Recombinant Escherichia coli O157:H7 Potassium-transporting ATPase C chain (kdpC)
Description
Overview
The Escherichia coli O157:H7 Potassium-transporting ATPase C chain (KdpC) is a subunit of the KdpFABC complex, which is a high-affinity ATP-driven potassium transport system in E. coli . This complex is crucial for maintaining potassium homeostasis within the bacterial cell, especially under conditions of potassium limitation . The KdpFABC complex comprises four membrane-bound subunits: KdpF, KdpA, KdpB, and KdpC .
Structure and Function
The KdpC subunit interacts with the nucleotide-binding loop of KdpB in an ATP-dependent manner near the ATP-binding pocket . This interaction enhances the ATP-binding affinity through the formation of a transient KdpB/KdpC/ATP ternary complex . KdpC contains a conserved glutamine residue that is essential for high-affinity nucleotide binding to the KdpFABC complex .
Research Findings
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A study involving truncations of the kdpC gene revealed that only a derivative lacking the base pairs coding for the four C-terminal amino acids could complement the chromosomal deletion of kdpC .
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Complementation experiments showed that kdpC from Mycobacterium tuberculosis could complement the E. coli deletion strain, but kdpC from Clostridium acetobutylicum or Synechocystis sp. PCC6803 could not .
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Hybrid constructs between kdpC of E. coli and C. acetobutylicum showed that the N-terminal transmembrane segment and the C-terminal third of the protein could be exchanged individually, but not simultaneously .
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The KdpC subunit interacts with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, increasing the ATP-binding affinity by forming a transient KdpB/KdpC/ATP ternary complex .
Role in Virulence
E. coli O157:H7 causes attaching and effacing (A/E) lesions on intestinal epithelial cells, which are mediated by various virulence factors . While KdpC itself is not directly implicated as a primary virulence factor, the general function of potassium transport systems can be important for bacterial survival and pathogenesis . Specifically, the study showed that metabolically active bacteria and intact lipid rafts are necessary for the recruitment of protein kinase C (PKC), which plays a role in A/E lesion formation .
Product Specs
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Target Background
The Recombinant Escherichia coli O157:H7 Potassium-transporting ATPase C chain (KdpC) is a component of the high-affinity ATP-driven potassium transport (Kdp) system. This system catalyzes ATP hydrolysis, coupled with the electrogenic transport of potassium ions into the cytoplasm. KdpC acts as a catalytic chaperone, enhancing the ATP-binding affinity of the ATP-hydrolyzing subunit KdpB through the formation of a transient KdpB/KdpC/ATP ternary complex.
KEGG: ecf:ECH74115_0789
Q&A
What is E. coli O157:H7 and how does it differ from non-pathogenic E. coli strains?
E. coli O157:H7 is an enterohemorrhagic pathogen that expresses somatic (O) antigen 157 and flagella (H) antigen 7. It has several distinguishing characteristics, including delayed D-sorbitol fermentation (>24 h) and inability to produce β-glucuronidase, which are used for its identification on Sorbitol MacConkey (SMAC) agar supplemented with MUG .
The genome of E. coli O157:H7 is approximately 5.5 Mb, with a 4.1 Mb backbone sequence conserved across all E. coli strains. Genome comparison between E. coli O157:H7 and non-pathogenic E. coli K12 shows that 0.53 Mb of DNA is missing in O157:H7, suggesting genomic reduction has played a role in its evolution . The majority of E. coli O157:H7-specific DNA sequences (1.4 Mb) are horizontally transferred foreign DNAs, particularly prophage and prophage-like elements. E. coli O157:H7 contains 463 phage-associated genes compared to only 29 in E. coli K-12 .
What is the function of kdpC in E. coli O157:H7?
The kdpC protein is a subunit of the Kdp-ATPase complex, a high-affinity potassium transport system that plays a critical role in maintaining potassium homeostasis, especially under potassium-limited conditions. In the Kdp-ATPase complex, kdpC functions as a stabilizing subunit that works in conjunction with other components (KdpF, KdpA, and KdpB) to facilitate potassium uptake.
The kdpC protein is structurally similar to the C chain of potassium-transporting ATPase seen in E. coli O81, as referenced in search result , though strain-specific variations may exist. While not directly mentioned in the search results, research indicates that the Kdp system is essential for bacterial survival under osmotic stress conditions and may contribute to virulence and stress response in pathogenic strains like E. coli O157:H7.
What expression systems are most effective for producing recombinant E. coli O157:H7 kdpC?
Based on the available data for recombinant kdpC production, E. coli expression systems have proven effective for expressing recombinant potassium-transporting ATPase C chain. As seen in the case of E. coli O81 kdpC, the protein can be expressed with an N-terminal His tag in E. coli expression systems .
For optimal expression of recombinant E. coli O157:H7 kdpC, researchers should consider the following methodology:
Table 1: Optimization Parameters for Recombinant kdpC Expression
| Parameter | Recommended Conditions | Considerations |
|---|---|---|
| Expression Vector | pET series with T7 promoter | Provides tight control of expression |
| Host Strain | BL21(DE3) or derivatives | Strains lacking lon and ompT proteases reduce protein degradation |
| Fusion Tag | N-terminal His tag | Facilitates purification via immobilized metal affinity chromatography |
| Induction Conditions | 0.1-0.5 mM IPTG, 16-25°C | Lower temperatures may improve protein folding |
| Medium | LB or TB supplemented with potassium | Potassium supplementation may stabilize the protein |
| Harvest Time | 4-16 hours post-induction | Optimization required for each specific construct |
How can digital PCR improve the detection and quantification of kdpC gene expression?
Digital PCR (dPCR) offers significant advantages for detecting and quantifying kdpC gene expression in E. coli O157:H7, particularly from complex samples. Based on the information from search result , dPCR provides exceptional precision and sensitivity in nucleic acid detection and quantification.
The methodology for optimizing dPCR for kdpC gene expression analysis involves:
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Sample partitioning: In dPCR, the sample is partitioned into individual reaction vessels or compartments, each containing a single molecule or few molecules of the target nucleic acid .
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Absolute quantification: The ratio between PCR-positive and PCR-negative partitions determines the total amount of the target based on Poisson's distribution, allowing for the easy detection of rare molecules .
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Platform selection: Researchers can choose between droplet digital PCR (ddPCR) and chip-based dPCR platforms, each with distinct advantages. While ddPCR may offer higher sensitivity for detection of rare target molecules, dPCR platforms may be more amenable to integration with other analytical techniques .
Table 2: Comparison of dPCR Platforms for kdpC Analysis
| Feature | Droplet Digital PCR (ddPCR) | Chip-based Digital PCR |
|---|---|---|
| Sensitivity | Higher sensitivity for rare targets | Potentially lower sensitivity |
| Sample Handling | Manual droplet generation may increase contamination risk | Automated systems minimize contamination risk |
| PCR Inhibitor Effects | Better mitigation of PCR inhibitors | Variable depending on platform |
| Integration Capabilities | Limited options for workflow customization | More amenable to integration with other techniques |
| Analysis Workflow | Specialized software for droplet analysis | Platform-specific analysis tools |
How does oxidative stress affect kdpC function in E. coli O157:H7?
Under oxidative stress conditions, E. coli O157:H7 significantly upregulates several genes, including regulatory genes responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance . Though kdpC isn't specifically mentioned, potassium transport systems like the Kdp-ATPase complex are often implicated in bacterial stress responses.
The response to oxidative agents involves different reaction rates with amino acid residues in proteins. For instance, the reaction rate of H₂O₂ with free thiol groups in Cys residues is about 2.9 M⁻¹s⁻¹ at pH 7.4 to 7.6, whereas the active chlorine OCl⁻ reacts much faster . This suggests that kdpC function may be differentially affected depending on the specific oxidative agent.
What methods are most effective for studying the structure-function relationship of kdpC?
To effectively study the structure-function relationship of kdpC in E. coli O157:H7, researchers can employ a combination of experimental approaches:
Table 3: Methodologies for kdpC Structure-Function Analysis
| Methodology | Application | Advantages | Limitations |
|---|---|---|---|
| X-ray Crystallography | Determining high-resolution protein structure | Provides atomic-level resolution | Requires protein crystallization |
| Cryo-Electron Microscopy | Visualizing the entire Kdp complex | Works with membrane proteins in near-native states | Lower resolution than X-ray crystallography |
| Site-Directed Mutagenesis | Identifying critical residues for function | Direct assessment of residue importance | May disrupt protein folding |
| Molecular Dynamics Simulations | Studying protein dynamics | Reveals conformational changes | Requires experimental validation |
| Isothermal Titration Calorimetry | Measuring binding affinities | Quantitative binding parameters | Requires purified components |
| Electrophysiology | Assessing ion transport | Direct functional measurement | Technical complexity |
How can researchers identify strain-specific variations in the kdpC gene across different E. coli O157:H7 isolates?
E. coli O157:H7 strains show genomic diversity, with nearly identical virulence-associated genes (99% similarity) between sequenced strains . To identify strain-specific variations in the kdpC gene:
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Whole Genome Sequencing: Use next-generation sequencing technologies to sequence multiple E. coli O157:H7 isolates.
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Comparative Genomics: Align the kdpC gene sequences from different isolates to identify single nucleotide polymorphisms (SNPs), insertions, deletions, or other variations.
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Digital PCR for Variant Detection: As described in search result , digital PCR offers exceptional precision for detecting genetic variants. This method partitions the sample into discrete units, allowing for absolute quantification of specific kdpC variants even in mixed populations.
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Functional Validation: Assess whether identified variations affect protein function through recombinant protein expression and functional assays.
The G+C content analysis may also be informative, as changes in G+C content can indicate genomic regions acquired by horizontal transfer , potentially affecting the kdpC gene or its regulatory elements.
How does transcriptomic profiling help understand kdpC regulation under different environmental conditions?
Transcriptomic profiling provides valuable insights into kdpC regulation under different environmental conditions. Drawing from the methodology used in search result to study E. coli O157:H7's response to oxidative stress:
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Experimental Design: Expose E. coli O157:H7 to various conditions relevant to its lifecycle (low potassium, different pH levels, oxidative stress, etc.).
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RNA Extraction and Analysis: Extract total RNA from treated and control cultures, followed by transcriptome analysis using RNA-Seq or microarray technology.
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Data Analysis: Identify differentially expressed genes, particularly kdpC and related genes in the kdp operon.
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Validation: Confirm expression changes using RT-qPCR or digital PCR for more precise quantification .
In the study of E. coli O157:H7's response to oxidative stress, over 380 genes were found to be differentially expressed after exposure to low levels of chlorine or hydrogen peroxide . Similar approaches can be applied to study kdpC regulation.
What experimental designs are most appropriate for studying the role of kdpC in E. coli O157:H7 pathogenesis?
Based on search result , several experimental designs can be effectively applied to study the role of kdpC in E. coli O157:H7 pathogenesis:
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Randomized Controlled Trials (RCTs): These can be used to evaluate the effect of kdpC mutations or overexpression on E. coli O157:H7 virulence in appropriate model systems .
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Factorial or Fractional-Factorial Designs: These are particularly useful when studying multiple factors that might influence kdpC function, such as environmental conditions, host factors, and genetic background .
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Interrupted Time Series (ITS): This design is valuable for studying the temporal dynamics of kdpC expression during infection progression .
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Stepped Wedge Designs: These can be used when all experimental units need to receive the intervention (e.g., a kdpC inhibitor) but in a staggered fashion .
When designing these experiments, researchers should consider:
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Clear definition of primary research questions
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Appropriate control conditions
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Sample size calculations for adequate statistical power
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Methods to minimize bias and confounding factors
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Validation strategies for key findings
How can researchers optimize purification protocols for obtaining functional recombinant kdpC protein?
Based on the information about recombinant kdpC protein production in search result , the following methodology can be used to optimize purification protocols:
Table 4: Optimization Parameters for Recombinant kdpC Purification
Critical considerations include:
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Maintaining the native conformation of the membrane protein
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Selecting appropriate detergents for solubilization
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Including potassium in buffers to stabilize the protein
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Careful optimization of elution conditions to maximize yield and purity
What are the challenges and solutions in studying kdpC interactions with other components of the Kdp-ATPase complex?
Studying protein-protein interactions within the Kdp-ATPase complex presents several challenges:
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Membrane Protein Complexity: The Kdp-ATPase complex consists of multiple membrane-embedded components, making traditional interaction studies difficult.
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Maintaining Complex Integrity: The complex may dissociate during purification, leading to loss of important interactions.
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Transient Interactions: Some interactions within the complex may be transient or dependent on specific conditions.
Methodological solutions include:
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Co-expression Systems: Express multiple components of the Kdp complex simultaneously to promote proper assembly.
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Crosslinking Approaches: Use chemical crosslinkers to capture transient interactions before purification.
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Native PAGE and Blue Native PAGE: These techniques allow analysis of intact protein complexes.
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Cryo-Electron Microscopy: This can provide structural information about the entire complex without requiring crystallization.
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Förster Resonance Energy Transfer (FRET): This can detect interactions between tagged proteins in live cells.
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Surface Plasmon Resonance (SPR): This allows measurement of binding kinetics between purified components.
By employing these advanced techniques, researchers can overcome the challenges inherent in studying membrane protein complexes like the Kdp-ATPase system in E. coli O157:H7.
