onto the structural model

  • Compare with related proteins from different species to identify conserved structural elements

  • Consider the native environment of S. amazonensis (temperature, salt concentration, pH) when interpreting results

This comprehensive approach enables researchers to establish detailed structure-function relationships for S. amazonensis atpE, contributing to the broader understanding of ATP synthase mechanics and bacterial bioenergetics.

What are the common challenges in working with recombinant S. amazonensis atpE and how can they be addressed?

Working with recombinant S. amazonensis atpE presents several technical challenges that researchers should anticipate and address:

• Expression challenges:

• Purification difficulties:

  • Detergent selection: Screen multiple detergents (DDM, LDAO, Triton X-100) for optimal solubilization without denaturation

  • Protein aggregation: Add glycerol (5-10%) to all buffers; maintain samples at 4°C; centrifuge samples before chromatography

  • His-tag accessibility: Consider using different tag positions (C-terminal vs. N-terminal) if initial purification yields are low

  • Co-purifying contaminants: Include additional wash steps with slightly higher imidazole concentrations; consider dual-tagging approach

• Stability issues:

  • Limited stability in solution: Store in optimal buffer conditions with glycerol; prepare fresh samples for critical experiments

  • Activity loss during storage: Aliquot and flash-freeze samples; avoid repeated freeze-thaw cycles

  • Conformational heterogeneity: Consider protein stabilization techniques like nanodiscs or amphipols

• Functional analysis challenges:

  • Difficulty in assessing function: Develop simplified assays focusing on specific aspects of atpE function (e.g., proton binding)

  • Reconstitution inefficiency: Optimize lipid composition for liposome reconstitution; consider native lipid extracts from S. amazonensis

  • Complex interactions: Use stepwise reconstitution of subcomplexes to understand individual contributions

By anticipating these challenges and implementing the suggested solutions, researchers can significantly improve their success in working with this challenging but important protein.

How can researchers validate the structural integrity and functional activity of purified recombinant S. amazonensis atpE?

Validating both the structural integrity and functional activity of purified recombinant S. amazonensis atpE is essential for ensuring reliable experimental results:

• Structural integrity assessment:

• Functional activity validation:

  • Proton binding assay: pH-dependent fluorescence changes using environment-sensitive probes

  • Reconstitution with partial ATP synthase complex to measure specific functionalities

  • Lipid bilayer incorporation and electrical measurements to assess ion channel-like activity

  • Assembly assays to evaluate interaction with other ATP synthase components

• In situ validation approaches:

  • Complementation studies in ATP synthase-deficient bacterial strains

  • Microscopy techniques to assess membrane localization when expressed in cells

  • Energy coupling measurements in reconstituted systems

• Comparative analysis:

  • Benchmarking against commercially available ATP synthase components

  • Comparison with native ATP synthase complex isolated from S. amazonensis

  • Side-by-side testing with atpE from well-characterized species (e.g., E. coli)

These validation methods provide comprehensive assessment of the purified protein's quality and functionality, establishing a solid foundation for subsequent research applications.

How can recombinant S. amazonensis atpE be utilized in studies of bacterial adaptation to environmental stress?

Recombinant S. amazonensis atpE serves as a valuable tool for investigating bacterial adaptation to environmental stressors, particularly in challenging conditions:

• Salt stress adaptation studies:

  • S. amazonensis thrives in environments with 1-3% NaCl and shows distinct stress responses to changes in salt concentration

  • The atpE protein can be used to investigate how ATP synthase structure and function adapt to osmotic challenges

  • Comparative studies between S. amazonensis atpE and homologs from non-halotolerant bacteria can reveal adaptive mechanisms

  • Experiments examining how salt stress affects proton gradients and ATP synthesis efficiency provide insights into bioenergetic adaptation

• Experimental approaches for stress response characterization:

• Systems biology applications:

  • Integration of atpE functional data with proteomics studies of stress responses

  • Correlation between ATP synthase activity and expression of stress response genes

  • Modeling of bioenergetic networks during adaptation to changing environments

  • Investigation of how S. amazonensis balances energy production with stress response requirements

• Ecological and evolutionary implications:

  • Insights into how ATP synthase adaptations contribute to the ecological success of Shewanella species in diverse environments

  • Understanding of evolutionary trade-offs between ATP synthesis efficiency and stress tolerance

  • Potential applications in predicting bacterial responses to changing environments, including climate change scenarios

These approaches position S. amazonensis atpE as a model system for studying the fundamental mechanisms by which bacteria adapt their energy metabolism to environmental challenges.

What insights can comparative studies of S. amazonensis atpE provide for understanding ATP synthase evolution and adaptation?

Comparative studies of S. amazonensis atpE offer valuable insights into ATP synthase evolution and adaptation across diverse bacterial lineages:

• Evolutionary context:

  • S. amazonensis belongs to the Gammaproteobacteria class but has adapted to specific ecological niches

  • Comparative sequence analysis between S. amazonensis atpE and other bacterial homologs reveals both conserved functional domains and adaptive variations

  • Phylogenetic analysis using both 16S rRNA and gyrB genes places S. amazonensis in a distinct position within the Shewanella genus, providing context for its ATP synthase evolution

• Structure-function evolutionary analysis:

• Physiological adaptations:

  • S. amazonensis thrives at 35°C with 1-3% NaCl and pH 7-8, suggesting its ATP synthase has adapted to these specific conditions

  • The exceptional ability of S. amazonensis to reduce iron, manganese, and sulfur compounds likely influences its bioenergetic requirements

  • Understanding how atpE structure accommodates these metabolic capabilities provides insights into co-evolution of energy production and utilization pathways

• Biotechnological implications:

  • Identification of naturally evolved adaptations in atpE can inspire biomimetic approaches for designing robust biological energy systems

  • Engineering of hybrid ATP synthases incorporating features from extremophile organisms

  • Development of ATP synthase variants with enhanced stability for biotechnological applications

These comparative studies enhance our understanding of how this essential molecular machine has evolved to support life across diverse and challenging environments.

What are the emerging research directions and unanswered questions regarding S. amazonensis atpE and bacterial ATP synthases?

Despite significant advances in understanding S. amazonensis atpE, several important research directions and questions remain to be addressed:

• Structural biology frontiers:

  • High-resolution structures of S. amazonensis ATP synthase remain to be determined, particularly in different functional states

  • The precise arrangement of c-subunits in the complete c-ring and their stoichiometry in S. amazonensis is not fully characterized

  • Structural adaptations that enable function under S. amazonensis's preferred environmental conditions require further investigation

• Bioenergetic questions:

  • How does the efficiency of ATP synthesis in S. amazonensis compare to other bacterial species, particularly in relation to its metal-reducing capabilities?

  • What is the precise mechanism coupling proton translocation through the c-ring to ATP synthesis in the F1 domain?

  • How does S. amazonensis ATP synthase maintain functionality during environmental stress conditions without changing membrane fatty acid composition?

• Ecological and adaptive significance:

  • How does atpE contribute to S. amazonensis's exceptional ability to reduce iron, manganese, and sulfur compounds?

  • What role does ATP synthase play in the organism's adaptation to its specific ecological niche in Amazon River delta sediments?

  • How has horizontal gene transfer influenced the evolution of ATP synthase components in Shewanella species?

• Emerging methodological approaches:

  • Single-molecule studies to directly observe the rotary mechanism of ATP synthase

  • In-cell structural biology techniques to study ATP synthase in its native environment

  • Systems biology approaches integrating proteomics, metabolomics, and bioenergetics

  • Computational modeling of ATP synthase function across varying environmental conditions

• Biotechnological applications:

  • Potential use of S. amazonensis ATP synthase components in synthetic biology applications

  • Development of biosensors based on ATP synthase function

  • Biomimetic energy systems inspired by bacterial ATP synthases

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, biophysics, microbiology, and computational biology, ultimately advancing our understanding of this fundamental bioenergetic system.

How might advances in our understanding of S. amazonensis atpE contribute to broader fields in microbiology and bioenergetics?

Advances in our understanding of S. amazonensis atpE have the potential to make significant contributions to multiple scientific disciplines:

• Fundamental bioenergetics:

  • Enhanced understanding of the universal principles governing biological energy conversion

  • Insights into the diversity of ATP synthesis mechanisms across different ecological niches

  • Expanded knowledge of structure-function relationships in rotary molecular machines

  • Deeper comprehension of the evolutionary adaptations in energy-transducing systems

• Environmental microbiology:

  • Better understanding of how energy metabolism supports microbial survival in diverse environments

  • Insights into bioenergetic adaptations to changing environmental conditions, including climate change

  • Improved models of microbial community energetics in complex ecosystems

  • Knowledge to enhance bioremediation applications utilizing metal-reducing bacteria like S. amazonensis

• Synthetic biology and biotechnology:

  • Design principles for engineering robust ATP synthases for biotechnological applications

  • Development of hybrid energy-transducing systems with enhanced efficiency or stability

  • Creation of novel biosensors based on ATP synthase components

  • Potential therapeutic targets for antimicrobial development

• Systems biology:

  • Integration of ATP synthase function into whole-cell models of bacterial metabolism

  • Understanding of regulatory networks connecting energy production to cellular processes

  • Quantitative frameworks for predicting bacterial responses to environmental perturbations

  • Multi-scale modeling from molecular dynamics to ecological interactions

The study of S. amazonensis atpE thus serves as a model system that bridges molecular mechanisms with ecological adaptations, potentially yielding insights with broad implications for understanding and harnessing biological energy systems in both natural and engineered contexts.