Recombinant Shewanella putrefaciens ATP synthase subunit c (atpE)

Intermediate Research Questions

• How does Shewanella putrefaciens ATP synthase subunit c (atpE) contribute to the organism's energy metabolism and adaptation to diverse environments?

Shewanella putrefaciens ATP synthase subunit c (atpE) plays a crucial role in the organism's energy metabolism as part of the F1F0-ATP synthase complex. This complex is central to energy transduction, utilizing the proton motive force across the membrane to synthesize ATP.

In Shewanella putrefaciens, this energy system has unique adaptations related to the organism's remarkable respiratory versatility:

Experimental evidence from Shewanella species shows they can use various electron acceptors including Fe³⁺, which connects to their ATP synthesis capabilities . The ATP synthase complex helps maintain energy homeostasis across diverse environmental conditions, enabling Shewanella putrefaciens to thrive in environments like the Iberian Pyrite Belt with low pH and high metal concentrations .

Research methodologies to study these adaptations include:

• Growth experiments under varied electron acceptor conditions

• ATP production measurement in different environmental conditions

• Membrane potential analysis using fluorescent probes

• Comparative transcriptomics/proteomics of ATP synthase components under stress conditions

• What experimental approaches can effectively investigate the relationship between ATP synthase function and extracellular electron transfer in Shewanella putrefaciens?

To investigate the relationship between ATP synthase and extracellular electron transfer (EET) in Shewanella putrefaciens, researchers can employ these methodological approaches:

Genetic manipulation strategies:

• Create atpE knockout or conditional mutants

• Perform site-directed mutagenesis of key residues

• Develop fluorescently tagged ATP synthase for localization studies

Bioenergetic measurement techniques:

• Oxygen consumption rate determination using respirometry

• Membrane potential measurements with potential-sensitive dyes

• ATP synthesis rate quantification under different EET conditions

EET activity assessment methods:

• Chronoamperometry using poised electrodes

• Ferric iron reduction assays

• Cyclic voltammetry for redox characterization

Gene expression analysis:

• RT-qPCR targeting ATP synthase and EET genes

• RNA-seq for global transcriptional response

• Protein quantification by targeted proteomics

These experiments should be designed to establish causality between ATP synthase function and EET capabilities. For example, examining if atpE mutations affect the expression of key EET genes like omcA and mtrCAB that are present in the Shewanella putrefaciens genome and essential for its EET capability .

• How can researchers design experiments to study the role of ATP synthase in Shewanella putrefaciens adaptation to extreme environments like the Iberian Pyrite Belt?

Designing experiments to study ATP synthase's role in adaptation to extreme environments requires multi-faceted approaches:

Field-to-laboratory experimental pipeline:

• Collect environmental samples from the Iberian Pyrite Belt (known habitat of Shewanella putrefaciens)

• Isolate and characterize native strains

• Compare ATP synthase structure and function between environmental isolates and laboratory strains

Environmental simulation methodologies:

• Develop bioreactors that mimic Iberian Pyrite Belt conditions (low pH, high metal content)

• Monitor growth rates and ATP production under simulated conditions

• Measure proton pumping efficiency at different pH values

Comparative genomics and proteomics approaches:

• Sequence atpE genes from multiple environmental isolates

• Identify adaptive mutations in ATP synthase genes

• Perform structural modeling to predict functional consequences of adaptations

Experimental validation techniques:

• Site-directed mutagenesis to introduce or revert adaptive mutations

• Competition assays between wild-type and mutant strains

• Long-term evolution experiments under selective pressure

This research design allows investigation of how ATP synthase adaptations contribute to Shewanella putrefaciens survival in extreme environments with high metal concentrations, which is relevant considering the organism's documented ability to reduce metal ions like Fe³⁺ .

• What methodological considerations are important when investigating the potential interaction between ATP synthase function and iron metabolism in Shewanella putrefaciens?

When investigating interactions between ATP synthase function and iron metabolism in Shewanella putrefaciens, researchers should consider these methodological approaches:

Experimental design considerations:

• Control iron availability precisely using chelators and supplementation

• Monitor both ferrous (Fe²⁺) and ferric (Fe³⁺) iron species

• Account for abiotic oxidation/reduction reactions

• Design time-course experiments to capture dynamic interactions

Analytical techniques:

• ICP-MS for precise quantification of iron species

• Ferrozine assays for measuring Fe²⁺ concentration

• EPR spectroscopy for detecting iron-protein interactions

• Transcriptomics focusing on iron regulation and ATP synthase genes

Since Shewanella spp. have evolved unique physiological characteristics to maintain iron homeostasis and can perform extracellular electron transfer to reduce Fe³⁺ , investigating how ATP synthase operation integrates with these processes could reveal important bioenergetic adaptations. Research should examine whether ATP synthase activity affects siderophore production, which is important for iron acquisition in Shewanella .

Advanced Research Questions

• How can advanced structural biology techniques be applied to resolve uncertainties in the molecular mechanism of proton translocation through Shewanella putrefaciens ATP synthase subunit c?

Resolving the molecular mechanism of proton translocation through Shewanella putrefaciens ATP synthase subunit c requires sophisticated structural biology approaches:

Cryo-electron microscopy methodology:

• Sample preparation optimization for membrane proteins

• High-resolution single-particle analysis

• Classification algorithms to capture different conformational states

• Sub-particle refinement focusing on the c-ring

X-ray crystallography strategies:

• Lipidic cubic phase crystallization for membrane proteins

• Heavy atom derivatives for phase determination

• Micro-crystallography at synchrotron beamlines

• Time-resolved crystallography for capturing intermediates

Integrative structural approaches:

• Hydrogen-deuterium exchange mass spectrometry to map proton accessibility

• Solid-state NMR to determine critical residue orientations

• Molecular dynamics simulations based on experimental structures

• FTIR spectroscopy to monitor protonation states

These methods should specifically investigate unique features of the Shewanella putrefaciens ATP synthase c subunit, with its amino acid sequence (METVISFTAIAVAIMIGLAALGTAIGFAILGGKFLEASARQPELAPALQIKMFIVAGLLDA ISMIAVGVALFFVFANPFLAQLG) potentially containing adaptations for function in extreme environments.

• What experimental approaches can resolve contradictions in the literature regarding the role of ATP synthase in supporting diverse respiratory pathways in Shewanella species?

To resolve contradictions regarding ATP synthase's role in diverse respiratory pathways of Shewanella species, researchers should implement these experimental strategies:

Standardized methodological framework:

• Establish consistent growth conditions across studies

• Define precise measurements for respiratory activity

• Use isogenic strains to minimize genetic variation

• Develop standardized bioenergetic parameter reporting

Multi-technique validation approach:

• Genetic manipulation (knockout, complementation, point mutations)

• Direct bioenergetic measurements (ATP levels, proton gradients)

• Respiratory activity quantification with diverse electron acceptors

• Isotope labeling to track electron and energy flow

Contradictory findings resolution strategies:

• Perform meta-analysis of existing literature

• Reproduce key experiments from contradictory studies

• Identify context-dependent factors that explain discrepancies

• Develop mathematical models that can accommodate apparently contradictory data

This experimental framework should specifically investigate how ATP synthase supports the unique respiratory versatility of Shewanella putrefaciens, which possesses genes for multiple respiratory pathways including nitrate reduction (napAB), hydrogen production (hydAB), and reduction of various sulfur compounds (sirA, phsABC, ttrABC) .

• How should experiments be designed to investigate the impact of post-translational modifications on Shewanella putrefaciens ATP synthase subunit c structure and function?

Investigating post-translational modifications (PTMs) of Shewanella putrefaciens ATP synthase subunit c requires a systematic experimental approach:

PTM identification methodology:

• High-resolution mass spectrometry with multiple fragmentation techniques

• Enrichment strategies for specific modifications (phosphorylation, acetylation)

• Top-down proteomics to maintain intact protein context

• Comparative analysis between different growth conditions

Functional impact assessment:

• Site-directed mutagenesis to mimic or prevent specific PTMs

• Enzymatic assays comparing modified and unmodified protein

• Structural analysis of PTM effects on protein conformation

• Molecular dynamics simulations predicting PTM effects

Biological context investigation:

• Identify environmental conditions that trigger PTMs

• Study temporal dynamics of modifications

• Examine enzyme systems responsible for PTM addition/removal

• Assess conservation of modification sites across Shewanella species

Data validation framework:

• Develop antibodies specific to identified PTMs

• Use chemical biology approaches to selectively modify the protein

• Create genetic tools to control PTM systems

• Perform in vitro reconstitution with defined modification states

This experimental design can help determine whether environmental factors specific to Shewanella putrefaciens habitats, such as metal-rich environments like the Iberian Pyrite Belt , induce PTMs that optimize ATP synthase function under these challenging conditions.

• What methodological strategies can effectively elucidate the co-evolution of ATP synthase and respiratory chain components in Shewanella putrefaciens adaptation to specialized ecological niches?

To investigate the co-evolution of ATP synthase and respiratory chain components in Shewanella putrefaciens, researchers should employ these methodological strategies:

Comparative genomics framework:

• Sequence ATP synthase and respiratory chain genes from diverse Shewanella isolates

• Analyze selection pressures (dN/dS ratios) across ecological gradients

• Identify co-evolving residues using mutual information analysis

• Reconstruct ancestral sequences to trace evolutionary trajectories

Experimental evolution approach:

• Design selective pressures mimicking natural environments

• Monitor genetic changes over generations using deep sequencing

• Assess fitness effects of co-occurring mutations

• Perform genetic reconstruction experiments to verify adaptive significance

Structure-function correlation analysis:

• Map evolutionary changes onto protein structures

• Identify interaction interfaces between complexes

• Perform cross-linking experiments to capture complex interactions

• Develop computational models of respiratory supercomplex evolution

Ecological validation:

• Sample microbiomes from diverse habitats

• Correlate genetic variants with environmental parameters

• Assess functional consequences in environmental isolates

• Develop fitness landscape models across ecological gradients

This research strategy would leverage Shewanella putrefaciens' unique adaptations, including its extracellular electron transfer capabilities (facilitated by genes like omcA and mtrCAB) and its ATP synthase, to understand how these systems co-evolved to enable survival in challenging environments like the metal-rich Iberian Pyrite Belt with low pH and high metal concentrations .