Recombinant Vibrio cholerae serotype O1 ATP synthase subunit delta (atpH)

What is the role of ATP synthase subunit delta (atpH) in the energy metabolism of V. cholerae?

The ATP synthase subunit delta (atpH) in V. cholerae functions as a critical connector between the F1 (catalytic) and F0 (membrane-embedded) portions of the F1F0-ATPase complex. It forms part of the central stalk that transmits energy from proton translocation to ATP synthesis. Recent evidence has established that V. cholerae F1F0 ATPase is specifically a proton (H+) pump, not a sodium (Na+) pump as previously hypothesized .

To investigate atpH function experimentally:

• Create deletion mutants (ΔatpH) and assess effects on growth rates

• Measure ATP synthesis capacity in membrane vesicles

• Perform complementation studies with wild-type and mutant atpH variants

• Use fluorescent probes to measure proton translocation efficiency

How does pH influence the function of ATP synthase and atpH in V. cholerae?

The function of ATP synthase in V. cholerae appears optimized for alkaline environments (pH 7-8), which correlates with the bacterium's ecological niche in the lower small intestine (ileum). Experimental evidence shows:

• V. cholerae demonstrates enhanced motility and mucus penetration at alkaline pH (7-8)

• The ileum, where V. cholerae preferentially colonizes, maintains pH 7-8

• ATP-dependent proton pumping was measured in inside-out membrane vesicles at both pH 7.5 and 8.5

• ATP-dependent membrane potential generation was similar at pH 7.5 and 8.5

Research methodologies to study pH effects include:

• Comparative ATP synthesis assays across pH ranges

• Membrane potential measurements using potentiometric dyes

• pH gradient assessment with fluorescent indicators like acridine orange

• Growth rate comparisons at different pH values

What experimental approaches can determine if atpH is essential for V. cholerae survival?

Determining the essentiality of atpH requires multiple complementary approaches:

• Gene deletion studies:

  • Create a clean atpH deletion mutant

  • Compare with other F1F0-ATPase mutants (e.g., ΔatpE)

  • Assess growth on fermentable vs. non-fermentable carbon sources

• Growth condition analysis:

  • Test survival under various pH conditions

  • Examine growth with different carbon sources

  • Measure fitness during aerobic vs. anaerobic conditions

• Physiological measurements:

  • Membrane potential formation

  • Intracellular ATP levels

  • Proton gradient establishment

From previous research on the AtpE subunit, we know that V. cholerae F1F0-ATPase deletion mutants display a classical "unc" phenotype, growing only on fermentable substrates like glucose and not on non-fermentable substrates like succinate or glycerol . This suggests that functional ATP synthase is essential for respiratory metabolism but not for fermentation.

How is atpH expression regulated in response to environmental conditions?

While direct data on atpH regulation in V. cholerae is limited, we can make evidence-based inferences from related studies:

• pH regulation:

  • OmpR is induced at alkaline pH in V. cholerae and acts as a virulence repressor

  • Environmental pH might similarly influence ATP synthase gene expression

• Growth phase regulation:

  • ATP synthase components are typically highly expressed during exponential growth

  • Expression often decreases during stationary phase

• Oxygen and metabolic regulation:

  • Respiratory chain components and ATP synthase genes are often co-regulated

  • Low oxygen conditions may alter expression patterns

To study atpH regulation experimentally:

• Use qRT-PCR to measure mRNA levels under various conditions

• Construct reporter gene fusions (atpH promoter driving GFP expression)

• Perform RNA-seq to identify co-regulated genes

• Western blot analysis with anti-atpH antibodies

How can researchers distinguish between the roles of proton motive force and sodium motive force in V. cholerae bioenergetics?

Understanding the relative contributions of proton and sodium gradients in V. cholerae energetics requires careful experimental design:

• Ion specificity determination:

  • Generate inside-out membrane vesicles from wild-type and ATP synthase mutant strains

  • Measure ATP-dependent ion movements using specific fluorescent probes

  • Test effects of ionophores (CCCP for H+ or monensin for Na+)

• Buffer composition controls:

  • Compare ATP-dependent gradient formation in Na+-containing vs. Na+-free buffers

  • Observe effects of secondary Na+/H+ antiporters on gradient formation

• Experimental evidence from published research:

  • ATP-dependent proton uptake occurs in Na+-free buffer

  • Addition of Na+ partially dissipates ATP-generated proton gradient due to secondary Na+/H+ antiport

  • Protonophores like CCCP collapse ATP-generated membrane potential

What molecular techniques can identify critical residues in atpH for protein-protein interactions within the ATP synthase complex?

Identifying crucial residues in atpH requires a combination of structural, genetic, and biochemical approaches:

• Structure-guided mutagenesis:

  • Generate a homology model based on related bacterial ATP synthases

  • Identify conserved surface residues likely involved in subunit interactions

  • Create alanine scanning mutants at these positions

• Protein-protein interaction assays:

  • Bacterial two-hybrid or split-GFP complementation assays

  • Co-immunoprecipitation followed by mass spectrometry

  • Surface plasmon resonance with purified components

  • Crosslinking studies followed by MS/MS analysis

• Functional validation:

  • Complement ΔatpH strains with mutant variants

  • Measure ATP synthesis/hydrolysis activities

  • Assess assembly of the ATP synthase complex

This approach mirrors successful strategies used to identify critical residues in other V. cholerae proteins, such as the comprehensive scanning alanine mutagenesis of ToxT that revealed key residues for dimerization, DNA binding, and transcriptional activity .

How does atpH contribute to V. cholerae motility and mucus penetration at different pH values?

The relationship between ATP synthesis, motility, and mucus penetration in V. cholerae presents an intriguing research area:

• Energetic requirements for motility:

  • ATP generated by F1F0-ATPase provides energy for flagellar rotation

  • Proton motive force directly drives some flagellar motors

• pH-dependent effects on motility:

  • V. cholerae swimming speed increases at alkaline pH (7-8)

  • Alkaline pH improves mucus penetration capability

  • This pH range coincides with optimal ileum colonization conditions

• Experimental approaches:

  • Compare swimming behavior of wild-type and atpH mutants using single-cell tracking

  • Measure flagellar rotation speeds at different pH values

  • Assess mucus penetration using ex vivo intestinal mucus models

  • Correlate ATP synthesis rates with motility parameters

Research has shown that the proportion of swimming cells and swimming speeds for both Classical O395 and El Tor C6706 strains increase as pH increases from 6 to 8 . This pH-dependent enhancement of motility may facilitate colonization of the slightly alkaline lower intestine.

What is the relationship between atpH function and V. cholerae's virulence regulation?

While ATP synthase is primarily involved in energy metabolism, its function likely impacts virulence regulation indirectly:

• Energy provision for virulence:

  • ATP is required for synthesis and secretion of virulence factors

  • Flagellar motility, powered by ATP, is critical for initial colonization

• pH sensing and adaptation:

  • V. cholerae uses various two-component systems like ToxRS to sense bile acids and pH

  • OmpR is induced at alkaline pH and represses virulence gene expression

  • Proper ATP synthase function may be critical for maintaining intracellular pH

• Metabolic regulation:

  • Energy metabolism status influences global regulatory networks

  • ATP levels may impact cyclic nucleotide signaling pathways that regulate virulence

• Research approaches:

  • Measure virulence gene expression in ATP synthase mutants

  • Assess colonization efficiency in animal models

  • Investigate the effects of pH and ATP synthase inhibitors on virulence factor production

The intimate connection between pH adaptation, energy metabolism, and virulence is illustrated by the finding that CBS-derived H₂S enhances V. cholerae resistance to oxidative stress and promotes host colonization , demonstrating how metabolic adaptations support pathogenesis.

What expression systems produce high yields of functional recombinant V. cholerae atpH protein?

Optimizing recombinant expression of V. cholerae atpH requires careful consideration of several factors:

• Expression host selection:

  • E. coli BL21(DE3) for standard cytoplasmic expression

  • C41(DE3) or C43(DE3) for potentially toxic proteins

  • Cell-free systems for difficult-to-express proteins

• Vector design considerations:

  • Codon optimization for the expression host

  • Appropriate promoter selection (T7, tac, or nirB)

  • Fusion tags for improved solubility and purification

  • Cleavable tags with protease recognition sites

• Culture conditions optimization:

  • Induction temperature (typically lower temperatures improve folding)

  • Inducer concentration titration

  • Growth media composition

  • Aeration levels (low aeration was beneficial for nirB promoter in V. cholerae)

• Step-wise optimization protocol:

  • Small-scale expression screening of multiple constructs

  • SDS-PAGE and Western blot analysis

  • Solubility assessment

  • Scale-up of optimized conditions

A similar approach was successfully used for expressing heterologous proteins like tetanus toxin fragment C (TetC) in V. cholerae, where the E. coli nirB promoter yielded high expression levels when bacteria were grown with low aeration .

What purification strategies maintain the structural integrity of recombinant atpH?

Purifying atpH while preserving its native structure requires a carefully designed protocol:

• Initial capture strategies:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Affinity chromatography appropriate for fusion tags (MBP, GST)

  • Ion exchange chromatography as an alternative approach

• Critical buffer components:

  • pH selection: maintain pH 7.5-8.0 based on V. cholerae preference for alkaline conditions

  • Salt concentration: typically 150-300 mM NaCl

  • Stabilizing additives: 5-10% glycerol, 1-5 mM DTT or TCEP

  • Consider mild detergents if membrane association is suspected

• Advanced purification steps:

  • Size exclusion chromatography for homogeneity

  • Tag removal using site-specific proteases

  • Additional polishing steps based on protein properties

• Quality control assessments:

  • SDS-PAGE for purity

  • Dynamic light scattering for homogeneity

  • Circular dichroism for secondary structure integrity

  • Thermal shift assay for stability

  • Activity assays to confirm functionality

A typical purification workflow with expected outcomes:

How can researchers assess the functional activity of purified recombinant atpH?

Evaluating atpH function requires both direct binding assays and functional reconstitution approaches:

• Protein-protein interaction assays:

  • Surface plasmon resonance (SPR) with other ATP synthase subunits

  • Isothermal titration calorimetry (ITC) for binding thermodynamics

  • Pull-down assays with partner subunits

  • Fluorescence anisotropy for interaction kinetics

• Structural integrity assessments:

  • Circular dichroism spectroscopy for secondary structure

  • Fluorescence spectroscopy for tertiary structure

  • Limited proteolysis to probe folding

  • Thermal stability assays

• Functional reconstitution:

  • In vitro assembly with purified partner subunits

  • Reconstitution into liposomes with other ATP synthase components

  • ATP synthesis or hydrolysis activity measurements

• In vivo complementation:

  • Transform atpH deletion mutants with recombinant atpH

  • Assess restoration of growth on non-fermentable substrates

  • Measure membrane potential restoration

These approaches parallel those used to study AtpE function in V. cholerae, where inside-out membrane vesicles were used to measure ATP-dependent proton uptake using fluorescent probes like acridine orange .

What controls should be included when studying the effects of pH on atpH function?

• Buffer system controls:

  • Use overlapping buffer systems to verify that observed effects are pH-dependent rather than buffer-dependent

  • Maintain consistent ionic strength across pH values

  • Control for potential buffer interactions with the protein

• Enzyme activity controls:

  • Include pH-insensitive enzymes as internal controls

  • Measure multiple parameters (binding, catalysis) to distinguish direct from indirect effects

  • Include enzyme kinetics across the pH range

• Structural stability controls:

  • Monitor protein stability at each pH using thermal shift assays

  • Verify that observed functional changes aren't due to protein denaturation

  • Assess reversibility of pH effects

• Physiological relevance controls:

  • Compare in vitro pH optima with known physiological pH of V. cholerae habitats

  • Include physiologically relevant ions (Na+, K+, Ca2+, Mg2+)

  • Consider the effects of pH on interaction partners

When studying pH effects on V. cholerae motility, researchers confirmed that changes in pH between 6 and 8 had little effect on the viscoelastic properties of mucus, indicating that enhanced motility at alkaline pH was due to bacterial adaptation rather than environmental changes .

How should conflicting data on atpH function from different V. cholerae strains be reconciled?

When faced with contradictory results across V. cholerae strains, a systematic approach to reconciliation is necessary:

• Strain-specific differences:

  • El Tor vs. Classical biotypes may have different ATP synthase properties

  • Toxigenic vs. non-toxigenic strains may show metabolic differences

  • Laboratory-adapted vs. clinical isolates might display distinct phenotypes

• Experimental condition variations:

  • pH values significantly affect V. cholerae behavior (pH 6-8)

  • Growth media composition can alter metabolism

  • Growth phase differences may affect ATP synthase expression

• Resolution strategies:

  • Direct side-by-side comparisons under identical conditions

  • Genetic complementation between strains

  • Sequencing of atpH and related genes to identify polymorphisms

  • Consider epistatic interactions with strain-specific genetic backgrounds

This approach is supported by findings that V. cholerae strains show different responses to environmental triggers. For example, bile acids can either suppress or enhance virulence factor production depending on strain background and specific promoters .

What are common pitfalls in recombinant atpH expression and how can they be overcome?

Recombinant expression of ATP synthase components often presents challenges that require systematic troubleshooting:

• Low expression levels:

  • Problem: Poor translation or rapid degradation

  • Solution: Optimize codon usage, reduce expression temperature, use fusion tags, or try different promoters

• Inclusion body formation:

  • Problem: Improper folding leads to aggregation

  • Solution: Lower induction temperature (16-20°C), co-express chaperones, use solubility-enhancing tags like MBP or SUMO

• Loss of activity during purification:

  • Problem: Denaturation or loss of essential interactions

  • Solution: Include stabilizing additives, minimize purification steps, maintain appropriate pH (7.5-8.0 for V. cholerae proteins)

• Failed interaction studies:

  • Problem: Improper folding or blocked interaction surfaces

  • Solution: Try alternative tag positions, use tag-free protein, include known binding partners during purification

• Troubleshooting decision tree:

  • Check expression using Western blot → Optimize solubility → Assess protein quality → Verify activity → Refine purification

Drawing parallels from other V. cholerae protein expression studies, researchers expressing TcfA found that the highest expression levels were achieved when using the nirB promoter under low aeration conditions , demonstrating the importance of optimizing expression conditions.

How can researchers distinguish between effects on ATP synthase assembly versus catalytic function when studying atpH?

Differentiating between assembly defects and functional defects requires complementary experimental approaches:

• Assembly analysis techniques:

  • Blue native PAGE to visualize intact ATP synthase complexes

  • Size exclusion chromatography to assess complex formation

  • Immunoprecipitation of tagged subunits to identify interaction partners

  • Confocal microscopy with fluorescently labeled subunits to visualize localization

• Functional assessment:

  • ATP synthesis/hydrolysis assays with membrane vesicles

  • Proton pumping measurements using pH-sensitive fluorescent dyes

  • Membrane potential formation using potentiometric dyes

  • Growth assays on fermentable vs. non-fermentable carbon sources

• Experimental design for differentiation:

  • Compare wild-type and mutant atpH under identical conditions

  • Perform complementation with different atpH variants

  • Develop partial assembly assays focusing on specific subcomplexes

  • Use chemical cross-linking to capture transient assembly intermediates

This differentiation is exemplified in studies of AtpE, where membrane vesicles from wild-type and ΔatpE mutants were compared for their ability to generate pH gradient and membrane potential in response to ATP , directly linking protein presence to function.

What are the key considerations when interpreting pH effects on ATP synthase function in V. cholerae?

Interpreting pH effects on ATP synthase requires careful consideration of multiple factors:

Research has shown that alkaline pH enhances V. cholerae motility and mucus penetration , while OmpR expression is induced at alkaline pH to repress genes involved in acid tolerance and virulence . These findings suggest that V. cholerae ATP synthase likely functions optimally in slightly alkaline environments that match its ecological niche.