Recombinant Dichelobacter nodosus ATP synthase subunit b (atpF)
Description
Introduction to Dichelobacter nodosus and ATP Synthase
Dichelobacter nodosus is a gram-negative anaerobic bacterium and the primary causative agent of ovine footrot, a debilitating disease in sheep that leads to lameness, separation of the hoof from underlying soft tissue, and significant economic losses in the sheep industry worldwide . This pathogen is classified into multiple serogroups (A through I and M) based on fimbrial antigens, with serogroup B being predominant in virulent footrot cases, particularly in regions like Jammu & Kashmir, India, where it accounts for approximately 92.46% of footrot cases .
ATP synthase, also known as F-type ATPase, is a ubiquitous enzyme complex found in the bacterial plasma membrane, mitochondria, and chloroplasts. This remarkable molecular machine plays a pivotal role in cellular energy production by synthesizing adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate through chemiosmotic coupling of proton translocation across biological membranes . The enzyme consists of two main domains: the membrane-embedded F₀ sector, which forms the proton channel, and the catalytic F₁ sector, which projects into the cytoplasm or matrix compartment.
Molecular Properties
The ATP synthase subunit b (atpF) is a critical component of the F₀ sector of the ATP synthase complex. In Dichelobacter nodosus, this protein is characterized by specific molecular properties that contribute to its function within the ATP synthase complex. According to the available data, the recombinant D. nodosus ATP synthase subunit b protein is identified with UniProt accession number A5EXJ9, derived from the Dichelobacter nodosus strain VCS1703A .
Expression Systems
The recombinant Dichelobacter nodosus ATP synthase subunit b protein is produced using heterologous expression in Escherichia coli . This approach allows for efficient production of the protein for research and analytical purposes. The protein is expressed as a partial sequence rather than the full-length protein, which may facilitate better expression yields or improve stability for research applications.
Physical and Chemical Properties
The recombinant protein demonstrates high purity, with values exceeding 85% as determined by SDS-PAGE analysis . This level of purity makes it suitable for various biochemical and immunological studies. The table below summarizes the key characteristics of the commercially available recombinant D. nodosus ATP synthase subunit b:
| Property | Specification |
|---|---|
| Product Code | CSB-EP002358DHZ1 |
| UniProt Accession | A5EXJ9 |
| Source Organism | Dichelobacter nodosus (strain VCS1703A) |
| Expression System | E. coli |
| Protein Length | Partial |
| Purity | >85% (SDS-PAGE) |
| Recommended Storage | -20°C/-80°C |
| Liquid Form Shelf Life | 6 months at -20°C/-80°C |
| Lyophilized Form Shelf Life | 12 months at -20°C/-80°C |
| Recommended Reconstitution | Deionized sterile water to 0.1-1.0 mg/mL with 5-50% glycerol |
Role in ATP Synthesis
The ATP synthase complex in D. nodosus, like in other bacteria, plays a critical role in energy metabolism. The atpF-encoded subunit b functions as a key structural component of the F₀ sector, which forms the proton channel across the bacterial membrane. This channel allows protons to flow down their electrochemical gradient, driving the rotation of the complex and catalyzing ATP synthesis.
While the specific details of atpF function in D. nodosus have not been extensively characterized, it is reasonable to infer its importance from the conservation of ATP synthase structure and function across bacterial species. The presence of this gene in the D. nodosus genome, with its retention through evolutionary processes, suggests its essential role in the bacterium's energy metabolism.
Relationship to Bacterial Survival and Virulence
The energy metabolism of pathogenic bacteria is intimately linked to their virulence and survival within host environments. In D. nodosus, efficient ATP production would be crucial for supporting various cellular processes, including the expression and assembly of virulence factors such as type IV fimbriae and extracellular proteases, which are known to contribute to the pathogenesis of ovine footrot .
D. nodosus genome analysis has revealed various metabolic subsystems, with protein metabolism (164 genes), cofactors, vitamins, prosthetic groups, pigments (70 genes), DNA metabolism (45 genes), stress response (25 genes), and virulence, disease, and defense (17 genes) being particularly important . The ATP synthase complex, including the atpF-encoded subunit b, would provide the energy required for these various metabolic processes.
Basic Research Applications
The recombinant D. nodosus ATP synthase subunit b serves as a valuable tool for basic research into bacterial bioenergetics and the structure-function relationships of ATP synthases. Studies using this recombinant protein could contribute to our understanding of how variations in ATP synthase components affect enzyme efficiency and bacterial metabolism.
Diagnostic and Therapeutic Potential
Given the importance of D. nodosus as a livestock pathogen, components of its cellular machinery, including ATP synthase, may have potential applications in disease diagnosis or treatment. While not specifically established for atpF, other D. nodosus components have been investigated as vaccine candidates. For instance, serogroup B isolates of D. nodosus have been identified as potential vaccine candidates to mitigate ovine footrot in India, where this serogroup is associated with the majority of virulent footrot cases .
The ATP synthase complex, being essential for bacterial energy metabolism, represents a potential target for novel antimicrobial strategies. Compounds that specifically interfere with the function of bacterial ATP synthases could inhibit bacterial growth with potentially minimal effects on host cells if sufficient selectivity can be achieved.
Genomic Context
The D. nodosus genome has been sequenced and analyzed, revealing a genome size of approximately 1.31 Mb with a GC content of 44.38% . The genome contains about 1,215 protein-coding genes, 44 tRNA and 7 rRNA genes . While the specific genomic context of the atpF gene is not detailed in the available search results, it would typically be part of the atp operon that encodes the various subunits of ATP synthase.
Interaction with Virulence Mechanisms
D. nodosus pathogenicity involves several key virulence factors, including type IV fimbriae and extracellular proteases . The expression of these virulence factors requires energy, which is provided by metabolic processes dependent on ATP synthase function.
The regulation of virulence in D. nodosus involves various systems, including a two-component signal transduction system (PilR/S) and an alternative sigma factor (σ54) that regulate fimbrial biogenesis . Additionally, iron regulation through the ferric uptake regulator (Fur) protein affects various aspects of metabolism and potentially virulence . While direct regulatory links between these systems and ATP synthase expression have not been established in the available data, the energy requirements for these processes underscore the importance of ATP synthase function in supporting bacterial virulence.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order. We will accommodate your request whenever possible.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: dno:DNO_1146
STRING: 246195.DNO_1146
Q&A
What is the genomic context of the atpF gene in Dichelobacter nodosus?
The atpF gene in D. nodosus is part of the ATP synthase operon, which encodes components of the F1F0-ATP synthase complex essential for energy metabolism. Based on genomic analysis of D. nodosus JKS-07B (serogroup B), the complete genome is approximately 1,311,533 bp with a G+C content of 44.38% . While the specific genomic location of atpF was not directly identified in the available sequence data, it would be part of the 1,215 protein-coding genes identified in the genome annotation process. ATP synthase genes are typically classified under energy metabolism subsystems, which would be among the 190 subsystems identified in the functional annotation of D. nodosus .
How does the structure of ATP synthase subunit b in D. nodosus compare to other bacteria?
ATP synthase subunit b in bacteria typically features a transmembrane domain that anchors it to the membrane and a cytoplasmic domain that interacts with the F1 portion of the complex. While specific structural information for D. nodosus atpF is not available in the search results, comparative genomic analysis would likely place it among the highly conserved components of bacterial energy metabolism. Researchers studying this protein should employ structural prediction tools that incorporate the unique genomic features of D. nodosus, which include a high level of diversity with low recombination rates as indicated by cgMLST and wgMLST analyses of the D. nodosus PubMLST database containing 171 isolates with 115 sequence types .
What expression systems are most effective for producing recombinant D. nodosus ATP synthase subunit b?
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Codon optimization accounting for the 44.38% G+C content of D. nodosus genome
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Use of expression vectors containing promoters compatible with the D. nodosus transcriptional machinery
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Incorporation of suitable affinity tags that don't interfere with the protein's structure
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Expression conditions that account for D. nodosus being an anaerobic organism
Expression in E. coli BL21(DE3) with the pET system typically provides good yields, though optimization of induction temperature (usually 25-30°C) and IPTG concentration (0.1-0.5 mM) is recommended for functional expression.
How can we determine if D. nodosus atpF contributes to virulence in footrot infections?
To investigate the potential role of atpF in D. nodosus virulence:
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Generate atpF knockout mutants using homologous recombination or CRISPR-Cas9 systems
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Perform comparative virulence studies between wild-type and atpF-mutant strains
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Assess changes in thermostable protease activity, as proteases (AprV2, AprV5, and BprV) are known virulence factors in D. nodosus
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Examine energy metabolism changes and their impact on virulence gene expression
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Conduct infection studies using appropriate animal models scored according to Stewart and Claxton protocol
The methodology should incorporate virulence characterization techniques similar to those used for protease genes, where single nucleotide polymorphisms have been associated with virulence differences . Since D. nodosus produces thermostable proteases in virulent strains, researchers should analyze whether atpF disruption affects this phenotype using gelatin gel assays.
What methodologies are most reliable for studying protein-protein interactions involving D. nodosus atpF in the ATP synthase complex?
| Methodology | Application to atpF Research | Advantages | Limitations |
|---|---|---|---|
| Bacterial Two-Hybrid | Mapping interactions with other ATP synthase subunits | In vivo detection | Potential false positives |
| Co-immunoprecipitation | Confirming native complex formation | Detects physiological interactions | Requires specific antibodies |
| Surface Plasmon Resonance | Measuring binding kinetics | Quantitative binding data | Requires purified proteins |
| Crosslinking Mass Spectrometry | Identifying proximity within complex | Maps interaction interfaces | Complex data analysis |
| Cryo-EM | Structural analysis of complete ATP synthase | Visualizes intact complex | Technically demanding |
For D. nodosus atpF specifically, researchers should consider the anaerobic growth requirements and prepare samples under appropriate conditions. Interaction studies should focus on both the membrane-embedded region and the cytoplasmic domain that interacts with other subunits of the ATP synthase complex.
How can post-translational modifications of D. nodosus atpF be accurately characterized?
For comprehensive analysis of post-translational modifications (PTMs) in D. nodosus atpF:
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Express and purify recombinant atpF with minimal tags to avoid interference
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Perform mass spectrometry analysis using both bottom-up (peptide) and top-down (intact protein) approaches
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Apply enrichment strategies for specific PTMs (phosphorylation, acetylation, etc.)
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Compare PTM profiles between virulent and benign strains to identify potential regulatory mechanisms
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Validate findings using site-directed mutagenesis of modified residues
This approach should be informed by the understanding that D. nodosus thrives in anaerobic environments and may utilize PTMs to regulate ATP synthesis under various environmental conditions experienced during infection.
What approaches are most effective for analyzing selective pressure on the D. nodosus atpF gene across different strains?
To analyze selective pressure on the atpF gene:
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Collect sequence data from multiple D. nodosus isolates, including representatives from different serogroups (especially serogroup B which is predominant in virulent footrot cases in India)
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Calculate dN/dS ratios to determine if the gene is under purifying, neutral, or positive selection
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Perform codon-by-codon selection analysis to identify specific sites under selection
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Map selection patterns to structural features of the ATP synthase subunit b protein
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Compare selection patterns between virulent and benign strains
Given that D. nodosus has high genomic diversity with low recombination rates , researchers should consider phylogenetic methods that account for this population structure. The analysis could be incorporated into the existing PubMLST database framework that currently contains 171 isolates with 115 sequence types .
How can genomic context analysis help understand the evolution of atpF in D. nodosus?
Genomic context analysis of atpF should examine:
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Conservation of the ATP synthase operon structure across D. nodosus strains and related species
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Presence of mobile genetic elements near the atpF gene that might indicate horizontal gene transfer
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Comparative analysis with the 21 unique genes identified in D. nodosus JKS-07B serogroup B
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Examination of the G+C content of atpF relative to the genome average (44.38%)
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Analysis of synteny with closely related bacterial species
What are the most reliable methods for measuring ATP synthase activity in recombinant D. nodosus atpF preparations?
| Method | Measurement Principle | Application to atpF Research | Technical Considerations |
|---|---|---|---|
| ATP synthesis assay | Direct measurement of ATP production | Assessing functional reconstitution | Requires intact ATP synthase complex |
| ATP hydrolysis assay | Measurement of phosphate release | Testing reverse reaction | Simpler than synthesis assay |
| Proton pumping assay | pH-sensitive fluorescent probes | Testing coupling efficiency | Requires proteoliposomes |
| Membrane potential measurements | Voltage-sensitive dyes | Assessing ΔΨ generation | Can be performed in whole cells |
| Isothermal titration calorimetry | Heat changes during binding | Substrate binding energetics | Requires highly purified protein |
For D. nodosus atpF research, these assays must be performed under anaerobic conditions that mimic the natural environment of the bacterium. Researchers should also consider the impact of temperature on activity measurements, as D. nodosus produces thermostable enzymes .
How can mutagenesis approaches be optimized to study structure-function relationships in D. nodosus atpF?
For effective mutagenesis studies of D. nodosus atpF:
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Identify conserved residues through multiple sequence alignment with atpF from related species
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Design a complementation system where the native atpF is deleted and complemented with mutant versions
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Implement site-directed mutagenesis targeting:
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Residues in the transmembrane domain
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Residues at the interface with other ATP synthase subunits
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Potential regulatory sites identified by PTM analysis
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Assess the impact of mutations on:
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ATP synthase assembly
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Proton translocation
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ATP synthesis/hydrolysis
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Growth under various conditions
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This approach should be integrated with structural information from related ATP synthases to guide rational mutagenesis design. Researchers should examine whether mutations in atpF affect expression of virulence factors like the extracellular subtilases that are characteristic of D. nodosus (AprV5, AprV2, and BprV) .
Which computational tools are most appropriate for predicting functional domains in D. nodosus atpF?
For comprehensive domain prediction in D. nodosus atpF:
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Use integrated approaches combining:
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Sequence-based tools: InterPro, Pfam, SMART
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Structure prediction: AlphaFold2, I-TASSER
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Transmembrane domain prediction: TMHMM, Phobius
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Coiled-coil prediction: COILS, MultiCoil
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Apply D. nodosus-specific parameters:
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Validate predictions with experimental approaches:
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Limited proteolysis to identify domain boundaries
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Circular dichroism to assess secondary structure content
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Truncation analysis to test domain functionality
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These approaches should be informed by the GO analysis of D. nodosus JKS-07B, which revealed 340 terms enriched in biological processes, 50 terms in cellular components, and 547 terms in molecular function .
How can systems biology approaches integrate atpF function with the broader metabolic network of D. nodosus?
Systems biology approaches for integrating atpF function should:
This systems approach should incorporate the unique metabolic features of D. nodosus as an anaerobic organism, potentially utilizing the 21 unique genes identified in D. nodosus JKS-07B that were not found in the reference strain . The analysis should consider the functional categories identified in the genome annotation, including protein metabolism (164 genes), cofactors/vitamins/prosthetic groups/pigments (70 genes), DNA metabolism (45 genes), stress response (25 genes), and virulence/disease/defense (17 genes) .
How can recombinant D. nodosus atpF be evaluated as a potential vaccine component against footrot?
To evaluate recombinant D. nodosus atpF as a vaccine component:
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Express and purify recombinant atpF using methods that preserve native conformation
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Assess immunogenicity in appropriate animal models:
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Measure antibody responses (titer, isotype, neutralizing capacity)
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Evaluate cell-mediated immune responses
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Determine cross-reactivity against different D. nodosus serogroups
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Conduct challenge studies in sheep using virulent D. nodosus strains
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Compare efficacy with existing whole-cell killed vaccines that use D. nodosus serogroup B, which is predominant in virulent footrot cases in India
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Evaluate potential for combination with other antigens, particularly the virulence-associated proteases
The methodology should follow established protocols for footrot vaccine evaluation, including lesion scoring according to Stewart and Claxton . Researchers should consider that D. nodosus serogroup B is associated with 85-90% of virulent footrot cases in India .
What methodologies are most appropriate for studying the immunological properties of D. nodosus atpF epitopes?
For comprehensive epitope analysis of D. nodosus atpF:
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Apply computational prediction tools to identify:
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B-cell epitopes based on surface accessibility and hydrophilicity
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T-cell epitopes using MHC binding prediction algorithms
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Conserved epitopes across different D. nodosus serogroups
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Validate predicted epitopes experimentally:
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Synthesize peptide arrays covering the atpF sequence
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Test reactivity with sera from infected animals
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Assess T-cell responses to peptides using proliferation assays
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Characterize cross-reactivity:
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Test epitope recognition in sera from animals infected with different D. nodosus serogroups
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Assess potential cross-reactivity with host proteins
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This approach should be integrated with knowledge about D. nodosus pathogenesis, particularly its role in footrot which causes significant economic losses due to recurrent treatment costs and increased culling rates .
