Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B ATP synthase subunit b (atpF)

What is the genomic context of atpF in Yersinia enterocolitica serotype O:8 / biotype 1B?

ATP synthase subunit b (atpF) in Y. enterocolitica serotype O:8 / biotype 1B is part of the ATP synthase complex operon. In the reference strain 8081, this gene is arranged within the atp operon that includes other ATP synthase components such as atpB, atpE, atpA, atpG, atpD, and atpC . The genomic organization is conserved across Y. enterocolitica strains, though specific nucleotide variations may occur between different serotypes. Comparative genomic analysis with other Y. enterocolitica strains, such as 105.5R(r) (biotype 3/O:9), shows high conservation of the ATP synthase complex genes across biotypes, indicating their essential role in bacterial metabolism .

What is the role of ATP synthase subunit b (atpF) in Y. enterocolitica physiology?

The atpF gene encodes the b subunit of the F₀ portion of F₁F₀-ATP synthase, which is critical for energy production. This membrane-bound protein functions as part of the peripheral stalk that connects the F₁ catalytic domain to the membrane-embedded F₀ domain . The ATP synthase complex is essential for bacterial energy metabolism, synthesizing ATP through the proton motive force generated across the bacterial membrane. In Y. enterocolitica, this function is particularly important for adaptation to different environmental conditions, including temperature variations (4-37°C) that the bacterium encounters during its lifecycle outside and inside hosts .

What methods are commonly used to clone and express recombinant atpF from Y. enterocolitica?

For cloning and expression of recombinant atpF from Y. enterocolitica O:8/biotype 1B, researchers typically employ:

• PCR amplification of the atpF gene using primers designed based on the published genome sequence of strain 8081

• Cloning into expression vectors (commonly pET series for E. coli expression systems)

• Expression in E. coli BL21(DE3) or SHuffle T7 for proteins requiring disulfide bonds

• Induction using IPTG at lower temperatures (16-25°C) to improve solubility

• Purification using affinity chromatography with His-tags

The gene can be amplified using primers targeting conserved regions of atpF and cloned into vectors containing appropriate tags for purification and detection . Expression conditions often require optimization due to the hydrophobic nature of membrane-associated proteins.

How can I generate knockout mutants of atpF in Y. enterocolitica O:8/biotype 1B for functional studies?

To generate atpF knockout mutants in Y. enterocolitica O:8/biotype 1B, a targeted gene replacement approach is recommended:

• Design two sets of primers to amplify two different fragments from the atpF gene (e.g., atpFUP and atpFDOWN) .

• Digest both fragments with BamHI, purify, ligate, and amplify as a single PCR fragment.

• Clone into a suicide vector like pGEM-T Easy or similar vector containing a counter-selectable marker (e.g., sacB for sucrose sensitivity) .

• Introduce the plasmid into E. coli CC118-λpir, then mobilize into Y. enterocolitica 8081 by triparental conjugation using helper strain E. coli HB101/pRK2013.

• Select transconjugants on appropriate selective media (e.g., Yersinia selective agar with streptomycin).

• Culture positive colonies without antibiotics, then plate on media containing 10% sucrose to select for double crossover events.

• Confirm the gene deletion by PCR and Southern blotting.

This method has been successfully employed for generating various gene knockouts in Y. enterocolitica, including genes involved in ATP synthesis .

What are the optimal conditions for in vitro assays to study AtpF functionality in Y. enterocolitica?

For in vitro assays examining AtpF functionality in Y. enterocolitica, consider the following conditions:

For ATP synthesis assays, inverted membrane vesicles are typically prepared from Y. enterocolitica cells grown at different temperatures to assess the impact of temperature on ATP synthase activity. Activity can be measured by ATP production using luciferase-based assays or by proton translocation using pH-sensitive fluorescent dyes .

How can I evaluate the role of atpF in Y. enterocolitica antimicrobial susceptibility?

To assess atpF's role in antimicrobial susceptibility:

• Generate atpF deletion mutants as described in question 2.1.

• Complement these mutants with plasmid-expressed atpF to confirm specificity.

• Perform minimum inhibitory concentration (MIC) determinations using:

  • Standard antimicrobials: ampicillin, tetracycline, gentamycin, ciprofloxacin

  • Antimicrobial peptides (which may target membrane integrity)

  • Microcin PDI (particularly relevant as ATP synthase is required for its activity)

• Conduct time-kill assays comparing wild-type, ΔatpF mutant, and complemented strains.

• Assess membrane potential using fluorescent dyes like DiSC3(5) to determine if atpF deletion affects membrane energetics.

Research has shown that deletion of ATP synthase components (atpA, atpF, atpE, atpH) results in loss of susceptibility to microcin PDI , suggesting that ATP synthase functionality is critical for certain antimicrobial mechanisms. The MIC values for various antibiotics should be compared between wild-type Y. enterocolitica O:8 and the ΔatpF mutant to understand the broader implications of ATP synthase disruption on antibiotic susceptibility profiles .

How does temperature affect atpF expression and ATP synthase function in Y. enterocolitica O:8/biotype 1B?

Temperature regulation is critical for Y. enterocolitica pathogenesis, with many virulence factors being temperature-regulated. For atpF specifically:

• Expression analysis using quantitative RT-PCR shows that atpF expression in Y. enterocolitica is influenced by temperature, with higher expression observed at 25°C compared to 37°C in most strains .

• Transcriptomic analysis of Y. enterocolitica grown at 10°C versus 30°C reveals that ATP synthase components are part of the cold-responsive gene network, though not among the early cold shock genes like cspA and cspB .

• The ATP synthase complex appears to be critical for adaptation to environmental temperatures, with potential roles in:

  • Maintaining energy homeostasis during temperature transitions

  • Supporting membrane integrity at lower temperatures

  • Enabling growth at environmental temperatures outside the host

These findings suggest that atpF and other ATP synthase components play key roles in the adaptation of Y. enterocolitica to its diverse ecological niches, from environmental reservoirs to mammalian hosts, with differential regulation based on temperature cues .

What is the relationship between atpF and the Type III Secretion System (T3SS) in Y. enterocolitica virulence?

The relationship between ATP synthase components like atpF and the T3SS in Y. enterocolitica involves complex energetic and regulatory connections:

Further research using co-immunoprecipitation or bacterial two-hybrid systems could identify potential direct interactions between ATP synthase components and T3SS machinery .

How do structural variations in atpF across Y. enterocolitica strains correlate with pathogenicity?

Comparative genomic analyses between Y. enterocolitica strains reveal:

• The atpF gene is generally conserved across Y. enterocolitica strains, but subtle variations exist between high-pathogenicity (biotype 1B) strains like O:8/1B and lower-pathogenicity strains like biotype 2-5 strains .

• Specific amino acid substitutions in AtpF may affect:

  • Protein-protein interactions within the ATP synthase complex

  • Efficiency of ATP synthesis

  • Stability of the protein under various environmental conditions

• Genomic analyses comparing strain 8081 (1B/O:8, high pathogenicity) with strain 105.5R(r) (3/O:9, moderate pathogenicity) show that while ATP synthase genes are present in both lineages, there may be differences in regulatory elements affecting expression patterns .

• Evolutionary analyses suggest that pathogenic Y. enterocolitica strains have maintained ATP synthase genes like atpF through selective pressure, indicating their importance for virulence or fitness .

A systematic analysis of atpF sequences across a broader range of Y. enterocolitica isolates, coupled with functional characterization of the variants, would provide deeper insights into the relationship between atpF structure and pathogenic potential.

How can atpF be utilized as a target for novel antimicrobials against Y. enterocolitica?

ATP synthase subunit b (atpF) presents several opportunities as an antimicrobial target:

• ATP synthase inhibitors: Compounds targeting the F₀ portion of ATP synthase, specifically interactions involving the b subunit (AtpF), could disrupt energy production in Y. enterocolitica. The difference in structure between bacterial and mammalian ATP synthases offers potential selectivity.

• Peptide-based inhibitors: Design of peptides that mimic natural interaction partners of AtpF could disrupt ATP synthase assembly. These could be developed based on structural analysis of protein-protein interaction domains within the ATP synthase complex.

• Microcin PDI mechanism: Understanding how microcin PDI requires functional ATP synthase, including AtpF, provides insight into natural antimicrobial mechanisms that could be leveraged for therapeutic development .

• Combination therapies: ATP synthase inhibitors could potentially sensitize Y. enterocolitica to conventional antibiotics by reducing energy availability for efflux pumps and other resistance mechanisms.

• Phage-based approaches: Bacteriophages like phage X1, which have been shown effective against Y. enterocolitica in murine models , could potentially be engineered to target cells based on surface-exposed portions of the ATP synthase complex.

Research on the susceptibility patterns of atpF mutants could reveal synergistic drug combinations that exploit the energetic vulnerabilities created by ATP synthase disruption .

What methods are most effective for studying protein-protein interactions involving AtpF in Y. enterocolitica?

For studying protein-protein interactions involving AtpF in Y. enterocolitica, several complementary approaches can be employed:

• Bacterial Two-Hybrid System:

  • Fusion of atpF and potential interacting partners to complementary fragments of adenylate cyclase

  • Detection of interactions via reporter gene expression

  • Advantage: Can be performed in vivo in a bacterial system

• Co-Immunoprecipitation:

  • Expression of epitope-tagged AtpF in Y. enterocolitica

  • Precipitation using antibodies against the tag

  • Identification of co-precipitated proteins by mass spectrometry

  • Advantage: Detects native complexes from bacterial cells

• Cross-Linking Coupled with Mass Spectrometry:

  • Treatment of bacterial cells or membrane fractions with cross-linking agents

  • Isolation of AtpF-containing complexes

  • Identification of cross-linked peptides by tandem mass spectrometry

  • Advantage: Captures transient interactions

• Surface Plasmon Resonance:

  • Immobilization of purified recombinant AtpF on sensor chips

  • Measurement of binding kinetics with potential interaction partners

  • Advantage: Provides quantitative binding parameters

• Biolayer Interferometry:

  • Immobilization of His-tagged AtpF on Ni-NTA biosensors

  • Real-time measurement of association/dissociation with partner proteins

  • Advantage: Requires minimal sample volumes and allows rapid screening

These methods have been employed to study ATP synthase components in various bacteria and could be adapted specifically for Y. enterocolitica AtpF interaction studies .

How can structural analysis of AtpF contribute to understanding Y. enterocolitica pathogenesis?

Structural analysis of AtpF can provide critical insights into Y. enterocolitica pathogenesis through several approaches:

• Homology Modeling and Molecular Dynamics Simulations:

  • Generation of structural models based on crystal structures from related organisms

  • Simulation of protein dynamics under different environmental conditions (pH, temperature)

  • Identification of critical residues for stability and function

• X-ray Crystallography or Cryo-EM of the ATP Synthase Complex:

  • Resolution of the complete structure of Y. enterocolitica ATP synthase

  • Comparison with ATP synthases from other pathogens and non-pathogenic bacteria

  • Identification of unique structural features that may relate to pathogenesis

• Structure-Function Relationships:

  • Site-directed mutagenesis of key residues identified from structural analysis

  • Assessment of the impact on ATP synthesis, growth, and virulence

  • Correlation of structural elements with pathogenic capabilities

• Environmental Adaptations:

  • Structural changes in AtpF at different temperatures (10°C vs. 37°C)

  • Adaptation mechanisms related to Y. enterocolitica's ability to grow at refrigeration temperatures

  • Potential structural explanations for temperature-dependent virulence factor regulation

Understanding the structure of AtpF could reveal how Y. enterocolitica maintains energy homeostasis during host invasion and environmental persistence, potentially identifying unique adaptations that contribute to its pathogenicity and survival in diverse niches.

What are common challenges in expressing and purifying recombinant AtpF from Y. enterocolitica, and how can they be addressed?

Recombinant expression and purification of AtpF presents several challenges due to its membrane-associated nature:

When expressing membrane proteins like AtpF, it's also crucial to consider the formation of disulfide bonds. The use of E. coli strains with enhanced disulfide bond formation capability (SHuffle T7) can improve the yield of correctly folded protein .

How can I address genetic redundancy issues when studying atpF function in Y. enterocolitica?

Addressing genetic redundancy when studying atpF requires strategic approaches:

• Comprehensive Bioinformatic Analysis:

  • Conduct thorough genome analysis to identify all potential ATP synthase-related genes

  • Assess sequence similarity and predicted functional domains

  • Construct phylogenetic trees to understand evolutionary relationships among paralogous genes

• Multiple Gene Knockout Strategies:

  • Generate single, double, and multiple knockout combinations

  • Use systems like CRISPR-Cas9 or lambda Red recombination for efficient multiple gene editing

  • Create knock-down strains using antisense RNA when complete knockout is lethal

• Complementation Studies:

  • Perform cross-complementation with homologous genes

  • Use controlled expression systems to titrate gene expression levels

  • Analyze functional rescue to assess functional overlap

• Expression Analysis under Different Conditions:

  • Compare expression patterns of potential redundant genes

  • Use RNA-seq to identify conditions where specific paralogs are preferentially expressed

  • Study regulatory elements that might differentiate expression patterns

• Biochemical Characterization:

  • Purify individual proteins and test for biochemical activity

  • Compare substrate specificity and kinetic parameters

  • Identify unique features that may indicate specialized functions

These approaches have been successful in addressing functional redundancy in bacterial systems, including Yersinia species where multiple factors may contribute to similar phenotypes .

What considerations are important when using animal models to study atpF function in Y. enterocolitica pathogenesis?

When using animal models to study atpF function in Y. enterocolitica pathogenesis, researchers should consider:

• Model Selection:

  • BALB/c mice are commonly used for Y. enterocolitica infection studies

  • Different mouse strains vary in susceptibility to Y. enterocolitica infection

  • Consider both oral infection (natural route) and intraperitoneal infection (systemic model)

• Strain-Specific Considerations:

  • Y. enterocolitica serotype O:8/biotype 1B shows high virulence in mice

  • Ensure proper bacterial growth conditions (25-28°C) before infection to prime virulence factors

  • Control for plasmid stability, as the pYV virulence plasmid can be lost during laboratory culture

• Infection Parameters:

  • Standardize bacterial inoculum (typically 10^7-10^8 CFU for oral infection)

  • Monitor bacterial colonization in Peyer's patches, mesenteric lymph nodes, spleen, and liver

  • Track temperature-dependent gene expression in vivo using reporter constructs

• Controls and Complementation:

  • Include wild-type, ΔatpF mutant, and complemented strains

  • Consider conditional expression systems if complete atpF deletion is lethal

  • Use isogenic strains differing only in the target gene

• Readouts:

  • Bacterial load in tissues (CFU determination)

  • Histopathological examination of infected tissues

  • Cytokine profiles (IL-6, TNF-α, IL-1β)

  • Survival curves for virulence assessment

• Ethical Considerations:

  • Implement refinement techniques to minimize animal suffering

  • Consider alternative models where appropriate (tissue culture, organoids)

  • Obtain proper ethical approvals and follow institutional guidelines

Studies have shown that Y. enterocolitica infection in mice can result in enteritis, mesenteric lymphadenitis, and systemic spread to liver and spleen, modeling the human disease progression .

What emerging technologies show promise for studying ATP synthase dynamics in Y. enterocolitica?

Several emerging technologies hold promise for advancing our understanding of ATP synthase dynamics in Y. enterocolitica:

• Cryo-Electron Tomography:

  • Visualization of ATP synthase in its native membrane environment

  • Capture of different conformational states during the catalytic cycle

  • Resolution of structural changes induced by environmental factors

• Single-Molecule FRET:

  • Real-time monitoring of conformational changes during ATP synthesis/hydrolysis

  • Observation of rotational dynamics of the F₀-F₁ complex

  • Quantification of effects of inhibitors or mutations on molecular motion

• In-Cell NMR Spectroscopy:

  • Structural and dynamic information in living bacterial cells

  • Non-invasive monitoring of ATP synthase conformational changes

  • Detection of interactions with other cellular components

• Nanopore Technology:

  • Single-molecule analysis of ATP synthase components

  • Detection of structural variations across bacterial strains

  • Potential for rapid diagnostic applications

• CRISPR-Interference (CRISPRi) Systems:

  • Precise temporal control of atpF expression

  • Tunable repression to study partial loss of function

  • Combinatorial targeting of multiple ATP synthase components

• Advanced Mass Spectrometry Techniques:

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Cross-linking mass spectrometry for protein-protein interaction mapping

  • Native mass spectrometry for intact complex analysis

These technologies could provide unprecedented insights into the structure-function relationships of ATP synthase in Y. enterocolitica and its role in adaptation to different environmental conditions .

How might systems biology approaches enhance our understanding of atpF's role in Y. enterocolitica metabolism and pathogenesis?

Systems biology approaches offer comprehensive frameworks to understand atpF's role in the context of entire cellular networks:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate atpF expression with global metabolic changes

  • Identify indirect effects of atpF mutation on cellular physiology

• Metabolic Flux Analysis:

  • Trace isotope-labeled substrates through metabolic pathways

  • Quantify changes in energy metabolism upon atpF mutation

  • Measure ATP synthesis rates under different conditions

• Genome-Scale Metabolic Models:

  • Develop Y. enterocolitica-specific metabolic models

  • Simulate effects of atpF deletion on growth and virulence

  • Identify synthetic lethal interactions with atpF

• Network Analysis:

  • Construct protein-protein interaction networks centered on AtpF

  • Identify hub proteins affected by ATP synthase dysfunction

  • Map regulatory networks controlling atpF expression

• Comparative Systems Analysis:

  • Compare system-wide responses between different Y. enterocolitica strains

  • Analyze differences between host-adapted and environmental states

  • Identify strain-specific adaptations related to energy metabolism

• Machine Learning Approaches:

  • Develop predictive models for virulence based on metabolic signatures

  • Identify patterns in gene expression data related to ATP synthase function

  • Extract features from high-dimensional data for phenotype prediction

Systems biology approaches have revealed that genes active during systemic infection (sif genes) differ significantly from those active during Peyer's patch colonization, suggesting different metabolic requirements during various stages of infection .

What potential exists for developing atpF-based vaccines against Y. enterocolitica?

The potential for developing atpF-based vaccines against Y. enterocolitica involves several innovative approaches:

• Attenuated Live Vaccines:

  • Development of Y. enterocolitica strains with regulated atpF expression

  • Creation of temperature-sensitive atpF mutants that grow at 25°C but not 37°C

  • Engineering strains with metabolic attenuation through ATP synthase modification

• Subunit Vaccine Approaches:

  • Identification of immunogenic epitopes within AtpF

  • Design of recombinant proteins combining AtpF epitopes with strong immunogens

  • Development of nanoparticle-based delivery systems for AtpF antigens

• DNA Vaccine Strategies:

  • Construction of DNA vaccines encoding atpF and immunostimulatory sequences

  • Optimization of codon usage for enhanced expression in mammalian cells

  • Combination with genes encoding other Y. enterocolitica virulence factors

• Cross-Protection Potential:

  • Assessment of conservation of AtpF across Y. enterocolitica strains and related pathogens

  • Evaluation of cross-protective immunity against multiple Yersinia species

  • Identification of broadly conserved epitopes for universal vaccine development

• Delivery Systems:

  • Oral delivery systems to target intestinal immune responses

  • Microencapsulation to protect antigens during gastrointestinal transit

  • Adjuvant selection to enhance mucosal and systemic immunity