Recombinant Desulfovibrio vulgaris subsp. vulgaris ATP synthase subunit c (atpE)

What is Desulfovibrio vulgaris and what ecological niches does it occupy?

Desulfovibrio vulgaris is a gram-negative, anaerobic, sulfate-reducing bacterium belonging to the Desulfovibrionaceae family. It is ubiquitously present in various environments including soil, aquatic sediments, and the human gastrointestinal tract. In the gut microbiome, Desulfovibrio species represent approximately 66% of all colonic sulfate-reducing bacteria (SRB) . These bacteria are typically found in pH-neutral environments like the distal colon, where they participate in complex microbial communities. Desulfovibrio species play crucial roles in sulfur and carbon cycling by using sulfate as a terminal electron acceptor in anaerobic respiration, producing hydrogen sulfide (H₂S) as an end product. While they are minor components of a healthy gut microbiome, they can become opportunistic pathobionts when environmental conditions shift, potentially contributing to various intestinal and extra-intestinal diseases .

What is the function of ATP synthase subunit c (atpE) in Desulfovibrio's energy metabolism?

ATP synthase subunit c (encoded by the atpE gene) forms the c-ring of the Fo portion of ATP synthase, which is embedded in the cytoplasmic membrane. In Desulfovibrio vulgaris, this protein plays a critical role in the organism's energy conservation strategy during anaerobic respiration. The c-ring functions as a rotor that couples proton translocation across the membrane to ATP synthesis. When Desulfovibrio vulgaris performs dissimilatory sulfate reduction, it generates a proton motive force across the membrane. Protons flow through the Fo portion of ATP synthase (through the a-subunit and c-ring interface), causing the c-ring to rotate. This rotation drives conformational changes in the F1 portion of the enzyme, leading to ATP synthesis from ADP and inorganic phosphate.

The atpE protein's structure and function are particularly important in Desulfovibrio because of the bacterium's unique bioenergetic challenges in low-redox potential environments where it competes with other anaerobes like methanogens and acetogens for hydrogen . The efficient operation of ATP synthase is crucial for the organism's energy conservation during sulfate reduction, which requires substantial energy investment (activation of sulfate consumes ATP).

How does the sulfate reduction pathway in Desulfovibrio relate to ATP synthase function?

In Desulfovibrio vulgaris, the dissimilatory sulfate reduction pathway is intimately connected to ATP synthase function through bioenergetic coupling. The pathway begins with the activation of sulfate by ATP sulfurylase, forming adenosine-5′-phosphosulfate (APS) with the consumption of ATP . APS is then reduced to sulfite by APS reductase, and sulfite is further reduced to hydrogen sulfide by dissimilatory sulfite reductase (specifically desulfoviridin in Desulfovibrio) .

This respiratory process generates a proton gradient across the cytoplasmic membrane, which is utilized by ATP synthase for ATP production. The c-subunit (atpE) forms the proton-conducting channel that couples proton flow to rotational energy. The efficiency of this coupling is crucial because sulfate reduction has a relatively low energy yield compared to aerobic respiration, making energy conservation mechanisms particularly important for Desulfovibrio's survival in anaerobic environments.

The interplay between electron transport, proton translocation, and ATP synthesis allows Desulfovibrio to thrive in sulfate-rich, anaerobic environments by effectively coupling sulfate reduction to energy conservation.

What genomic features characterize the ATP synthase operon in Desulfovibrio vulgaris?

The ATP synthase operon in Desulfovibrio vulgaris follows the typical bacterial arrangement with genes encoding both the F₁ (catalytic) and F₀ (membrane) components of the complex. The operon typically includes genes encoding the δ, α, γ, β, ε, and a-c subunits. The atpE gene, which encodes the c subunit, is located within this operon.

The gene order and organization in the atp operon of Desulfovibrio vulgaris reflects evolutionary adaptations to its anaerobic lifestyle and energy conservation strategy. Comparative genomic analyses have revealed sequence variations in the c subunit that may relate to the organism's ability to function optimally in low-energy environments and maintain ATP synthesis efficiency despite the relatively low energy yield of sulfate reduction.

What are the optimal expression systems for recombinant Desulfovibrio vulgaris atpE production?

For recombinant expression of Desulfovibrio vulgaris atpE, several expression systems can be employed, each with specific advantages for different research objectives:

E. coli-based expression systems:

• BL21(DE3) with pET vectors: This system provides high expression levels for biochemical and structural studies. The T7 promoter system allows tight control of expression.

• C41(DE3) and C43(DE3): These strains are particularly suitable for membrane proteins like atpE, as they have adaptations that reduce toxicity of membrane protein overexpression.

• Lemo21(DE3): Allows tunable expression through modulation of T7 RNA polymerase activity, which can improve folding of membrane proteins.

Methodological considerations:

• Gene optimization: Codon optimization for E. coli is essential, as Desulfovibrio has different codon usage patterns.

• Fusion partners: Use of fusion tags like MBP (maltose-binding protein) can improve solubility.

• Expression temperature: Lower temperatures (16-20°C) after induction often improve proper folding.

• Inducer concentration: Lower IPTG concentrations (0.1-0.5 mM) can reduce inclusion body formation.

For structural studies requiring native-like membrane environments, alternative expression systems such as cell-free expression in the presence of lipids or nanodiscs can be considered to maintain the native conformation of the c-ring structure.

What purification strategies yield highest purity and stability for recombinant atpE?

Purification of recombinant Desulfovibrio vulgaris atpE presents challenges due to its hydrophobic nature and membrane integration. A successful purification strategy involves multiple steps:

• Membrane extraction:

  • Isolate cell membranes through differential centrifugation

  • Extract membrane proteins using detergents such as n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), or digitonin

• Affinity chromatography:

  • If expressed with a His-tag, use Ni-NTA chromatography

  • For MBP fusion constructs, amylose resin provides selective binding

  • Optimize detergent concentration in all buffers to prevent protein aggregation

• Size exclusion chromatography:

  • Critical for separating monomeric from oligomeric forms

  • Useful for detergent exchange and removal of aggregates

  • Buffer typically contains 10-20 mM Tris-HCl (pH 7.5-8.0), 100-150 mM NaCl, and detergent at CMC + 0.05%

• Quality assessment:

  • SDS-PAGE analysis under conditions that can distinguish monomeric c subunits

  • Blue native PAGE to analyze oligomeric state

  • Mass spectrometry to confirm protein identity and detect post-translational modifications

For studies requiring functional c-rings, maintaining the oligomeric structure during purification is crucial. This may require milder detergents and avoidance of harsh denaturants. Additionally, inclusion of lipids or amphipols during later purification stages can enhance stability of the purified complexes.

How can recombinant atpE be properly refolded and reconstituted into functional complexes?

Refolding and reconstitution of Desulfovibrio vulgaris atpE into functional complexes requires careful manipulation of lipid environments and protein concentrations:

Refolding protocol:

• If the protein is extracted from inclusion bodies, solubilize in 8M urea or 6M guanidinium hydrochloride

• Perform stepwise dialysis to gradually remove denaturant while introducing appropriate detergents

• Include phospholipids during later dialysis steps to promote proper folding

Reconstitution into liposomes:

• Prepare liposomes using E. coli polar lipid extract or synthetic lipids (POPC/POPE/POPG)

• Mix detergent-solubilized atpE with preformed liposomes

• Remove detergent using Bio-Beads or gradual dialysis

• Confirm incorporation by sucrose gradient centrifugation

Functional assessment:

• Proton translocation assays using pH-sensitive fluorescent dyes

• ATP synthesis activity when co-reconstituted with complete ATP synthase components

• Structural integrity assessment using circular dichroism spectroscopy

The optimal lipid-to-protein ratio typically ranges from 20:1 to 100:1 (w/w), depending on the specific experimental goals. The reconstitution efficiency can be monitored by freeze-fracture electron microscopy or by functional assays measuring proton conductance.

What analytical methods are most effective for verifying the structural integrity of recombinant atpE?

Multiple complementary analytical techniques should be employed to comprehensively verify the structural integrity of recombinant Desulfovibrio vulgaris atpE:

For the most definitive structural assessment, cryo-electron microscopy or X-ray crystallography would provide atomic-level details, though these methods require significant optimization for membrane proteins like atpE.

How can the proton translocation function of Desulfovibrio vulgaris atpE be measured experimentally?

Measuring proton translocation through recombinant Desulfovibrio vulgaris atpE requires specialized techniques that can detect proton movement across membranes:

Liposome-based assays:

• Reconstitute purified atpE into liposomes with controlled orientation

• Establish a pH gradient across the liposome membrane

• Monitor pH changes using:

  • pH-sensitive fluorescent dyes (ACMA, pyranine)

  • pH electrodes for direct measurement

  • Radioactive isotopes (³H⁺) for high-sensitivity measurements

Patch-clamp electrophysiology:

• Reconstitute atpE into planar lipid bilayers

• Apply voltage across the membrane

• Record single-channel currents at varying transmembrane potentials

• Analyze conductance properties and ion selectivity

Whole-cell measurements:

• Express atpE in appropriate host cells (E. coli with deleted native atpE)

• Measure membrane potential using potential-sensitive dyes

• Correlate ATP synthesis rates with proton translocation

For quantitative assessment, the stoichiometry of protons translocated per ATP synthesized can be determined by combining measurements of ATP synthesis rates with simultaneous monitoring of proton movement. This stoichiometry is particularly important for understanding the energetic efficiency of ATP synthase in Desulfovibrio vulgaris, which operates in energy-limited anaerobic environments.

What methods can determine the c-ring stoichiometry of Desulfovibrio vulgaris ATP synthase?

Determining the c-ring stoichiometry (number of c subunits per ring) is crucial for understanding the bioenergetics of Desulfovibrio vulgaris ATP synthase, as it directly affects the H⁺/ATP ratio. Several complementary approaches can be used:

Structural methods:

• Cryo-electron microscopy:

  • Directly visualizes the c-ring structure

  • Allows counting of individual c subunits through symmetry analysis

  • Requires highly purified, homogeneous samples

• X-ray crystallography:

  • Provides high-resolution structural data if crystals can be obtained

  • Has been successfully used for c-rings from other bacteria

• Atomic force microscopy:

  • Can resolve individual c subunits in membrane-reconstituted samples

  • Provides topographical information about c-ring arrangement

Biochemical methods:

• Cross-linking and mass spectrometry:

  • Chemical cross-linking followed by SDS-PAGE can reveal oligomeric states

  • Mass spectrometry of intact c-rings can determine exact molecular weight and thus subunit count

• Native mass spectrometry:

  • Analyzes intact c-rings to determine precise molecular mass

  • Requires specialized instrumentation for membrane protein complexes

Functional approaches:

• Measure the H⁺/ATP ratio through simultaneous monitoring of proton translocation and ATP synthesis

• Compare with theoretical calculations based on different c-ring stoichiometries

The c-ring stoichiometry in bacteria varies between species (typically 10-15 subunits) and may reflect adaptation to specific bioenergetic constraints. For Desulfovibrio vulgaris, which operates in energy-limited environments performing sulfate reduction, the c-ring stoichiometry would provide valuable insights into its energy conservation strategy.

How does pH affect the stability and function of recombinant Desulfovibrio vulgaris atpE?

The pH dependence of Desulfovibrio vulgaris atpE stability and function is important to characterize, particularly given the organism's adaptation to various environmental conditions:

pH stability profile:

• Structural stability assessment:

  • Circular dichroism spectroscopy across pH range 4.0-9.0

  • Thermal denaturation studies at different pH values

  • Differential scanning calorimetry to measure transition temperatures

• Aggregation behavior:

  • Dynamic light scattering to monitor particle size distribution

  • Turbidity measurements to detect precipitation

  • Size exclusion chromatography to assess oligomeric state changes

Functional pH dependence:

• Proton translocation activity:

  • Measure proton conductance in reconstituted systems across pH range

  • Determine pH optimum for proton translocation

  • Assess effect of pH gradients on directionality of proton movement

• ATP synthase activity (when part of the complete complex):

  • Measure ATP synthesis/hydrolysis rates as a function of pH

  • Determine effect of ΔpH on ATP synthesis efficiency

Desulfovibrio vulgaris typically inhabits pH-neutral environments in the distal colon , but some Desulfovibrio species can tolerate moderately acidic conditions. The pH dependence of atpE function likely reflects adaptations to the organism's ecological niche and may differ from that of non-sulfate-reducing bacteria. Understanding this pH dependence provides insights into how ATP synthase contributes to Desulfovibrio's energy conservation strategy under various environmental conditions.

What inhibitors specifically target Desulfovibrio ATP synthase, and how can they be used in research?

Research on inhibitors targeting Desulfovibrio ATP synthase can provide valuable tools for studying its function and potential therapeutic applications, particularly given the pathogenic potential of Desulfovibrio species :

Known ATP synthase inhibitors and their effects on Desulfovibrio:

• General F-type ATP synthase inhibitors:

  • Oligomycin: Binds to the c-ring/a-subunit interface

  • DCCD (N,N'-dicyclohexylcarbodiimide): Covalently modifies the critical glutamate residue in the c subunit

  • Venturicidin: Interacts with the c-ring

• Development of Desulfovibrio-specific inhibitors:

  • Structure-based design targeting unique features of Desulfovibrio atpE

  • High-throughput screening approaches using purified components

  • Whole-cell assays measuring growth inhibition in conjunction with ATP synthesis inhibition

Methodological applications in research:

• Functional studies:

  • Use inhibitors as tools to block specific aspects of ATP synthase function

  • Titrate inhibitor concentrations to determine affinity differences

  • Compare inhibition patterns between Desulfovibrio and other bacterial ATP synthases

• Structural studies:

  • Co-crystallization with inhibitors to identify binding sites

  • Use of photo-affinity labeled inhibitors to map interaction surfaces

• Therapeutic research:

  • Target Desulfovibrio in gut dysbiosis conditions

  • Develop selective inhibitors that don't affect beneficial microbiota

  • Test in animal models of inflammatory bowel disease where Desulfovibrio overgrowth is observed

Given the emerging role of Desulfovibrio species in various diseases including inflammatory bowel disease and Parkinson's disease , developing specific inhibitors of its ATP synthase could have significant therapeutic potential while also providing valuable research tools.

How does the atpE subunit contribute to Desulfovibrio's adaptation to energy-limited environments?

Desulfovibrio vulgaris has evolved specific adaptations in its ATP synthase c subunit (atpE) to optimize energy conservation in anaerobic, energy-limited environments:

Structural and functional adaptations:

• c-ring stoichiometry optimization:

  • The number of c subunits per ring likely reflects a balance between energy conservation efficiency and growth rate

  • A higher c/a ratio would allow ATP synthesis at lower proton motive force but require more protons per ATP

• Proton binding site modifications:

  • Specific amino acid substitutions around the critical glutamate residue may alter pKa values

  • These modifications can optimize proton binding/release at the prevailing pH and membrane potential

• Subunit-subunit interactions:

  • Specific residues at the interface between adjacent c subunits may enhance stability

  • Altered interactions with the a-subunit could improve coupling efficiency

Physiological significance:

• Desulfovibrio competes with methanogens and acetogens for hydrogen in anaerobic environments, with the presence of sulfate being crucial to this competition

• ATP synthase efficiency directly impacts the energetic cost of sulfate reduction

• Adaptations in atpE contribute to Desulfovibrio's ability to thrive in environments with low redox potential

Understanding these adaptations requires comparative studies between Desulfovibrio and other bacteria, along with directed mutagenesis experiments to test the functional consequences of specific amino acid substitutions. Such research provides insights into how energy conservation mechanisms have evolved in response to ecological constraints.

What is the relationship between atpE function and hydrogen metabolism in Desulfovibrio vulgaris?

The relationship between ATP synthase function (particularly the c subunit) and hydrogen metabolism in Desulfovibrio vulgaris represents a fascinating aspect of the organism's bioenergetics:

Metabolic connections:

• Hydrogen cycling:

  • Desulfovibrio can use hydrogen as an electron donor for sulfate reduction

  • In mixed microbial communities, primary bacterial fermenters may rely on SRB to maintain efficient substrate oxidation through their consumption of H₂

  • ATP synthase activity supports the energetics of hydrogen oxidation

• Proton gradient utilization:

  • Hydrogen oxidation contributes to the proton motive force

  • The c-ring of ATP synthase harnesses this proton gradient for ATP synthesis

  • The efficiency of this coupling affects the organism's ability to grow on hydrogen

• Reverse electron transport:

  • Under certain conditions, Desulfovibrio can produce hydrogen

  • This process may involve reverse electron transport requiring ATP

  • The c subunit's properties affect the energetic efficiency of this process

Experimental approaches to study this relationship:

• Growth experiments comparing wild-type and atpE mutants on hydrogen versus other electron donors

• Measurement of hydrogen production/consumption rates in relation to ATP synthesis

• Membrane potential measurements during hydrogen metabolism

• Isotope labeling to track proton/hydrogen exchange between metabolism and ATP synthase

This research area is particularly relevant for understanding Desulfovibrio's ecological role in anaerobic environments and its potential applications in biohydrogen production or bioremediation processes.

How can atpE mutants be generated and screened to investigate structure-function relationships?

Creating and screening atpE mutants provides valuable insights into structure-function relationships in Desulfovibrio vulgaris ATP synthase:

Mutant generation strategies:

• Site-directed mutagenesis:

  • Target conserved residues (the essential glutamate in the c subunit)

  • Modify residues at subunit interfaces

  • Alter residues predicted to affect proton binding/release

• Random mutagenesis approaches:

  • Error-prone PCR to generate libraries of atpE variants

  • DNA shuffling between atpE genes from different species

  • CRISPR-based techniques for genome editing in Desulfovibrio

Expression systems for mutant screening:

• Heterologous expression:

  • E. coli strain lacking endogenous atpE as a complementation system

  • Controlled expression levels to avoid toxicity

• Homologous expression:

  • Development of genetic tools for Desulfovibrio

  • Allelic exchange to replace native atpE with mutant versions

Screening methodologies:

• Growth-based screens:

  • Comparative growth under various energy sources

  • Competition assays between mutants

• Biochemical assays:

  • ATP synthesis/hydrolysis rates

  • Proton translocation efficiency

  • Inhibitor sensitivity profiles

• Structural characterization:

  • Stability of c-ring assembly

  • Conformational changes using spectroscopic methods

• In silico analysis:

  • Molecular dynamics simulations of mutant structures

  • Prediction of proton transfer pathways

By systematically analyzing the effects of mutations on ATP synthase function, researchers can identify critical residues and structural features that contribute to the unique properties of Desulfovibrio vulgaris ATP synthase, potentially revealing adaptations specific to sulfate-reducing bacteria.

What computational approaches are valuable for modeling atpE structure and dynamics?

Computational modeling provides powerful tools for understanding Desulfovibrio vulgaris atpE structure, dynamics, and function when experimental structural data is limited:

Structural modeling approaches:

• Homology modeling:

  • Use experimentally determined structures of c subunits from other bacteria as templates

  • Refine models based on Desulfovibrio-specific sequence features

  • Validate models using experimental constraints from biochemical data

• Ab initio and threading methods:

  • For regions with low sequence similarity to known structures

  • Predict transmembrane topology and helical packing

  • Rosetta membrane protocol for modeling membrane protein structures

• c-ring assembly modeling:

  • Symmetry-based modeling of the complete c-ring

  • Prediction of subunit-subunit interaction surfaces

  • Interface refinement using molecular mechanics

Dynamic simulations:

• Molecular dynamics simulations:

  • All-atom MD simulations in explicit lipid bilayers

  • Study conformational flexibility and lipid interactions

  • Long-timescale simulations to capture protonation/deprotonation events

• Coarse-grained simulations:

  • MARTINI force field for extended timescale dynamics

  • Study large-scale motions of the c-ring

  • Investigate interactions with other ATP synthase components

• Quantum mechanics/molecular mechanics (QM/MM):

  • Detailed modeling of proton transfer reactions

  • Calculate energy barriers for critical steps in proton translocation

  • Identify key residues involved in proton coordination

Functional predictions:

• Calculation of pKa values for the critical glutamate residue

• Prediction of proton pathways through the protein

• Virtual screening for potential inhibitors targeting unique features

These computational approaches complement experimental studies and can provide hypotheses for targeted experimental validation, particularly valuable for understanding atomic-level details of proton translocation mechanisms.

How might Desulfovibrio vulgaris atpE be utilized in synthetic biology applications?

The unique properties of Desulfovibrio vulgaris atpE present interesting opportunities for synthetic biology applications:

Bioenergetic engineering:

• Modified ATP synthases:

  • Engineering hybrid ATP synthases with altered c-ring stoichiometry

  • Creating chimeric c subunits combining features from different species

  • Optimizing ATP production efficiency in synthetic systems

• Artificial energy conservation systems:

  • Integration of Desulfovibrio ATP synthase components into designer microorganisms

  • Creation of minimal synthetic cells with customized bioenergetics

  • Coupling novel electron transport chains to ATP synthesis

Biotechnological applications:

• Biofuel production:

  • Engineering systems for hydrogen production using modified ATP synthases

  • Development of organisms with enhanced ability to grow on waste substrates

  • Optimizing energy conservation for improved biofuel yields

• Environmental applications:

  • Engineered systems for metal recovery based on Desulfovibrio's capabilities

  • Design of biosensors using atpE-based components

  • Bioremediation applications targeting contaminated sites

• Nanobiotechnology:

  • Using c-rings as nanoscale rotary motors

  • Development of ATP synthase-powered nanodevices

  • Creation of bioelectronic interfaces using ATP synthase components

Design considerations:

• Careful analysis of structure-function relationships to identify modules for engineering

• Development of appropriate expression and assembly systems

• Methods for testing and validating engineered constructs

• Consideration of stability and activity in different environments

The ability of Desulfovibrio to thrive in anaerobic environments and its unique bioenergetic mechanisms make its ATP synthase components particularly valuable for synthetic biology applications targeting energy-limited or anaerobic conditions.

What are the current research frontiers regarding Desulfovibrio vulgaris atpE?

Research on Desulfovibrio vulgaris ATP synthase subunit c (atpE) continues to evolve across several exciting frontiers:

These research frontiers represent opportunities for significant advances in our understanding of bacterial bioenergetics, with potential applications in biotechnology, medicine, and environmental science. The unique adaptations of Desulfovibrio vulgaris ATP synthase to anaerobic energy conservation continue to yield valuable insights into fundamental principles of biological energy transduction.

What interdisciplinary approaches might advance our understanding of Desulfovibrio vulgaris atpE?

Advancing research on Desulfovibrio vulgaris atpE will likely require interdisciplinary approaches that combine techniques and perspectives from multiple scientific fields:

• Integration of structural biology with biophysics:

  • Combining cryo-EM, X-ray crystallography, and NMR spectroscopy

  • Single-molecule techniques to study c-ring rotation

  • Advanced spectroscopic methods to track conformational changes

• Systems biology and metabolic engineering:

  • Multi-omics approaches to understand ATP synthase in the context of cellular metabolism

  • Metabolic flux analysis to quantify energy flows

  • Genome-scale modeling of energy conservation

• Computational science and bioinformatics:

  • Advanced molecular simulation techniques

  • Machine learning approaches for structure prediction

  • Evolutionary analysis across diverse sulfate-reducing bacteria

• Synthetic biology and bioengineering:

  • Development of genetic tools for Desulfovibrio

  • Creation of minimal ATP synthase systems

  • Design of hybrid energy transduction mechanisms

• Clinical and environmental microbiology:

  • Understanding the role of Desulfovibrio ATP synthase in host-microbe interactions

  • Environmental adaptation of energy conservation mechanisms

  • Targeted approaches for managing Desulfovibrio in clinical and environmental contexts