Recombinant Shewanella oneidensis ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c (atpE) in Shewanella oneidensis?

ATP synthase subunit c (atpE) is a critical component of the F0 portion of the F0F1 ATP synthase complex in S. oneidensis MR-1. This complex is involved in ATP production through oxidative phosphorylation. The c subunit forms the membrane-embedded proton channel that facilitates proton movement across the membrane, which drives the rotary mechanism of ATP synthesis.

Interestingly, studies have shown that inactivation of the F0F1 ATP synthase operon in S. oneidensis MR-1 resulted in only minor growth defects under certain anaerobic conditions, suggesting that substrate-level phosphorylation may play a more important role in ATP generation under these conditions . This distinguishes S. oneidensis from many other bacteria where ATP synthase is absolutely essential for growth.

How does atpE in S. oneidensis compare to atpE in other bacteria?

• Sequence conservation: While the core structure is conserved, sequence variations exist between S. oneidensis atpE and those of other bacteria, particularly those from different phyla.

• Functional adaptations: S. oneidensis atpE may have adaptations that allow ATP synthase to function efficiently under the diverse respiratory conditions that characterize this organism, including metal reduction and electrode-based respiration .

• Regulatory differences: The expression and regulation of atpE in S. oneidensis likely reflects its metabolic flexibility, particularly its ability to switch between aerobic and various anaerobic respiratory pathways.

Unlike Mycobacterial atpE that has been targeted by antibiotics such as bedaquiline , there is no evidence of similar targeting in S. oneidensis atpE, reflecting structural differences between these proteins.

What are the standard methods for recombinant expression of S. oneidensis atpE?

Recombinant expression of S. oneidensis atpE typically follows these methodological steps:

• Gene amplification: PCR amplification of the atpE coding sequence using specific primers designed for the S. oneidensis genome.

• Cloning strategy: The amplified atpE gene can be cloned into expression vectors like pMV261 (as demonstrated for other atpE genes) or vectors specific for S. oneidensis work.

• Expression system selection:

  • E. coli expression systems (BL21, DH5α) are commonly used for initial protein production

  • Homologous expression in S. oneidensis WM3064 via conjugation can be employed for functional studies

• Purification approach: Histidine-tagged constructs allow for nickel affinity chromatography purification of the recombinant protein.

• Validation methods: Western blotting, mass spectrometry, and functional assays to confirm identity and activity of the recombinant protein.

The transformation of S. oneidensis requires specialized protocols, typically using E. coli WM3064 as a donor strain for conjugation rather than direct transformation methods .

How does the function of atpE change under different respiratory conditions in S. oneidensis?

The function of atpE and the entire ATP synthase complex in S. oneidensis shows remarkable adaptability across different respiratory conditions:

Experimental approaches to study these changes typically involve:

• Comparative proteomics under different respiratory conditions

• ATP synthesis rate measurements

• Membrane potential analysis

• Isotope labeling to track proton flow through the F0 complex

Research has shown that S. oneidensis can survive through pyruvate fermentation when electron acceptors are unavailable, though this doesn't support growth. In such conditions, the ATP synthase likely shifts from synthesis to maintenance of proton gradients .

What structural modifications to recombinant atpE affect its proton conductance and ATP synthesis efficiency?

Structural modifications that impact proton conductance and ATP synthesis efficiency in recombinant atpE include:

• Transmembrane domain mutations: Alterations in the critical residues of the transmembrane helices can significantly impact proton translocation. Key residues include:

  • Conserved acidic residues in the proton channel

  • Residues involved in subunit-subunit interactions

  • The essential carboxyl groups that participate in proton transfer

• c-ring stoichiometry modifications: The number of c subunits in the ring affects the H⁺/ATP ratio and therefore the bioenergetic efficiency of the complex.

• Interface modifications: Changes at the interface between c subunits and other components of the F0 complex (particularly subunit a) can alter rotational coupling.

Methodological approaches to study these modifications include:

• Site-directed mutagenesis to introduce specific amino acid changes

• Reconstitution of modified subunit c into proteoliposomes for proton conductance assays

• Structural analysis using cryo-EM or X-ray crystallography

• ATP synthesis assays using inverted membrane vesicles

• Molecular dynamics simulations to predict functional impacts of mutations

Such studies could reveal unique adaptations in S. oneidensis atpE that contribute to its metabolic versatility across different respiratory conditions .

How can genetic engineering of atpE contribute to enhancing extracellular electron transfer in S. oneidensis?

Genetic engineering of atpE can potentially enhance extracellular electron transfer (EET) in S. oneidensis through several approaches:

• Proton motive force (PMF) optimization: Modifying atpE to alter ATP synthase efficiency could redistribute energy between growth and EET processes.

• Integration with electron transfer pathways:

  • Coupling ATP synthase function more directly to the Mtr pathway

  • Engineering atpE to respond to electrode potentials in bioelectrochemical systems

• Relationship with flavin pathways: Research has shown that synthetic flavin pathways can enhance bidirectional electron transfer in S. oneidensis . Engineered atpE variants could potentially be optimized to work synergistically with these pathways.

• Metabolic engineering strategies:

  • Adjusting ATP demand by modifying atpE to redirect carbon flow toward specific pathways

  • Coordinating atpE expression with engineered production pathways for chemicals like glutamate or itaconic acid

Methodological approaches:

• Genome editing using CRISPR-Cas9 systems adapted for S. oneidensis

• Directed evolution of atpE under EET-selective conditions

• Construction of chimeric ATP synthase complexes with components from other EET-capable organisms

• Bioelectrochemical screening systems to quantify EET enhancement

Research has demonstrated that ATP production in S. oneidensis involves a complex interplay between substrate-level phosphorylation and oxidative phosphorylation, with the former sometimes dominating during anaerobic growth . This unique energy landscape provides opportunities for atpE engineering that may not exist in other bacterial systems.

What are the optimal conditions for assessing atpE function in membrane preparations from S. oneidensis?

Optimizing conditions for assessing atpE function in membrane preparations requires careful consideration of multiple factors:

Methodological protocol:

• Cell cultivation: Grow S. oneidensis under defined respiratory conditions (aerobic, fumarate-reducing, or metal-reducing) .

• Membrane isolation: Use French press or sonication followed by differential centrifugation.

• Vesicle preparation: Prepare right-side-out vesicles for proton pumping assays or inverted vesicles for ATP synthesis assays.

• Activity measurements:

  • ATP synthesis: Monitor ATP production using luciferase-based assays

  • Proton pumping: Use pH-sensitive fluorescent probes

  • Membrane potential: Employ potentiometric dyes like DiSC3(5)

Critical controls must include:

• Inhibitor controls (e.g., DCCD to specifically inhibit F0 function)

• Comparison between wild-type and atpE mutant preparations

• Assessment of membrane integrity using appropriate markers

The unique respiratory versatility of S. oneidensis requires special attention to the electron transport chain components that interface with ATP synthase under different growth conditions .

How can isotope labeling be used to track the assembly and integration of recombinant atpE into the ATP synthase complex?

Isotope labeling provides powerful approaches to track assembly and integration of recombinant atpE:

• Pulse-chase labeling method:

  • Grow S. oneidensis in media containing ¹⁴N

  • Switch to ¹⁵N-containing media simultaneously with induction of recombinant atpE expression

  • Harvest cells at different time points

  • Isolate membrane fractions and purify ATP synthase complexes

  • Perform mass spectrometry to distinguish newly synthesized (¹⁵N-labeled) from pre-existing (¹⁴N) atpE

• SILAC approach (Stable Isotope Labeling with Amino acids in Cell culture):

  • Incorporate heavy isotope-labeled amino acids specifically into the recombinant atpE

  • Track integration using quantitative proteomics

  • Calculate incorporation rates based on heavy/light peptide ratios

• Time-resolved assembly tracking:

  • Use dual isotope labeling with different timing

  • Map the sequential incorporation of subunits into the ATP synthase complex

  • Determine if atpE integration is rate-limiting for complex assembly

Data analysis methods:

• Mass spectrometry to quantify labeled:unlabeled peptide ratios

• Blue native PAGE to separate intact complexes

• Correlation of assembly rates with functional development of ATP synthesis activity

This approach can reveal whether atpE in S. oneidensis has unique assembly kinetics compared to other organisms, potentially reflecting adaptations to its diverse respiratory capabilities .

What techniques can be used to study the effect of electron acceptor availability on atpE expression and ATP synthase assembly?

The relationship between electron acceptor availability and atpE expression/ATP synthase assembly can be studied through multiple complementary techniques:

• Transcriptional analysis:

  • RT-qPCR targeting atpE and other ATP synthase genes

  • RNA-seq to capture global transcriptional changes

  • Promoter-reporter fusions (e.g., atpE promoter-GFP) to monitor expression in real-time

• Protein level assessment:

  • Western blotting with antibodies specific to AtpE

  • Targeted proteomics (SRM/MRM) to quantify AtpE abundance

  • Pulse labeling with ³⁵S-methionine to measure synthesis rates

• Complex assembly monitoring:

  • Blue native PAGE to visualize intact ATP synthase complexes

  • Crosslinking coupled with mass spectrometry

  • Fluorescence microscopy with tagged ATP synthase subunits

• Functional measurements:

  • ATP synthesis rates in membrane vesicles

  • Proton pumping activity

  • Growth yield measurements (Y_ATP)

Experimental design should include:

• Growth with various electron acceptors (O₂, fumarate, Fe(III), electrodes)

• Controlled transition experiments between acceptor types

• Comparison with mutants affecting electron transport pathways

S. oneidensis shows remarkable metabolic flexibility, with the proportion of ATP produced by substrate-level phosphorylation varying from 33% to 72.5% depending on the electron acceptor availability . This suggests that ATP synthase expression and assembly may be finely regulated in response to electron acceptor conditions.

What are the challenges in obtaining pure, functional recombinant atpE from S. oneidensis and how can they be overcome?

Obtaining pure, functional recombinant atpE presents several challenges with corresponding solutions:

• Membrane protein solubility:

  • Challenge: AtpE is highly hydrophobic with multiple transmembrane regions

  • Solutions:

    • Use specialized detergents (DDM, LDAO, or Fos-choline)

    • Express as fusion with solubility-enhancing tags (MBP, SUMO)

    • Consider cell-free expression systems with lipid nanodiscs

• Proper folding:

  • Challenge: Maintaining native conformation outside the membrane environment

  • Solutions:

    • Co-express with chaperones (GroEL/GroES)

    • Use mild solubilization conditions

    • Reconstitute into proteoliposomes immediately after purification

• Expression toxicity:

  • Challenge: Overexpression may disrupt host membrane potential

  • Solutions:

    • Use tightly regulated inducible promoters

    • Lower induction temperature (16-18°C)

    • Consider expression in S. oneidensis rather than E. coli

• Functional validation:

  • Challenge: Confirming activity of isolated subunit c

  • Solutions:

    • Reconstitution with other ATP synthase subunits

    • Proton conductance assays in liposomes

    • Binding assays with known inhibitors

• Yield optimization:

  • Challenge: Low expression levels common for membrane proteins

  • Solutions:

    • Codon optimization for expression host

    • Screen multiple constructs with varying tags/fusion partners

    • Optimize growth media and induction parameters

A particularly effective approach combines genetic techniques similar to those used for site-directed mutagenesis in Mycobacterial atpE with the specialized conjugation methods developed for S. oneidensis , adapted for membrane protein expression.

How can cryo-electron microscopy be optimized for structural determination of S. oneidensis ATP synthase with focus on the c-ring?

Optimizing cryo-electron microscopy (cryo-EM) for structural determination of S. oneidensis ATP synthase c-ring requires:

• Sample preparation refinements:

  • Purification strategy:

    • Two-step affinity purification using His-tagged subunit β

    • Size exclusion chromatography to ensure complex integrity

    • Amphipol or nanodisc reconstitution to maintain native-like environment

  • Grid preparation:

    • Optimize protein concentration (typically 1-3 mg/ml)

    • Test multiple grid types (Quantifoil R1.2/1.3, UltrAuFoil)

    • Glow discharge conditions adjusted for membrane proteins

• Data collection parameters:

  • Microscope settings:

    • 300kV acceleration voltage

    • Energy filter (20eV slit)

    • K3 direct electron detector in counting mode

    • Dose fractionation (40-50 frames)

    • Total dose limitation (~40-50 e⁻/Ų)

  • Collection strategy:

    • Tilted data collection (30° and 40°) to overcome preferred orientation

    • Beam-tilt pair collection for improved 3D reconstruction

• Image processing workflow:

  • Motion correction optimized for membrane proteins

  • CTF estimation with programs sensitive to astigmatism

  • Particle picking strategies that account for side views

  • 3D classification focused on the c-ring

  • Local refinement of the c-ring region

  • Post-processing with optimized B-factor sharpening

• Validation approaches:

  • Resolution assessment by gold-standard FSC

  • Model validation by independent refinement of half-datasets

  • Comparison with existing ATP synthase structures

This methodology leverages the experience with other bacterial ATP synthases while addressing the specific challenges of S. oneidensis, particularly the potential structural adaptations related to its unique energy metabolism under diverse respiratory conditions .

What are the most effective approaches for studying the interaction between atpE and other components of the electron transport chain in S. oneidensis?

Studying interactions between atpE and the electron transport chain (ETC) components requires integrative approaches:

• In vivo interaction mapping:

  • Proximity-based labeling:

    • BioID or TurboID fused to atpE to identify neighboring proteins

    • APEX2 tagging for spatial proteomics in the membrane

  • Crosslinking approaches:

    • Photo-activatable crosslinkers incorporated at specific positions

    • Chemical crosslinking followed by mass spectrometry (XL-MS)

    • In vivo formaldehyde crosslinking to capture physiological interactions

• Functional coupling analysis:

  • Bioenergetic measurements:

    • Membrane potential determinations under different respiratory conditions

    • Relationship between proton motive force and ATP synthesis

    • Response to inhibitors targeting specific ETC components

  • Mutant phenotyping:

    • Construction of strains with mutations in both atpE and ETC components

    • Analysis of synthetic genetic interactions

    • Suppressor screens to identify functional relationships

• Structural organization studies:

  • Super-resolution microscopy:

    • PALM/STORM imaging of tagged ATP synthase and ETC components

    • Quantification of co-localization patterns

  • Native membrane organization:

    • Atomic force microscopy of native membranes

    • Lipid raft association using detergent resistance

• System-level approaches:

  • Flux balance analysis incorporating ATP synthase and ETC

  • Integrative modeling of the complete electron and proton transfer network

  • Multi-omics correlation of ATP synthase with ETC component expression

These approaches are particularly relevant for S. oneidensis given its remarkable respiratory versatility and the documented importance of both substrate-level phosphorylation and oxidative phosphorylation in its energy metabolism . The potential connection between ATP synthase function and extracellular electron transfer pathways represents a unique aspect of S. oneidensis physiology that distinguishes it from many other model organisms .

How should researchers interpret conflicting data on the essentiality of atpE in S. oneidensis under different growth conditions?

When faced with conflicting data regarding atpE essentiality across different growth conditions, researchers should apply these interpretative frameworks:

• Methodological reconciliation:

  • Examine differences in gene inactivation approaches:

    • Complete deletion vs. point mutations

    • Polar effects on other ATP synthase subunits

    • Compensation time allowed before phenotyping

  • Growth condition standardization:

    • Precise oxygen availability (fully aerobic vs. microaerobic)

    • Carbon source considerations (lactate vs. pyruvate metabolism differences)

    • Electron acceptor concentration and accessibility

• Functional redundancy analysis:

  • Assessment of substrate-level phosphorylation capacity:

    • Activity of enzymes like acetate kinase (Ack) and phosphotransacetylase (Pta)

    • Ability to redirect metabolic flux to compensate for ATP synthase deficiency

  • Proton motive force (PMF) utilization:

    • Alternative PMF consumers in S. oneidensis

    • PMF requirements under different respiratory modes

• Adaptive response characterization:

  • Temporal dimension:

    • Short-term vs. long-term essentiality (compensatory mutations)

    • Growth phase-dependent requirements

  • Compensatory pathways:

    • Upregulation of substrate-level phosphorylation

    • Altered expression of electron transport chain components

    • Metabolic rewiring to reduce ATP demand

• Data integration framework:

  • Construct a condition-dependent essentiality matrix

  • Apply statistical approaches to weight contradictory findings

  • Develop testable hypotheses to resolve discrepancies

Research has demonstrated that S. oneidensis shows unusual flexibility in ATP generation, with substrate-level phosphorylation contributing significantly to growth under anaerobic conditions, while ATP synthase inactivation shows only minor growth defects in some conditions . This suggests a context-dependent essentiality model where atpE importance varies across S. oneidensis' diverse metabolic modes.

What statistical approaches are most appropriate for analyzing the impact of atpE mutations on ATP synthesis rates and growth phenotypes?

Appropriate statistical approaches for analyzing atpE mutation effects include:

• For ATP synthesis rate analysis:

  • Enzyme kinetics modeling:

    • Michaelis-Menten parameters (Vmax, Km) comparison between wild-type and mutants

    • Inhibitor kinetics (Ki determination) for structure-function studies

  • Time-series analysis:

    • Repeated measures ANOVA for temporal profiles

    • Area under the curve (AUC) comparisons

    • Rate constant derivation and comparison

  • Multiple condition testing:

    • Two-way ANOVA with mutation and condition as factors

    • Post-hoc tests with appropriate correction for multiple comparisons

    • Interaction effect quantification between mutations and conditions

• For growth phenotype analysis:

  • Growth curve parameter extraction:

    • Nonlinear regression to extract lag phase, growth rate, and carrying capacity

    • Bootstrapping for parameter confidence intervals

    • Principal component analysis for multidimensional phenotype comparison

  • Condition-dependent fitness calculation:

    • Competitive index determination

    • Relative fitness (w) calculation

    • Integration with genome-wide fitness datasets

• For integrating ATP synthesis and growth data:

  • Correlation analysis:

    • Spearman's rank correlation for non-parametric relationships

    • Partial correlation controlling for confounding variables

    • Mixed effects models accounting for batch variation

  • Predictive modeling:

    • Multiple regression models to predict growth from ATP synthesis parameters

    • Machine learning approaches for complex phenotype prediction

    • Flux balance analysis incorporating ATP synthesis constraints

• For handling biological variability:

  • Robust statistical approaches:

    • Median-based methods less sensitive to outliers

    • Bootstrapping for parameter uncertainty estimation

    • Bayesian frameworks incorporating prior knowledge

How can researchers differentiate between direct effects of atpE modification and indirect metabolic adaptations in engineered S. oneidensis strains?

Differentiating direct effects from indirect adaptations requires multi-level analysis:

• Temporal resolution approaches:

  • Immediate response characterization:

    • Rapid sampling after induction of atpE expression/mutation

    • Real-time monitoring using biosensors (ATP, pH, membrane potential)

    • Metabolic quenching techniques to capture instantaneous metabolic state

  • Adaptive response tracking:

    • Time-series omics (transcriptomics, proteomics, metabolomics)

    • Rate-of-change analysis to identify primary vs. secondary responses

    • Clustering of temporal patterns to separate immediate from adaptive effects

• Genetic dissection strategies:

  • Epistasis analysis:

    • Combined mutations in atpE and potential adaptation pathways

    • Genetic background simplification by removing known adaptation mechanisms

    • Suppressor mutation identification and characterization

  • Controlled expression systems:

    • Titrated expression levels of wild-type vs. modified atpE

    • Orthogonal inducible systems for controlled timing

    • Complementation assays with variant atpE forms

• Bioenergetic isolation techniques:

  • Direct parameter measurement:

    • Proton pumping assays in isolated vesicles

    • ATP synthesis measurement in controlled systems

    • Membrane potential quantification with specific probes

  • In situ activity determination:

    • Single-cell analysis of membrane potential

    • Subcellular ATP imaging

    • Microfluidic techniques for rapid environmental shifts

• Metabolic flux discrimination:

  • ¹³C metabolic flux analysis:

    • Isotope tracing to map redirected carbon flows

    • Metabolic network modeling to quantify flux changes

    • Distinction between altered ATP production vs. consumption

  • Comparative fluxomics:

    • Integration with constraint-based models

    • Identification of altered metabolic node activities

    • Energy charge homoeostasis mechanisms

This comprehensive approach can disentangle the complex interplay between ATP synthase function and metabolic adaptation in S. oneidensis, particularly important given its metabolic flexibility and unique energy conservation strategies across different respiratory conditions .

What are the most promising applications of engineered S. oneidensis atpE variants in bioelectrochemical systems?

The most promising applications of engineered atpE variants in bioelectrochemical systems include:

• Enhanced bioelectricity production:

  • ATP synthase variants optimized for:

    • Improved coupling between electron transport chain and ATP synthesis

    • Reduced proton leakage to maximize energy capture efficiency

    • Altered c-ring stoichiometry to optimize H⁺/ATP ratio for electrode-based respiration

  • Potential outcomes:

    • Higher current densities in microbial fuel cells

    • Improved stability of bioelectrochemical performance

    • Lower internal resistance in bioelectrochemical systems

• Bidirectional electron transfer optimization:

  • Engineering atpE to support:

    • Enhanced cathode-driven ATP synthesis

    • Coordination with synthetic flavin pathways for improved electron transfer

    • Optimized energy conservation during both oxidation and reduction processes

  • Applications:

    • Microbial electrosynthesis of value-added compounds

    • Bioelectrochemical CO₂ reduction systems

    • Bioelectrochemical remediation technologies

• Bioelectrochemical sensing platforms:

  • atpE variants engineered for:

    • Specific response to target analytes through altered proton translocation

    • Coupling ATP synthesis to biosensor output signals

    • Integration with S. oneidensis electron transfer pathways

  • Implementation in:

    • Environmental monitoring systems

    • Metabolite detection platforms

    • Real-time bioprocess monitoring

• Bio-hybrid energy systems:

  • ATP synthase engineering for:

    • Light-driven proton pumping integration

    • Temperature-responsive activity profiles

    • Interface with artificial photosynthetic systems

Research demonstrates S. oneidensis can be metabolically engineered for producing compounds like glutamate and itaconic acid , suggesting that atpE engineering could create specialized strains that efficiently couple bioelectrochemical energy capture with valuable chemical production.

What novel insights could be gained by comparing atpE function across Shewanella species with different respiratory capabilities?

Comparative analysis of atpE across Shewanella species could reveal:

• Evolutionary adaptations in energy conservation:

  • Sequence and structural variations correlating with:

    • Respiratory diversity (number and types of electron acceptors utilized)

    • Ecological niche (marine vs. freshwater, free-living vs. biofilm-associated)

    • Environmental pressure adaptations (psychrophilic, piezophilic species)

  • Functional implications:

    • Species-specific c-ring stoichiometry

    • Proton binding site variations

    • Interface adaptations with other ATP synthase subunits

• Respiratory flexibility mechanisms:

  • Correlation between atpE characteristics and:

    • Metal reduction capabilities across species

    • Extracellular electron transfer efficiency

    • Survival under electron acceptor limitation

  • Detailed comparison between:

    • S. oneidensis MR-1 (model organism with diverse respiratory capabilities)

    • S. violacea and S. benthica (piezophilic species)

    • Other species with specialized metabolic capabilities

• Environmental adaptation signatures:

  • atpE modifications related to:

    • pH tolerance ranges

    • Temperature adaptation

    • Pressure resistance (relevant for deep-sea Shewanella species)

  • Regulatory differences:

    • Promoter architecture

    • Expression response to environmental signals

    • Post-translational modifications

• Biotechnological application potential:

  • Identification of naturally optimized variants for:

    • Extreme condition bioenergy applications

    • Specialized bioelectrochemical functions

    • Enhanced survival in engineered systems

This comparative approach could reveal how ATP synthase has evolved within the Shewanella genus to support their remarkable respiratory diversity, including some species' ability to survive extreme pressures (>1.5 GPa) , providing insights impossible to gain from studying S. oneidensis alone.

How might CRISPR-Cas9 genome editing techniques be optimized for creating precise atpE modifications in S. oneidensis?

Optimizing CRISPR-Cas9 for precise atpE modifications requires specialized approaches:

• Delivery system refinement:

  • Vector optimization:

    • Development of S. oneidensis-specific plasmid backbones

    • Integration with conjugation-based delivery systems

    • Inducible expression control for Cas9 and guide RNAs

  • Alternative delivery methods:

    • Electroporation protocols optimized for S. oneidensis

    • Conjugation from specialized E. coli donor strains like WM3064

    • Potential application of cell-penetrating peptides for Cas9-gRNA ribonucleoprotein delivery

• Guide RNA design considerations:

  • S. oneidensis-specific parameters:

    • GC content optimization for the AT-rich genome

    • PAM site accessibility in the membrane protein-encoding atpE

    • Off-target analysis against the S. oneidensis genome

  • Multiplex editing strategies:

    • Simultaneous targeting of multiple ATP synthase subunits

    • Combined modifications of atpE and electron transport components

    • Orthogonal guide RNA systems for complex engineering

• Repair template optimization:

  • Homology-directed repair enhancement:

    • Optimal homology arm length determination (typically 500-1000 bp)

    • Strand bias investigation for template design

    • Strategic selection of silent mutations to prevent re-cutting

  • Selection marker strategies:

    • Transient selection systems to avoid stable marker integration

    • Counter-selection approaches for marker removal

    • Scarless editing techniques adaptation

• Screening and validation pipeline:

  • High-throughput phenotyping:

    • Growth-based screens under various respiratory conditions

    • ATP synthesis activity assays

    • Membrane potential measurement in candidate clones

  • Molecular verification:

    • Optimized PCR-based screening strategies

    • Sequencing approaches for confirming precise edits

    • Expression verification of modified atpE

These approaches build upon the genetic manipulation methods demonstrated for S. oneidensis while addressing the specific challenges of modifying the essential membrane protein-encoding atpE gene within the ATP synthase operon.