Recombinant Edwardsiella ictaluri Probable intracellular septation protein A (NT01EI_1681)
Description
Introduction to NT01EI_1681
Probable intracellular septation protein A, encoded by the NT01EI_1681 gene (also known as yciB), is a membrane protein found in Edwardsiella ictaluri, a gram-negative bacterium primarily known as a pathogen affecting freshwater fish. The recombinant form of this protein has been developed for research purposes and is commercially available from several biotechnology suppliers. This protein belongs to the YciB family of bacterial proteins that are involved in cellular division processes, specifically in intracellular septation mechanisms . The protein is identified in the UniProt database with the accession number C5BDB0 and is classified as an inner membrane-spanning protein .
Current evidence suggests that this protein, like its homologs in other bacterial species, participates in the complex process of bacterial cell division. The increasing interest in bacterial cell division mechanisms has highlighted the importance of understanding proteins like NT01EI_1681, particularly in the context of developing novel antimicrobial strategies and comprehending fundamental bacterial biology.
Physical and Chemical Properties
The recombinant versions of this protein are typically produced with affinity tags to facilitate purification. The most common form is His-tagged, which adds a string of histidine residues to either the N-terminus or C-terminus of the protein . The exact tag configuration may vary depending on the manufacturer and intended application. When analyzed by SDS-PAGE, the protein demonstrates a purity level typically exceeding 85-90% in commercial preparations .
| Property | Characteristic |
|---|---|
| Protein Length | Full Length (1-180 amino acids) |
| Molecular Weight | Approximately 20 kDa (varies with tag) |
| Purity | >85% or >90% (SDS-PAGE) |
| Format | Lyophilized powder or liquid |
| Tags | His-tag (N-terminal) or as specified |
| Expression System | E. coli |
Role in Cell Division
NT01EI_1681 is primarily characterized as an intracellular septation protein involved in bacterial cell division processes . While the specific mechanisms of action for this protein in E. ictaluri are not fully elucidated, insights can be gained from studies of its homologs in other bacterial species, particularly the YciB protein in Escherichia coli.
Research on the E. coli homolog has revealed that YciB interacts directly with ZipA, an essential component of the divisome complex that orchestrates bacterial cell division. This interaction suggests that YciB, and by extension NT01EI_1681, may play a crucial role in the formation of the septum during cell division .
Membrane Association and Cell Envelope Synthesis
The protein's predicted transmembrane domains and association with the inner membrane indicate its involvement in processes related to the cell envelope. Studies with the E. coli homolog demonstrated that deletion of the yciB gene resulted in shorter cells compared to wild type, while overexpression caused cell elongation. These phenotypic changes suggest that the protein influences cell morphology, likely through its role in cell envelope synthesis .
Furthermore, the E. coli YciB protein appears to be involved in a ZipA-directed pathway of cell envelope synthesis that operates independently of PBP3 (Penicillin Binding Protein 3), a key enzyme in peptidoglycan synthesis during cell division . This independence from PBP3 suggests a unique pathway for cell wall modification, which may represent an additional or alternative mechanism for bacterial cell division.
Recombinant Expression Systems
Recombinant NT01EI_1681 protein is typically produced using E. coli expression systems, which provide efficient production of bacterial proteins . The gene encoding the protein is cloned into appropriate expression vectors, often with affinity tags to facilitate purification. The expression constructs are then transformed into E. coli host strains optimized for protein production.
The choice of expression system and conditions significantly impacts the yield and quality of the recombinant protein. Factors such as temperature, induction conditions, and host strain characteristics are carefully controlled to maximize protein production while maintaining proper folding and activity.
Purification Methods
Purification of the recombinant protein typically involves affinity chromatography, leveraging the included affinity tags. For His-tagged versions, immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins is the predominant method . This approach allows for selective binding of the tagged protein, followed by washing steps to remove contaminants and elution with imidazole or pH changes to recover the purified protein.
Following initial purification, additional chromatographic steps may be employed to achieve higher purity. The final product is typically assessed using SDS-PAGE to confirm purity levels, which commercial preparations generally report as exceeding 85-90% .
Antibody Production and Immunoassays
The availability of purified recombinant protein facilitates the development of antibodies specific to NT01EI_1681, which can be valuable tools for detecting and studying the native protein in E. ictaluri. These antibodies can be used in immunoassays such as Western blotting, immunoprecipitation, or immunohistochemistry to investigate the expression, localization, and interactions of the protein in bacterial cells.
Drug Discovery and Antimicrobial Development
Given the essential role of cell division proteins in bacterial survival, NT01EI_1681 and its homologs represent potential targets for antimicrobial development. Recombinant protein can be used in high-throughput screening assays to identify compounds that inhibit its function, potentially leading to the development of novel antibiotics with activity against E. ictaluri and related pathogens.
YciB in Escherichia coli
The most well-studied homolog of NT01EI_1681 is the YciB protein in E. coli. Research has revealed several key aspects of YciB function that likely apply to NT01EI_1681 as well:
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Deletion mutants (ΔyciB) exhibit shorter cell length compared to wild-type E. coli, suggesting a role in cell elongation or division .
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Overexpression of yciB causes cell elongation, further supporting its involvement in cell growth and division processes .
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YciB directly interacts with ZipA, an essential divisome protein, indicating integration into the cell division machinery .
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The septum localization of ZipA is disturbed in yciB deletion mutants, suggesting that YciB influences the spatial organization of division proteins .
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YciB appears to function in a pathway of cell envelope synthesis that is independent of PBP3, potentially representing an alternative or complementary mechanism for cell wall remodeling during division .
These findings provide a framework for understanding the likely functions of NT01EI_1681 in E. ictaluri, although species-specific differences may exist.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is specifically requested and pre-arranged. Additional fees apply for dry ice shipping.
The tag type is finalized during production. If you require a specific tag, please inform us, and we will prioritize its implementation.
Target Background
KEGG: eic:NT01EI_1681
STRING: 634503.NT01EI_1681
Q&A
What is Edwardsiella ictaluri Probable intracellular septation protein A?
Edwardsiella ictaluri Probable intracellular septation protein A (NT01EI_1681) is a membrane-associated protein expressed by Edwardsiella ictaluri, a Gram-negative facultative intracellular pathogen primarily known for causing enteric septicemia in catfish (ESC). The protein consists of 180 amino acids and is thought to play a role in bacterial cell division processes during intracellular growth . E. ictaluri is particularly significant as a pathogen in aquaculture settings, where it can cause substantial economic losses in catfish farming, though it has also been identified in other fish species including zebrafish .
How does E. ictaluri infection manifest in fish species?
Edwardsiella ictaluri primarily causes enteric septicemia in catfish (ESC), characterized by a systemic infection that can lead to high mortality rates. In experimental studies, mortality rates of 46.91% have been observed with wild-type strains . Clinical signs may include:
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Small areas of raised scales
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Tissue necrosis, particularly near the caudal peduncle
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Systemic infection leading to acute mortality (up to 30% within days in some outbreaks)
Susceptibility to infection increases with environmental stressors, including:
While initially identified as a pathogen in catfish species, E. ictaluri has been documented to naturally infect non-ictalurid fishes, including zebrafish (Danio rerio), which has implications for research colonies utilizing these model organisms .
What expression systems are optimal for recombinant production of NT01EI_1681?
For recombinant expression of NT01EI_1681, E. coli expression systems have proven effective, particularly when the protein is fused to an N-terminal His tag . The methodology should consider:
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Vector selection: Vectors containing strong promoters (T7, tac, etc.) with N-terminal His-tag fusion sites are recommended
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E. coli strain optimization: BL21(DE3) or similar strains lacking proteases are preferred
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Expression conditions: Induction parameters should be optimized for membrane proteins:
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Lower temperatures (16-25°C) to prevent inclusion body formation
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Reduced IPTG concentrations (0.1-0.5 mM)
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Extended induction times (overnight)
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When working with membrane proteins like NT01EI_1681, it may also be beneficial to consider specialized E. coli strains engineered for membrane protein expression that contain extra copies of rare codons or chaperones to facilitate proper folding .
What are the recommended storage and handling procedures for recombinant NT01EI_1681?
For optimal stability and activity of recombinant NT01EI_1681, the following storage and handling procedures are recommended:
Short-term storage:
Long-term storage:
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Store at -20°C/-80°C upon receipt
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Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles
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Use Tris/PBS-based buffer with 6% Trehalose, pH 8.0 as a storage buffer
Reconstitution protocol:
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Briefly centrifuge the vial prior to opening to bring contents to the bottom
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Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C
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The recommended default final concentration of glycerol is 50%
Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of activity.
How does iron availability affect E. ictaluri virulence mechanisms?
While specific information on NT01EI_1681's relationship with iron metabolism is not established, research on E. ictaluri shows that iron acquisition is critical for its virulence:
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During initial infection, E. ictaluri encounters iron starvation stress in the gastric environment of fish hosts
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The TonB energy transducing system supports active transport of scarce resources including iron, which is essential for bacterial virulence
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Deletion of the tonB gene (locus tag = NT01EI_RS07425) in E. ictaluri results in:
| Condition | Wild-type E. ictaluri | E. ictaluri ΔtonB mutant |
|---|---|---|
| Mortality rate | 46.91% | 21.69% |
| Growth in iron-depleted medium | Normal | Significantly reduced |
| Growth with ferric iron supplementation | Normal | Similar to wild-type |
| Protective immunity in surviving fish | 40.47% survival upon challenge | 100% survival upon challenge |
These findings indicate that iron acquisition systems are important virulence determinants in E. ictaluri, and proteins involved in iron metabolism or regulation may be potential targets for vaccine development .
What molecular techniques can be used to study NT01EI_1681 function?
To investigate the function of NT01EI_1681, researchers can employ several molecular techniques:
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Gene deletion strategies:
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Gene splicing by overlap extension method as demonstrated with the tonB gene
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Amplification of upstream and downstream fragments of the target gene
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Fusion of these fragments followed by cloning into a suitable vector (e.g., pMEG-375)
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Transfer into a donor strain and mobilization into E. ictaluri by conjugation
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Selection for double crossovers using sucrose selection
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Protein localization techniques:
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Fluorescent protein fusion constructs
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Immunofluorescence microscopy with antibodies against the His-tag
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Subcellular fractionation followed by Western blotting
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Protein-protein interaction studies:
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Bacterial two-hybrid systems
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Co-immunoprecipitation with tagged proteins
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Cross-linking studies followed by mass spectrometry
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Expression analysis:
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qRT-PCR under various growth conditions
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Proteomics approaches to identify differential expression
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These methodologies can provide insights into when and where NT01EI_1681 functions in the bacterial cell cycle and during infection .
How can NT01EI_1681 be utilized in vaccine development against E. ictaluri infections?
NT01EI_1681 could potentially be incorporated into vaccine strategies against E. ictaluri through several approaches:
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Subunit vaccine development:
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Recombinant NT01EI_1681 protein, properly folded and purified, could be evaluated as an antigen
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Combination with appropriate adjuvants to enhance immunogenicity
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Delivery routes (injection, immersion, oral) should be evaluated for efficacy in fish
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Live attenuated vaccine approaches:
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Generation of E. ictaluri strains with modified NT01EI_1681 expression
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Similar to the ΔtonB mutant approach, which showed protective immunity (100% survival in vaccinated fish vs. 40.47% in naïve fish upon challenge)
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Safety assessment is crucial as complete gene deletion may not sufficiently attenuate virulence
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DNA vaccine strategies:
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Plasmid constructs encoding NT01EI_1681 for in vivo expression
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Evaluation of immune response profiles (humoral vs. cell-mediated)
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Research has shown that fish surviving infection with attenuated E. ictaluri strains can develop protective immunity against subsequent challenge with virulent strains, suggesting the feasibility of vaccine approaches targeting membrane-associated proteins .
What analytical techniques are most effective for studying membrane proteins like NT01EI_1681?
Membrane proteins present unique challenges for structural and functional studies. For NT01EI_1681, consider these analytical approaches:
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Structural characterization:
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Circular dichroism (CD) spectroscopy to assess secondary structure
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Nuclear magnetic resonance (NMR) for smaller domains
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Cryo-electron microscopy for larger complexes
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X-ray crystallography following optimization of crystallization conditions
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Functional assays:
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Liposome reconstitution systems to assess membrane integration
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Electrophysiology if channel or transport functions are suspected
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Bacterial growth complementation studies with mutant strains
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Protein-lipid interactions:
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Differential scanning calorimetry
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Fluorescence resonance energy transfer (FRET) with labeled lipids
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Native mass spectrometry with nanodiscs
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Computational approaches:
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Molecular dynamics simulations to predict membrane interaction
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Homology modeling based on related proteins with known structures
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Protein-protein and protein-ligand docking simulations
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These techniques can help overcome the inherent difficulties in working with hydrophobic membrane proteins like NT01EI_1681 .
How does E. ictaluri infection in zebrafish compare to infection in catfish species?
E. ictaluri has been documented to infect both catfish and zebrafish, but with some notable differences:
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Clinical presentation:
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Mortality rates:
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Detection methods:
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Research implications:
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Zebrafish infection represents a significant concern for research facilities
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Highlights the importance of quarantine practices when introducing new fish
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Provides a potential alternative model organism for studying E. ictaluri pathogenesis
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The occurrence of E. ictaluri in zebrafish research colonies emphasizes the need for rigorous health monitoring programs and preventive measures in laboratory settings using these model organisms .
What research gaps exist in our understanding of NT01EI_1681 and related proteins?
Several knowledge gaps remain in our understanding of NT01EI_1681 and warrant further research:
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Functional characterization:
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Precise molecular function during cell division and septation
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Role in bacterial survival during intracellular growth phases
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Potential interactions with host cell components
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Structure-function relationships:
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Three-dimensional structure determination
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Identification of critical functional domains
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Membrane topology and orientation
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Expression regulation:
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Environmental triggers affecting expression levels
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Potential co-regulation with virulence factors
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Response to stress conditions encountered in the host
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Evolutionary conservation:
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Comparative analysis across Edwardsiella species and strains
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Functional conservation of homologs in other bacterial pathogens
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Identification of unique features that could be exploited for targeted interventions
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Therapeutic potential:
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Evaluation as a diagnostic marker for early detection of infection
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Assessment as a vaccine component or drug target
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Investigation of cross-protective immunity against different strains
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Addressing these research gaps would significantly advance our understanding of E. ictaluri pathogenesis and potentially lead to improved control strategies for both aquaculture and research settings .
