CA1 E.Coli
Description
Functional Role in CO₂ Metabolism
Carbonic anhydrases (CAs) catalyze the reversible hydration of CO₂ to bicarbonate and protons, a reaction critical for pH regulation and metabolic processes . In E. coli, CA1 is one of two native carbonic anhydrases (alongside CynT) involved in maintaining intracellular CO₂ levels during gluconate metabolism . Studies using E. coli knockout strains (e.g., ΔcynT Δcan) have demonstrated that CA1 deficiency disrupts CO₂-concentrating mechanisms, impairing bacterial growth under low-CO₂ conditions .
Production and Applications in Research
CA1 E.Coli is produced via heterologous expression in E. coli BL21(DE3) strains, leveraging optimized codon usage and chromatographic purification . Its recombinant form is utilized in:
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Enzyme Kinetics: Testing inhibitors or activators of carbonic anhydrase activity.
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Structural Studies: Crystallography to resolve active-site interactions with zinc ions .
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Metabolic Engineering: Reconstituting CO₂-fixing pathways in synthetic biology projects .
For example, engineered E. coli cocultures using CA1-enabled pathways have been employed to produce industrial compounds like muconic acid, demonstrating the enzyme’s utility in biomanufacturing .
Research Findings and Comparative Insights
Recent studies highlight CA1’s role in bacterial stress responses and pathogenicity:
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Stress Adaptation: E. coli lacking CA1 (Δcan) show reduced survival under oxidative stress, linking CA activity to redox homeostasis .
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Host-Pathogen Interactions: While CA1 itself is not directly implicated in virulence, related carbonic anhydrases in pathogenic E. coli strains contribute to colonization and immune evasion .
Industrial and Environmental Relevance
CA1 E.Coli’s stability and catalytic efficiency make it a candidate for:
Product Specs
Carbonic anhydrase (CA) is an enzyme that catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and protons (H+). This enzyme plays a crucial role in maintaining acid-base balance in the blood and other tissues. CA contains a zinc ion (Zn) at its active site, which is essential for its catalytic activity. One of the primary functions of CA is to facilitate the transport of CO2 from tissues to the lungs for exhalation. CA works in conjunction with Carbonic Anhydrase I to carry out this process.
Recombinant CA1 from E. coli is a single, non-glycosylated polypeptide chain. It consists of 240 amino acids, with amino acids 1-220 representing the CA1 protein, and has a molecular weight of 27.0 kDa. The protein is expressed in E. coli and purified using proprietary chromatographic methods. A 20 amino acid His-Tag is fused to the N-terminus to aid in purification.
CA1 E.Coli protein is supplied at a concentration of 1mg/ml in a buffer consisting of 20mM Tris-HCl (pH 8.0), 1mM DTT, and 10% glycerol.
For short-term storage (up to 2-4 weeks), the product can be stored at 4°C. For extended storage, it is recommended to freeze the product at -20°C. The addition of a carrier protein such as HSA or BSA (0.1%) is advised for long-term storage. Avoid repeated freezing and thawing of the product.
The purity of the CA1 E.Coli protein is greater than 95%, as determined by SDS-PAGE analysis.
Carbonate dehydratase, CAN, ECK0125, JW0122, yadF, CA 1, CA I, CA1, CAI, Car 1, Car1, Carbonate dehydratase I, Carbonic anhydrase 1, Carbonic anhydrase B.
MGSSHHHHHH SSGLVPRGSH MKDIDTLISN NALWSKMLVE EDPGFFEKLA QAQKPRFLWI GCSDSRVPAE RLTGLEPGEL FVHRNVANLV IHTDLNCLSV VQYAVDVLEV EHIIICGHYG CGGVQAAVEN PELGLINNWL LHIRDIWFKH SSLLGEMPQE RRLDTLCELN VMEQVYNLGH STIMQSAWKR GQKVTIHGWA YGIHDGLLRD LDVTATNRET LEQRYRHGIS NLKLKHANHK.
Q&A
What is the K1 capsule in E. coli and why is it significant in research?
The K1 capsule is a specific polysaccharide extracellular barrier that surrounds certain E. coli strains. This particular capsule type is significant because it allows bacteria to mimic molecules already present in human tissues, enabling them to enter the body undetected by the immune system. E. coli strains possessing the K1 capsule are known to cause invasive diseases such as bloodstream infections, kidney infections, and meningitis, particularly in newborns . The K1 capsule represents a critical virulence factor that enables the bacterium to evade host immune responses, making it an important target for therapeutic interventions.
How does E. coli with K1 capsule differ from other pathogenic E. coli strains?
E. coli with K1 capsule differs from other pathogenic strains primarily through its enhanced ability to cause extraintestinal infections. While many E. coli strains like O157:H7 primarily cause intestinal disease, K1 capsule-containing strains can penetrate the bloodstream and cross the blood-brain barrier. This is due to molecular mimicry—the K1 capsule contains polysialic acid that resembles host neural cell adhesion molecules, allowing it to remain "invisible" to the immune system. Additionally, K1 strains have been associated with a mortality rate as high as 40% in neonatal meningitis cases . These strains also demonstrate different evolutionary pathways compared to enteric pathogenic E. coli strains.
What research models are most appropriate for studying K1 capsule E. coli infections?
Research models for studying K1 capsule E. coli infections include:
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In vitro cellular models: Human brain microvascular endothelial cell lines are commonly used to study blood-brain barrier penetration.
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Animal models: Sprague-Dawley rats have been successfully used for in vivo research on implantation and hydrogen evolution, as demonstrated in studies of magnesium-based alloys that include observations relevant to infection models . Additionally, neonatal rat models have proven effective for studying E. coli infection impacts on neurobehavioral outcomes .
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Evolutionary models: The E. coli long-term evolution experiment (LTEE) methodology, while not specifically developed for K1 research, provides valuable protocols for studying bacterial adaptation and evolution that can be adapted for K1 capsule studies .
The choice of model depends on research objectives—cellular models for mechanistic studies, animal models for pathogenesis and treatment efficacy, and evolutionary models for adaptation studies.
What are the most sensitive methods for detecting K1 E. coli in clinical and environmental samples?
Several methodologies have demonstrated high sensitivity for detecting K1 E. coli:
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Immunomagnetic separation (IMS) combined with cyanoditolyl tetrazolium chloride (CTC) and antibody staining: This method has shown excellent results with detection limits of approximately 10 CFU/g in ground beef and <10 CFU/ml in liquid samples . The procedure involves:
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Incubating samples with paramagnetic beads coated with anti-O157 specific antibody
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Staining attached cells with CTC to identify metabolically active bacteria
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Counter-staining with fluorescein-conjugated anti-O157 antibody
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Enumeration by microscopy or solid-phase laser cytometry
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Comparative detection efficiency: The IMS-CTC-FAb method demonstrated superior recovery compared to traditional culture methods:
| Sample Type | IMS-CTC-FAb vs. Sorbitol MacConkey Agar Recovery Ratio |
|---|---|
| Beef | 6.0 times higher |
| Peptone | 3.0 times higher |
| Water | 2.4 times higher |
This enhanced recovery allows for faster detection (5-7 hours) compared to traditional plating methods .
While this data specifically references O157 detection, the methodological approach can be adapted for K1 capsule detection with appropriate antibodies specific to K1 antigens.
How should researchers optimize experimental conditions when working with K1 capsule E. coli?
Optimal experimental conditions for K1 capsule E. coli research include:
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Growth media selection: The use of defined minimal media with controlled glucose concentrations (such as DM25 with 25 mg/L glucose) provides reproducible conditions similar to those used in evolution experiments . This reduces clonal interference while allowing for careful monitoring of adaptive changes.
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Temperature control: Maintain cultures at 37°C, which represents the optimal growth temperature for these bacteria and mimics human physiological conditions .
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Incubation protocols for detection methods: When using CTC staining for respiratory activity:
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Sample preparation considerations: For complex matrices like food:
These conditions ensure optimal growth, detection sensitivity, and reproducibility across experiments.
What genetic mechanisms contribute to K1 capsule regulation and expression in E. coli?
The genetic regulation of the K1 capsule involves multiple levels of control:
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Capsule synthesis gene cluster: The K1 capsule is encoded by a specific gene cluster that includes genes for polysialic acid synthesis, export, and assembly at the cell surface.
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Evolutionary adaptations: Based on evolutionary studies of E. coli, we understand that regulatory mechanisms can undergo significant changes over time. The LTEE has demonstrated that E. coli can acquire new metabolic capabilities through genomic changes, suggesting similar mechanisms may apply to virulence factor regulation in K1 strains .
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Phenotypic consequences: Mutations affecting capsule production can significantly alter virulence properties. These genetic changes have been mapped in evolutionary timelines, revealing how K1 capsule-producing strains have evolved their virulence mechanisms .
Research targeting these genetic mechanisms has shown promise for developing new therapeutic approaches that could disrupt capsule formation or regulation, potentially reducing the virulence of these strains in clinical settings.
How does the K1 capsule contribute to neuropathogenesis and what are the molecular mechanisms involved?
The K1 capsule contributes to neuropathogenesis through several mechanisms:
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Neuroinflammatory responses: E. coli infection triggers significant inflammatory responses in the brain, as evidenced by increased expression of inflammatory markers. Studies have demonstrated time-dependent increases in CD11b mRNA levels (increased 24h and 48h post-infection) and IL-1β mRNA in brain tissue following E. coli infection .
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Neurobehavioral impacts: Neonatal E. coli infection leads to measurable neurobehavioral deficits, including:
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Blood-brain barrier penetration: The K1 capsule enables E. coli to penetrate the blood-brain barrier through molecular mimicry, as the polysialic acid in the capsule resembles neural cell adhesion molecules.
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Immune evasion: The capsule helps the bacterium evade immune recognition, allowing it to persist and proliferate within the central nervous system.
These mechanisms collectively explain why K1 capsule-expressing E. coli strains are particularly dangerous in neonatal infections, with mortality rates as high as 40% in meningitis cases .
How should researchers analyze and interpret data from evolutionary studies of E. coli with K1 capsule?
Data analysis for evolutionary studies of E. coli with K1 capsule should follow these methodological guidelines:
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Long-term experimental design: The E. coli Long-Term Evolution Experiment (LTEE) provides a valuable framework for evolutionary studies. This approach involves:
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Statistical approaches for fitness analysis:
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Genomic data interpretation:
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Phenotypic data correlation:
When analyzing contradictory results, researchers should consider differences in selective pressures, genetic backgrounds, and experimental conditions, as these can dramatically impact evolutionary outcomes.
What methodological approaches are most effective for studying capsule-targeting therapeutics?
Effective methodological approaches for studying capsule-targeting therapeutics include:
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In vitro screening assays:
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Capsule inhibition assays measuring changes in capsule thickness or production
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Growth inhibition assays in the presence of potential inhibitors
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Biofilm formation assays to assess the impact on bacterial communities
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Ex vivo models:
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Human blood survival assays to assess immune evasion properties
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Serum resistance testing to measure complement sensitivity
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In vivo efficacy studies:
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Animal infection models assessing bacterial load reduction
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Survival studies measuring therapeutic impact on mortality
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Analysis of inflammatory markers to assess disease progression
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Combination approaches:
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Testing capsule-targeting agents with conventional antibiotics
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Evaluating immune-stimulating approaches alongside capsule inhibition
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Imperial College London research has demonstrated that targeting the K1 capsule can be an effective treatment approach, particularly for bloodstream infections . This strategy addresses the core virulence mechanism that allows these strains to cause invasive disease.
How can researchers reconcile contradictory findings in E. coli evolution studies?
Researchers can address contradictory findings in E. coli evolution studies through these methodological approaches:
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Examining experimental conditions: The LTEE research demonstrates how different selective pressures can dramatically impact evolutionary outcomes. For example, studies on citrate utilization showed that:
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Considering population dynamics:
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Defining evolutionary contingency:
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Standardizing methodologies:
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Using consistent growth conditions and selective pressures
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Establishing common phenotypic and genotypic assessment methods
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Creating standardized reporting formats for evolutionary experiments
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These approaches can help researchers determine whether contradictory findings represent truly different biological phenomena or merely reflect methodological differences.
What are the most promising future research directions for combating K1 capsule E. coli infections?
The most promising future research directions include:
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Capsule-targeting therapeutics:
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Evolutionary-informed approaches:
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Rapid detection methodologies:
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Combination therapies:
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Targeting the capsule while simultaneously inhibiting other virulence factors
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Enhancing immune recognition of capsulated strains
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Developing adjunctive therapies to conventional antibiotics
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These approaches could address the urgent need for new strategies to combat hypervirulent and multi-drug resistant E. coli strains that have emerged over the past decade .
Product Science Overview
CAs are divided into several classes: α, β, γ, δ, ζ, η, θ, and ι. Each class has evolved to perform specific functions in different organisms. For instance, in Escherichia coli (E. coli), β-CA (CynT) catalyzes the hydration of CO₂ generated by cyanase, thus preventing final HCO₃⁻ depletion in bacteria resulting from degradation of cyanate and/or other metabolic processes .
Expressing recombinant proteins, including CAs, in E. coli is a common practice due to the bacterium’s well-understood genetics, rapid growth, and ability to express foreign proteins. However, the expression of CAs in E. coli can be challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications .
The ability to express CAs in E. coli has significant industrial implications, particularly for carbon capture, utilization, and storage (CCUS) processes. Identifying efficient and robust CAs and expressing them in model host cells like E. coli enables more efficient engineering of these enzymes for industrial CO₂ capture .
Recent studies have focused on streamlining the heterologous expression of top CAs in E. coli. For example, researchers have used bioinformatic tools to predict the solubility of various CA candidates and have successfully expressed high-solubility CAs in E. coli, leading to significantly higher protein yields . This approach not only enhances the efficiency of CA production but also provides insights into the phylogenetic clustering patterns of CA solubility and production yields .
In conclusion, the recombinant expression of Carbonic Anhydrase-1 in E. coli represents a promising avenue for both scientific research and industrial applications. The advancements in bioinformatics and experimental validation have paved the way for more efficient and robust production of these essential enzymes.
