Recombinant Desulfovibrio vulgaris ATP synthase subunit delta (atpH)

Basic Research Questions

• What is the functional role of ATP synthase delta subunit in Desulfovibrio vulgaris?

The ATP synthase delta subunit (atpH) in Desulfovibrio vulgaris serves as a critical component of the F-type ATP synthase complex, functioning primarily as a connecting element between the F1 (catalytic) and F0 (membrane-embedded) sectors. In D. vulgaris, this F-type ATP synthase operates in connection with sulfate respiration, generating ATP by utilizing the proton gradient established during anaerobic electron transport .

The delta subunit specifically contributes to:

• Structural stabilization of the F1F0 complex

• Proper coupling of proton translocation to ATP synthesis

• Regulation of rotational catalysis efficiency

Unlike facultative anaerobes, D. vulgaris relies on this ATP synthase as a primary mechanism for energy conservation under strictly anaerobic conditions, with its production levels comparable to those observed in aerobically cultured Escherichia coli .

• How is the ATP synthase complex organized in Desulfovibrio vulgaris, and where does the delta subunit fit?

The ATP synthase in D. vulgaris follows the typical F-type organization but with adaptations specific to anaerobic sulfate respiration. The complex consists of:

F1 sector (cytoplasmic):

• Alpha subunit (atpA): 488 amino acids

• Beta subunit (atpD): 471 amino acids

• Gamma subunit (atpG): 294 amino acids

• Delta subunit (atpH): 172 amino acids

• Epsilon subunit (atpC): 134 amino acids

F0 sector (membrane-embedded):

• Subunit a (atpB): 233 amino acids

• Subunit c (atpE): 82 amino acids

• Additional membrane components

The delta subunit occupies a peripheral position in the F1 sector, interacting with both the alpha/beta subunits and components of the F0 sector, thereby forming a critical structural bridge in the holoenzyme assembly .

• What experimental evidence confirms the presence of F-type ATP synthase in Desulfovibrio vulgaris?

Multiple lines of evidence confirm the presence of a functional F-type ATP synthase in D. vulgaris:

Genetic evidence:

• Cloning and sequencing of genes encoding ORFs 1-5 (corresponding to delta, alpha, gamma, beta, and epsilon subunits) revealed significant amino acid sequence identity with other known F-type ATPases

Biochemical evidence:

• Partial purification of ATPase activity from cytoplasmic membrane fractions

• N-terminal amino acid sequencing of three major polypeptides matching the predicted sequences

• Phenyl Sepharose column chromatography confirming enzymatic activity

Expression analysis:

• Differential regulation of ATP synthase genes under various stress conditions, including alkaline stress

• Coordinated expression with other energy metabolism genes

These complementary approaches definitively establish the presence and functional significance of F-type ATP synthase in D. vulgaris metabolism .

• How does energy conservation via ATP synthase in D. vulgaris differ from aerobic organisms?

D. vulgaris employs unique mechanisms for energy conservation compared to aerobic organisms:

Despite these differences, the amount of F-type ATP synthase produced in D. vulgaris cells is surprisingly similar to that in aerobically cultured E. coli, indicating its critical importance in anaerobic bioenergetics .

Advanced Research Questions

• What methodological approaches are most effective for expressing and purifying recombinant D. vulgaris ATP synthase delta subunit?

For optimal expression and purification of recombinant D. vulgaris ATP synthase delta subunit (atpH), the following methodological workflow is recommended:

Expression strategy:

• Vector selection: pET-based expression systems with N-terminal His-tag fusion (similar to available commercial constructs)

• Host strain: E. coli BL21(DE3) or Rosetta for addressing potential codon bias issues due to D. vulgaris' high GC content (65%)

• Induction conditions: IPTG concentration of 0.5-1.0 mM, at lower temperatures (20-25°C) to enhance proper folding

• Buffer considerations: Include reducing agents (DTT or β-mercaptoethanol) to maintain anaerobic protein characteristics

Purification protocol:

• Initial capture using Ni-NTA affinity chromatography

• Secondary purification via ion exchange chromatography

• Final polishing step using size exclusion chromatography

• Buffer optimization: Tris/PBS-based buffer containing 6% trehalose at pH 8.0

Quality assessment:

• SDS-PAGE analysis (expect >90% purity)

• Western blot confirmation

• Mass spectrometry verification

• Limited functional assays to verify folding

For long-term storage, addition of 50% glycerol and storage at -80°C is recommended to prevent protein degradation while maintaining structural integrity .

• How does nitrite stress impact ATP synthase expression and function in D. vulgaris, and what are the implications for delta subunit?

Nitrite stress elicits significant changes in ATP synthase expression in D. vulgaris with important implications for the delta subunit:

Gene expression changes under nitrite stress:

• Downregulation of genes encoding ATP synthase subunits, including epsilon subunit (atpC) with fold changes of 1.77-3.21 depending on exposure time

• This downregulation is part of a coordinated response affecting multiple pathways, including energy metabolism, nitrogen metabolism, and oxidative stress response

Metabolic implications:

• Decreased ATP synthesis capacity as electron flow shifts from respiratory phosphorylation to nitrite reduction

• Consequent reduction in ATP-dependent processes, including amino acid transport and protein synthesis

Research approaches to study delta subunit specifically:

• Targeted qRT-PCR measuring atpH expression during nitrite stress timeline

• Western blot analysis of delta subunit protein levels

• BN-PAGE analysis to assess F1F0 complex integrity during stress

• ATP synthesis activity assays comparing wild-type to delta subunit mutants

These findings suggest that under nitrite stress, D. vulgaris prioritizes detoxification mechanisms at the expense of ATP synthesis, with potential structural consequences for the ATP synthase complex that would directly impact the delta subunit's connecting function .

• What role does the ATP synthase delta subunit play in the hydrogen cycling model proposed for Desulfovibrio species?

The ATP synthase delta subunit has significant implications in the hydrogen cycling model proposed for Desulfovibrio species:

Hydrogen cycling model context:

• Odom and Peck proposed a chemiosmotic hydrogen cycling model as a general mechanism for energy coupling in Desulfovibrio species

• This model predicts generation of proton gradients through hydrogen molecule oxidation coupled to sulfate reduction

Delta subunit's potential functions:

• Structural adaptor: May facilitate specialized associations between ATP synthase and hydrogenase complexes unique to Desulfovibrio

• Regulatory element: Could function as a sensor responding to hydrogen availability or intracellular redox state

• Efficiency modulator: Might optimize proton coupling ratio under different hydrogen concentrations

Experimental evidence and research directions:

• Coordinated expression patterns between ATP synthase and hydrogenase genes under different growth conditions

• Periplasmic hydrogenases (particularly the [Fe] hydrogenase and [NiFeSe] hydrogenase) show differential expression based on hydrogen availability, with implications for ATP synthesis efficiency

• Future research should explore protein-protein interactions between ATP synthase components and hydrogenases using techniques like crosslinking, co-immunoprecipitation, or hydrogen deuterium exchange mass spectrometry

The delta subunit likely plays a critical role in adapting ATP synthase function to the unique energetic demands of the hydrogen cycling process in Desulfovibrio species .

• How do sequence and structural features of D. vulgaris ATP synthase delta subunit compare with those from other bacteria and what are the functional implications?

Comparative analysis of D. vulgaris ATP synthase delta subunit reveals important evolutionary adaptations:

Sequence analysis:
The ATP synthase delta subunit from D. vulgaris displays:

• Moderate sequence identity (approximately 30-45%) with delta subunits from other bacteria

• Conservation of key structural domains for F1-F0 interaction

• Unique sequence features potentially related to sulfate respiration and anaerobic lifestyle

• 172 amino acids in length, which is within the typical range for bacterial delta subunits

Structural predictions and implications:

• N-terminal domain: Likely contains a nucleotide-binding fold involved in interactions with F1 sector

• C-terminal domain: Probably features extended alpha-helical structures involved in F0 interaction

• Potential adaptations for functioning at lower proton motive force compared to aerobes

• Possible unique interaction surfaces for association with Desulfovibrio-specific energy complexes

Functional considerations:

• The delta subunit's sequence adaptations may enable D. vulgaris ATP synthase to maintain efficiency under the lower energy potential of sulfate respiration

• Specific residues may facilitate interactions with other bioenergetic complexes found in Desulfovibrio but not in model aerobes

• Conservation analysis suggests the most highly conserved regions correspond to essential structural roles while divergent regions likely represent adaptations to anaerobic lifestyle

These comparisons provide valuable insights into how D. vulgaris has adapted this critical bioenergetic component to thrive in its unique ecological niche .

• What approaches can be used to investigate the assembly pathway of ATP synthase in D. vulgaris and the specific role of the delta subunit?

Investigating ATP synthase assembly in D. vulgaris requires specialized approaches to accommodate its anaerobic nature:

Genetic approaches:

• Gene deletion and complementation:

  • Apply the λ red recombination system adapted for D. vulgaris to create atpH deletion strains

  • Complement with wild-type or modified versions to assess functionality

  • Evaluate growth phenotypes on different electron donors (lactate, hydrogen at 5% or 50%)

• Tagged protein expression:

  • Create chromosomal modifications encoding TAP-tagged delta subunit

  • Utilize the protocol established for high-throughput manipulation of D. vulgaris genome

  • Analyze assembly intermediates that accumulate in the absence of specific components

Biochemical and proteomic approaches:

• Assembly intermediate isolation:

  • Blue native PAGE separation of membrane complexes

  • Immunoprecipitation using antibodies against delta subunit

  • Mass spectrometry identification of assembly partners and chronology

• Time-resolved assembly analysis:

  • Pulse-chase experiments with isotopically labeled amino acids

  • Synchronize protein synthesis using inducible promoter systems

  • Monitor incorporation of subunits into the complex over time

Structural visualization:

• In situ localization:

  • Fluorescence microscopy using delta subunit fusion with fluorescent proteins

  • Immuno-gold electron microscopy to visualize assembly patterns within cells

• Cryo-EM analysis:

  • Visualize assembly intermediates and compare with complete complex

  • Map structural transitions during assembly process

These multidisciplinary approaches would reveal the unique features of ATP synthase assembly in anaerobic sulfate reducers and the specific contribution of the delta subunit to this process .

• How can recombinant D. vulgaris ATP synthase delta subunit be utilized in reconstitution experiments to understand subunit interactions and proton pumping mechanisms?

Reconstitution experiments with recombinant D. vulgaris ATP synthase delta subunit can provide crucial insights into its functional interactions:

Preparation of components:

• Protein expression and purification:

  • Express individual ATP synthase subunits as His-tagged recombinant proteins in E. coli

  • For delta subunit, utilize established protocols achieving >90% purity via affinity chromatography

  • Verify protein quality using SDS-PAGE and Western blotting

• Liposome preparation:

  • Generate proteoliposomes from purified phospholipids (POPC/POPE mixtures)

  • Consider incorporating lipids extracted from D. vulgaris membranes for native-like environment

  • Control size distribution through extrusion techniques

Reconstitution approaches:

• Stepwise assembly:

  • Begin with F1 subcomplex formation (alpha, beta, gamma, delta, epsilon)

  • Assess contribution of delta subunit through comparison of complexes with and without it

  • Measure ATP hydrolysis activity to verify functional assembly

• Proton pumping assessment:

  • Monitor proton translocation using pH-sensitive fluorescent dyes

  • Quantify proton/ATP ratios under different conditions

  • Evaluate delta subunit's role in maintaining coupling efficiency

Advanced biophysical measurements:

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to measure conformational changes

  • Optical tweezers to assess mechanical properties of the rotor assembly

  • High-speed atomic force microscopy to visualize rotational dynamics

• Structural analysis:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Crosslinking coupled with mass spectrometry to identify proximity relationships

  • Cryo-EM of reconstituted complexes to determine structural organization

These reconstitution experiments would provide mechanistic insights into how the delta subunit of D. vulgaris ATP synthase contributes to the unique bioenergetic adaptations required for life as an anaerobic sulfate reducer .

Research Methods and Protocols

• What are the optimal conditions for expressing and characterizing recombinant D. vulgaris ATP synthase delta subunit?

Optimal conditions for expressing and characterizing the recombinant delta subunit must address D. vulgaris' anaerobic nature and unique genetic features:

Expression optimization:

Purification strategy:

• Lysis in Tris/PBS buffer (pH 8.0) with 6% trehalose as stabilizer

• Ni-NTA affinity chromatography with imidazole gradient elution

• Size exclusion chromatography to ensure monodispersity

• Storage with 50% glycerol at -80°C

Functional characterization approaches:

• Binding assays: Surface plasmon resonance to measure interactions with other ATP synthase subunits

• Structural analysis: Circular dichroism to evaluate secondary structure in solution

• Stability assessment: Differential scanning fluorimetry to determine thermal stability

• Assembly contribution: In vitro reconstitution with other subunits followed by BN-PAGE

These optimized conditions account for the specific challenges of working with proteins from anaerobic sulfate-reducing bacteria while maximizing yield and maintaining native-like properties .

• How do environmental stressors affect ATP synthase expression and function in D. vulgaris and what methodologies can detect these changes?

Environmental stressors significantly impact ATP synthase expression and function in D. vulgaris, requiring specific methodologies to characterize these effects:

Key environmental stressors affecting ATP synthase:

Advanced methodological approaches:

• Systems biology integration:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Flux balance analysis to quantify energy conservation changes

  • Hierarchical clustering analysis of temporal gene expression profiles

• Protein-level assessment:

  • Targeted proteomics (MRM-MS) to quantify specific ATP synthase subunits

  • Activity assays measuring ATP synthesis/hydrolysis under stress conditions

  • Blue native PAGE to assess complex integrity during stress exposure

• In vivo measurements:

  • Membrane potential assessment using fluorescent probes

  • Intracellular ATP concentration determination

  • Growth rate analysis of wild-type vs. ATP synthase mutants under stress

These approaches provide complementary insights into how D. vulgaris modulates its bioenergetic machinery to adapt to environmental challenges, with particular relevance for understanding the role of the delta subunit in maintaining ATP synthase function during stress conditions .

• What techniques can identify potential post-translational modifications in D. vulgaris ATP synthase delta subunit and their functional significance?

Investigating post-translational modifications (PTMs) in D. vulgaris ATP synthase delta subunit requires specialized techniques adapted for anaerobic proteins:

Identification methods:

• Mass spectrometry-based approaches:

  • High-resolution LC-MS/MS with multiple fragmentation methods (HCD, ETD)

  • Enrichment strategies for specific modifications (phosphopeptides, redox-modified peptides)

  • Top-down proteomics to capture intact protein modifications

  • Targeted SRM/MRM assays for quantification of modified peptides

• Specialized redox PTM analysis:

  • OxiCAT methodology to capture in vivo redox states of cysteine residues

  • Biotin-switch technique for S-nitrosylation detection

  • Western blotting with anti-sulfenic acid antibodies

• Other PTM detection methods:

  • Phos-tag gel electrophoresis for phosphorylation

  • Western blotting with modification-specific antibodies

  • Metal-binding assays to detect metallation

Functional characterization approaches:

• Site-directed mutagenesis:

  • Replace modified residues with non-modifiable variants

  • Introduce phosphomimetic mutations (e.g., Ser→Asp) for phosphorylation

  • Create redox-insensitive variants by Cys→Ser substitutions

• Structure-function analysis:

  • Circular dichroism to assess structural changes upon modification

  • Fluorescence spectroscopy to monitor conformation alterations

  • Functional reconstitution assays comparing modified vs. unmodified protein

• In vivo significance assessment:

  • Construct D. vulgaris strains expressing modification-resistant variants

  • Evaluate growth and bioenergetic parameters under various conditions

  • Monitor ATP synthesis rates in response to environmental shifts

This comprehensive approach would reveal how PTMs of the delta subunit might serve as regulatory mechanisms for adapting ATP synthase function to the unique metabolic demands of anaerobic sulfate reduction .

• What is the relationship between ATP synthase function and electron transport chains in D. vulgaris, and how can this be experimentally investigated?

The relationship between ATP synthase and electron transport in D. vulgaris represents a unique anaerobic bioenergetic system that can be investigated through specialized approaches:

Current understanding:

• D. vulgaris utilizes sulfate as the terminal electron acceptor instead of oxygen

• Electron transport involves cytochromes, menaquinone, rubredoxin, ferredoxin, and flavoproteins

• The F-type ATP synthase captures energy from the proton gradient generated by this anaerobic electron transport

• A hydrogen cycling model has been proposed as an energy coupling mechanism

Experimental investigation strategies:

• Biochemical coupling analysis:

  • Measure proton translocation using pH-sensitive probes in membrane vesicles

  • Quantify H+/ATP ratios under different electron donor/acceptor conditions

  • Determine P/O ratios (ATP formed per oxygen equivalent in sulfate) during sulfate respiration

• Genetic dissection approaches:

  • Create deletion mutants in specific electron transport components

  • Engineer strains with modified ATP synthase subunits (including delta)

  • Evaluate growth phenotypes on different electron donors (lactate, hydrogen)

• Real-time bioenergetic measurements:

  • Simultaneous monitoring of membrane potential and ATP synthesis

  • Respirometry adapted for anaerobic systems measuring sulfate reduction rates

  • In vivo NMR to track metabolic fluxes through central carbon metabolism

• Localization and interaction studies:

  • Super-resolution microscopy to visualize spatial relationships between complexes

  • Crosslinking coupled with mass spectrometry to map protein-protein interactions

  • Co-purification studies to identify stable interactions between ATP synthase and electron transport components

These methodologies would provide mechanistic insights into how the ATP synthase delta subunit contributes to the integration of electron transport and ATP synthesis in the unique context of anaerobic sulfate respiration .

• How does the genomic context of atpH in D. vulgaris compare to other bacteria, and what can this reveal about its evolution and regulation?

The genomic context of atpH in D. vulgaris provides valuable insights into evolutionary adaptations specific to sulfate-reducing bacteria:

Genomic organization comparison:

Research methodologies for comparative analysis:

• Comparative genomic approaches:

  • Whole-genome alignment of multiple Desulfovibrio species and other delta-proteobacteria

  • Identification of conserved motifs in promoter regions

  • Analysis of selection pressure on ATP synthase genes using dN/dS ratios

• Transcriptional regulation studies:

  • Identification of transcription start sites using primer extension

  • Characterization of promoter elements through reporter gene assays

  • Identification of transcription factors binding to regulatory regions

• Evolutionary analysis:

  • Phylogenetic reconstruction using conserved protein sequences (rpoB and gyrB) to place D. vulgaris in evolutionary context

  • Comparative analysis with gene duplications found in D. gigas (which has both standard F-type ATP synthase and a V-type ATP synthase)

  • Examination of horizontal gene transfer events through anomalous sequence composition analysis

These approaches would reveal how the genomic context of atpH reflects adaptations to D. vulgaris' unique ecological niche and metabolic capabilities, providing insights into the evolution of bioenergetic systems in anaerobic environments .