Recombinant Aeromonas salmonicida ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its significance in Aeromonas salmonicida?

ATP synthase subunit c (atpE) is a critical component of the F₀ portion of F₁F₀-ATP synthase, forming the membrane-embedded proton channel that drives ATP synthesis. In A. salmonicida, a gram-negative psychrophilic pathogen responsible for furunculosis in marine and freshwater fish, the atpE protein functions within the bacterial cell membrane . This subunit helps facilitate proton translocation across the membrane, which drives the conformational changes in F₁ that enable ATP synthesis. Given A. salmonicida's adaptation to colder environments, its ATP synthase components potentially exhibit distinctive structural and functional characteristics compared to mesophilic bacteria.

The significance of atpE in A. salmonicida extends beyond basic energy metabolism. As a component of the essential ATP synthase complex, it represents a potential target for antimicrobial development. Additionally, energy metabolism plays a crucial role in bacterial virulence and survival under different environmental conditions, making atpE an important factor in understanding A. salmonicida pathogenicity mechanisms and environmental adaptations.

How does the atpE gene organize within the A. salmonicida genome and ATP synthase operon?

The atpE gene in A. salmonicida is typically organized within an atp operon containing eight or nine genes that encode the various subunits of the ATP synthase complex. Based on comparative genomic analyses of bacterial ATP synthase operons, the gene arrangement in A. salmonicida likely follows the structure:

atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC

In this arrangement, atpE is positioned between atpB and atpF genes. The proximity of these genes allows for coordinated expression of the ATP synthase components. Transcriptomic studies of A. salmonicida under various conditions have shown that environmental factors can influence the expression of genes within this operon, suggesting regulatory mechanisms that control ATP synthase production in response to changing conditions .

The genomic context of atpE may also provide insights into potential co-regulatory relationships with other metabolic genes or virulence factors in A. salmonicida. Researchers investigating atpE function should consider these genomic relationships when designing experiments to understand its role within the broader context of bacterial physiology.

What structural features characterize the atpE protein in A. salmonicida?

The ATP synthase subunit c (atpE) in A. salmonicida likely exhibits the characteristic hairpin structure observed in other bacterial species, consisting of two transmembrane α-helices connected by a polar loop region. Key structural features include:

• Two hydrophobic transmembrane α-helices that span the bacterial membrane

• A conserved carboxyl group (typically from an aspartic or glutamic acid residue) within the second transmembrane helix that participates in proton binding and translocation

• A polar loop region that faces the cytoplasmic side of the membrane

• Multiple copies of the subunit c that assemble into a ring structure within the membrane

The number of c-subunits in the ring can vary between bacterial species, typically ranging from 8-15 subunits, which affects the bioenergetic efficiency of ATP synthesis. A. salmonicida, as a psychrophilic organism, may possess specific structural adaptations in atpE that contribute to ATP synthase function at lower temperatures, such as altered amino acid composition in the transmembrane regions to maintain appropriate membrane fluidity and protein flexibility at lower temperatures .

What expression systems are optimal for recombinant A. salmonicida atpE production?

The optimal expression systems for recombinant A. salmonicida atpE production must address several challenges specific to membrane proteins. Based on experimental approaches for similar proteins, researchers should consider:

E. coli-based expression systems:

• BL21(DE3) with pET vector systems, optimized for membrane protein expression

• C41(DE3) and C43(DE3) strains, which are specifically designed for toxic and membrane protein expression

• Codon-optimized constructs to account for different codon usage between A. salmonicida and E. coli

• Fusion tags such as MBP or SUMO to improve solubility

Expression conditions optimization:

• Induction at lower temperatures (16-20°C) to improve proper folding

• Lower IPTG concentrations (0.1-0.5 mM) to reduce formation of inclusion bodies

• Addition of membrane-stabilizing agents such as glycerol (5-10%) to the growth medium

Commercial suppliers like CUSABIO TECHNOLOGY LLC have successfully produced recombinant A. salmonicida atpE, suggesting established protocols exist for its expression and purification . When designing expression constructs, researchers should consider whether to include the entire protein or focus on specific domains, depending on the intended application and downstream analyses.

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

Purification of recombinant atpE presents significant challenges due to its hydrophobic nature and membrane integration. Effective purification strategies include:

Membrane protein extraction:

• Cell lysis using mechanical disruption (French press or sonication)

• Membrane fraction isolation via differential centrifugation

• Solubilization with appropriate detergents (DDM, LDAO, or C₁₂E₈)

Chromatographic purification:

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

• Size exclusion chromatography to remove aggregates

• Ion exchange chromatography as a polishing step

Detergent considerations:

• Initial extraction with stronger detergents (e.g., SDS or Triton X-100)

• Transition to milder detergents (e.g., DDM or CHAPS) for functional studies

• Consideration of novel solubilization agents such as SMA copolymers for native-like extractions

For functional studies, researchers should consider reconstituting purified atpE into proteoliposomes or nanodiscs to provide a membrane-like environment. Quality control assessments should include SDS-PAGE, Western blotting, mass spectrometry, and circular dichroism to verify protein identity, purity, and secondary structure .

How can researchers verify functional integrity of purified recombinant atpE?

Verifying the functional integrity of purified recombinant atpE requires multiple complementary approaches:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy to confirm alpha-helical content characteristic of membrane proteins

• Limited proteolysis to verify proper folding

• Thermal stability assays using differential scanning fluorimetry

Functional assays:

• Proton translocation assays using pH-sensitive fluorescent dyes

• Reconstitution into proteoliposomes and measurement of membrane potential

• Assembly assays with other ATP synthase components

Interaction studies:

• Crosslinking experiments to verify oligomerization properties

• Pull-down assays to confirm interactions with other ATP synthase subunits

• Native gel electrophoresis to assess complex formation

Success in these functional verification steps provides confidence that the recombinant protein maintains native-like properties suitable for downstream research applications .

What experimental approaches effectively measure ATP synthase subunit c activity?

Measuring ATP synthase subunit c activity requires specialized techniques that address its role in proton translocation and ATP synthesis. Effective experimental approaches include:

Proton translocation measurements:

• Fluorescence-based assays using pH-sensitive probes (ACMA, pyranine)

• Potentiometric measurements with electrodes in reconstituted systems

• Isotope exchange studies using deuterium or tritium

ATP synthesis/hydrolysis coupling:

• Measurement of ATP synthesis rates in coupled systems

• Analysis of proton-motive force dissipation during ATP hydrolysis

• Determination of P/O ratios (ATP synthesized per oxygen consumed)

Inhibitor-based approaches:

• Oligomycin binding assays (oligomycin binds specifically to the c-ring)

• DCCD (dicyclohexylcarbodiimide) labeling studies

• Competitive inhibition assays with various inhibitors

The assembly of the c-ring can be studied using native PAGE, cross-linking followed by mass spectrometry, or electron microscopy of reconstituted systems. For A. salmonicida specifically, researchers should consider conducting these assays across a temperature range (4-28°C) to characterize the psychrophilic adaptations of the atpE protein .

How does atpE integrate with cold adaptation mechanisms in A. salmonicida?

A. salmonicida, as a psychrophilic pathogen, exhibits specific cold adaptation mechanisms that may involve atpE function. Integrative approaches to study this relationship include:

Comparative analysis:

• Sequence comparison of atpE between psychrophilic and mesophilic Aeromonas species

• Analysis of amino acid composition biases (increased glycine, decreased proline)

• Examination of hydrophobic core flexibility in transmembrane regions

Temperature-dependent studies:

• Growth and ATP synthesis rates at various temperatures (0-28°C)

• Thermal stability of isolated atpE and assembled c-rings

• Membrane fluidity measurements in wild-type vs. atpE mutants

Cold shock response analysis:

• Transcriptomic profiling of atp operon expression during cold shock

• Proteomic analysis of ATP synthase complex assembly at different temperatures

• Integration with cold shock protein (Csp) regulatory networks

Recent research on cold shock proteins in A. salmonicida has shown that CspB and CspD play crucial roles in the bacterium's adaptation to low temperatures and virulence . These cold shock proteins function as transcriptional regulators affecting numerous genes. Although direct regulation of atpE by Csp proteins has not been specifically documented, transcriptome analysis of ΔcspB, ΔcspD, and ΔcspBΔcspD mutants revealed differential expression of numerous genes involved in energy metabolism, suggesting potential regulatory connections between cold adaptation mechanisms and ATP synthase components .

What methods are used to study atpE's role in proton translocation?

Studying the role of atpE in proton translocation requires sophisticated biophysical and biochemical approaches:

Site-directed mutagenesis approaches:

• Mutation of the conserved carboxyl group in the c-subunit

• Alterations to the transmembrane helices to modify proton-binding properties

• Creation of cysteine mutants for chemical labeling studies

Biophysical measurements:

• Solid-state NMR to study protonation states in the assembled c-ring

• FTIR spectroscopy to analyze proton-binding residues

• Single-molecule FRET to monitor conformational changes during proton transport

Reconstitution systems:

• Proteoliposomes with defined lipid compositions

• Co-reconstitution with partial or complete ATP synthase complexes

• Nanodiscs for single-particle analysis

Computational approaches:

• Molecular dynamics simulations of proton movement through the c-ring

• Quantum mechanical calculations of proton affinity

• Homology modeling based on related bacterial ATP synthases

These methods can be particularly informative when applied to compare wild-type and mutant versions of atpE, or to examine how A. salmonicida atpE functions under different environmental conditions such as temperature, pH, or salinity that may be encountered during fish infection .

How might atpE function interconnect with virulence mechanisms in A. salmonicida?

The potential interconnection between atpE function and virulence mechanisms in A. salmonicida represents an emerging area of research with several intriguing hypotheses:

Energy-dependent virulence factor secretion:
ATP synthase activity provides the energy required for type III secretion systems (T3SS) and type VI secretion systems (T6SS), which are critical virulence mechanisms in A. salmonicida. The T3SS exports effector proteins including AexT, AopH, AopO, and AopP that disrupt host cell signaling and cytoskeleton . Disruptions in ATP synthesis through atpE mutations could potentially impair the function of these secretion systems.

Metabolic adaptation during infection:
During infection, A. salmonicida must adapt to changing environmental conditions within the host. The ATP synthase complex may undergo regulatory changes to optimize energy production under these conditions. Transcriptomic studies of A. salmonicida ΔcspBΔcspD mutants showed differential expression of 921 genes, including those involved in energy metabolism and virulence, suggesting potential regulatory connections .

Biofilm formation:
A. salmonicida forms biofilms that contribute to persistence and antibiotic resistance. ATP synthesis provides energy for biofilm formation processes, and disruptions in atpE function might affect biofilm development. Studies have shown that cold shock protein mutants (ΔcspD) exhibit defects in biofilm formation, suggesting a potential link between energy metabolism and biofilm development .

Resistance to host immune defenses:
The proper functioning of ATP synthase may be crucial for bacterial survival under stress conditions imposed by host defenses. Research has shown that A. salmonicida possesses virulence factors like VapA that help evade host immune responses . The energy required for producing these factors depends on functional ATP synthesis.

What experimental models best evaluate atpE function in the context of A. salmonicida pathogenesis?

Evaluating atpE function in the context of A. salmonicida pathogenesis requires appropriate experimental models that reflect the natural host-pathogen interaction:

In vitro cellular models:

• Fish cell lines (RTG-2 rainbow trout gonadal cells, ASK Atlantic salmon kidney cells)

• Primary fish macrophage cultures to study bacterial survival

• Cell invasion assays to measure bacterial internalization

Ex vivo tissue models:

• Fish gill explant cultures

• Intestinal tissue cultures to study mucosal interactions

• Perfused organ systems to maintain tissue viability

In vivo fish models:

• Lumpfish (Cyclopterus lumpus) infection models, which have been successfully used to study A. salmonicida virulence

• Atlantic salmon (Salmo salar) as a natural host for furunculosis

• Zebrafish as a more accessible genetic model system

Genetic approaches:

• Construction of atpE deletion mutants using suicide vectors like pMEG-375 or pR112, similar to approaches used for cold shock protein studies

• Complementation studies to verify phenotypes

• Site-directed mutagenesis to target specific functional residues

• Reporter gene fusions to monitor atpE expression during infection

When conducting these studies, researchers should measure multiple parameters including bacterial survival, dissemination, expression of virulence factors, host immune responses, and tissue pathology. Temperature conditions should reflect the psychrophilic nature of A. salmonicida, typically ranging from 4-20°C depending on the specific research question .

How do environmental stressors affect atpE expression and ATP synthase function in A. salmonicida?

A. salmonicida encounters various environmental stressors both in aquatic environments and during host infection. Understanding how these stressors affect atpE expression and ATP synthase function provides insights into bacterial adaptation mechanisms:

Temperature stress responses:

• Cold shock (0-4°C): May trigger increased expression of ATP synthase components to compensate for reduced enzymatic efficiency

• Heat stress (≥20°C): Often leads to altered ATP synthase expression and potential protein misfolding

• Studies with cold shock protein mutants (ΔcspB, ΔcspD) show differential growth patterns at 28°C, suggesting temperature-dependent regulatory mechanisms affecting energy metabolism

Oxidative stress:

• Reactive oxygen species can damage ATP synthase components

• Potential protective mechanisms may include altered expression or post-translational modifications

• Integration with other stress response systems like oxidative stress regulons

Nutrient limitation:

• Carbon source availability affects ATP synthase expression

• Iron limitation, common in host environments, may alter energy metabolism

• Amino acid starvation responses may integrate with ATP synthase regulation

pH and osmotic stress:

• Changes in environmental pH affect proton gradient and ATP synthase function

• Osmotic stress may alter membrane properties affecting c-ring rotation

• A. salmonicida must adapt to different osmolarities between freshwater and marine environments

Research on cold shock proteins has shown that ΔcspD mutants display reduced survival at low temperatures (0°C and 4°C), suggesting that cold adaptation mechanisms significantly impact A. salmonicida physiology and potentially ATP synthase function .

What are common challenges in atpE mutagenesis studies and how can they be overcome?

Mutagenesis studies of atpE present several technical challenges due to the protein's essential nature and membrane localization. Common challenges and solutions include:

Challenges in generating viable mutants:

• Complete deletion of atpE is often lethal due to its essential role in energy metabolism

• Solution: Use conditional mutants with inducible promoters or partial function mutations

• Approach: Similar to strategies used for cold shock protein studies, where defined in-frame deletions were created using suicide vectors like pMEG-375 or pR112

Plasmid stability issues:

• A. salmonicida may reject certain plasmids, as observed in attempts to complement cold shock protein mutants

• Solution: Test multiple vector backbones with different origins of replication and selection markers

• Approach: Consider chromosomal integration of complementation constructs rather than plasmid-based expression

Polar effects on the atp operon:

• Mutations in atpE may affect expression of downstream genes in the operon

• Solution: Design mutations that maintain the reading frame and don't disrupt operon structure

• Approach: Create defined deletions that "include the ATG start codon until one codon before the stop codon but do not include the TAG stop codon" as described for other A. salmonicida genes

Phenotype verification:

• Distinguishing direct effects of atpE mutation from secondary metabolic adaptations

• Solution: Conduct comprehensive phenotypic analysis including growth curves, biochemical profiling (e.g., API20NE), and virulence assays

• Approach: Compare results across multiple mutant strains and environmental conditions to identify consistent phenotypes

Technical strategies for successful mutagenesis:

• Use of counter-selectable markers like sacB for allelic exchange

• Two-step recombination processes with selection for single crossovers followed by counter-selection

• PCR verification of mutants using primers flanking the targeted region

• Biochemical characterization to confirm expected phenotypic changes

How can researchers interpret contradictory data in atpE functional studies?

When encountering contradictory data in atpE functional studies, researchers should employ systematic troubleshooting and analytical approaches:

Sources of experimental variability:

• Differences in genetic backgrounds of A. salmonicida strains

• Variations in growth conditions affecting ATP synthase expression

• Methodological differences in protein purification or activity assays

• Environmental factors like temperature, pH, or media composition

Reconciliation strategies:

• Strain verification: Confirm the genetic identity of strains using PCR, sequencing, or biochemical profiles (API20NE)

• Standardize experimental conditions: Establish consistent protocols for temperature, media, and growth phase

• Control for secondary mutations: Use whole genome sequencing to identify potential compensatory mutations

• Employ multiple complementary assays: Cross-validate findings using different methodological approaches

Analytical frameworks:

• Develop mathematical models to interpret seemingly contradictory data

• Consider threshold effects or non-linear responses in ATP synthase function

• Evaluate the possibility of redundant systems or metabolic adaptations

Case study approach:
When confronted with conflicting data, construct a detailed comparison table of experimental conditions, strain backgrounds, and methodological differences that might explain the discrepancies. For example, research on cold shock proteins showed strain-specific differences in growth at different temperatures and biofilm formation capacity , highlighting the importance of controlling for genetic background and environmental conditions in A. salmonicida studies.

What quality control measures ensure reliable atpE research outcomes?

Implementing rigorous quality control measures is essential for reliable atpE research outcomes:

Genetic verification:

• Whole genome sequencing to confirm mutant construction and detect secondary mutations

• PCR verification of gene deletions or modifications using primers outside the targeted region

• Restriction enzyme analysis to confirm plasmid constructs

Protein quality assessment:

• Multiple purification methods to verify consistent biochemical properties

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

• Circular dichroism to verify proper secondary structure

• Size exclusion chromatography to assess oligomerization state

Functional verification:

• Multiple complementary assays to measure ATP synthase activity

• Controls with known inhibitors (e.g., oligomycin, DCCD) to validate assay specificity

• Comparison with well-characterized ATP synthase systems from model organisms

Experimental design considerations:

• Include appropriate positive and negative controls in all experiments

• Perform biological triplicates to establish reproducibility

• Blind analysis of data where applicable to reduce bias

• Use statistical approaches appropriate for the data type and distribution

Documentation and reporting:

• Maintain detailed records of strain construction and verification

• Document all experimental conditions thoroughly

• Report negative or conflicting results alongside positive findings

• Follow field-specific guidelines for data presentation and analysis

Researchers studying A. salmonicida mutants have employed rigorous verification methods including PCR confirmation, biochemical characterization using standardized systems like API20NE, and phenotypic analysis under multiple conditions , providing a model for quality control in atpE studies.

How might atpE serve as a target for antimicrobial development against A. salmonicida?

ATP synthase subunit c represents a promising antimicrobial target due to its essential role in bacterial energy metabolism and the structural differences between bacterial and eukaryotic ATP synthases. Several approaches show potential for targeting atpE in A. salmonicida:

Structure-based drug design:

• Targeting the unique aspects of bacterial c-rings compared to eukaryotic equivalents

• Development of compounds that interfere with proton binding or c-ring rotation

• Modification of known ATP synthase inhibitors (oligomycin derivatives, DCCD analogs) for improved specificity

Antimicrobial peptides:

• Design of membrane-active peptides that specifically disrupt c-ring assembly or function

• Conjugation strategies to improve peptide delivery to the bacterial membrane

• Screening of natural antimicrobial peptides from fish for activity against ATP synthase

Cold-adapted inhibitor development:

• Designing inhibitors that maintain activity at temperatures relevant to A. salmonicida infection (4-15°C)

• Exploration of synergistic effects with cold stress

• Targeting cold-specific conformational states of the ATP synthase complex

A strategic advantage of targeting atpE is that ATP synthase inhibitors could potentially show synergy with existing antibiotics, particularly those requiring active transport or affecting energy-dependent processes. Additionally, the essential nature of ATP synthase makes the development of resistance less likely compared to non-essential targets, though careful assessment of resistance mechanisms would be necessary .

What emerging technologies might advance atpE research in A. salmonicida?

Emerging technologies offer exciting opportunities to advance atpE research in A. salmonicida:

Advanced genetic tools:

• CRISPR-Cas9 systems optimized for A. salmonicida to enable precise genome editing

• Inducible gene expression systems for conditional mutants

• Single-cell tracking technologies to monitor bacterial responses in real-time

Structural biology approaches:

• Cryo-electron microscopy for high-resolution structures of the complete ATP synthase

• Hydrogen-deuterium exchange mass spectrometry to map dynamic protein regions

• Integrative structural biology combining multiple experimental data types

Systems biology integration:

• Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

• Machine learning to identify patterns in large-scale data sets

• Network analysis to position ATP synthase within the broader context of A. salmonicida metabolism

Advanced biophysical techniques:

• Single-molecule microscopy to visualize ATP synthase rotation

• Nano-scale thermophoresis for interaction studies

• Advanced fluorescence techniques to monitor proton movement in real-time

In vivo imaging:

• Development of reporter systems to visualize ATP synthase activity in living bacteria

• Intravital microscopy to track A. salmonicida during fish infection

• Correlative light and electron microscopy to connect function with ultrastructure

Integration of these technologies with traditional microbiological approaches would provide a more comprehensive understanding of atpE function in A. salmonicida physiology and pathogenesis, potentially leading to novel intervention strategies for furunculosis control .