phrB E.Coli

What is the phrB gene in E. coli?

The phrB gene in Escherichia coli encodes a deoxyribodipyrimidine photo-lyase, which is a member of the DNA photolyase class-1 family. This protein takes part in the repair of UV radiation-induced DNA damage, specifically catalyzing the light-dependent monomerization (300-600 nm) of cyclobutyl pyrimidine dimers that form between adjacent bases on the same DNA strand upon exposure to ultraviolet radiation . The protein is also known by several synonyms including ECK0697, JW0698, phr, Deoxyribodipyrimidinephoto-lyase, DNA photolyase, and Photoreactivating enzyme (EC=4.1.99.3, b0708) .

What is the molecular structure of the phrB protein?

The phrB E. coli protein is a single, non-glycosylated polypeptide chain containing 472 amino acids with a molecular mass of approximately 56.1 kDa . Recombinant forms of the protein often include a His-tag at the N-terminus for purification purposes, resulting in a 495 amino acid protein (including the 23 amino acid His-tag) . The complete amino acid sequence is available in the product information and includes the functional domains necessary for its photolyase activity, including binding sites for DNA and chromophores that absorb light energy required for the repair process .

What experimental techniques are commonly used to isolate and characterize phrB protein?

Several techniques are used for isolation and characterization of phrB protein:

• Recombinant protein expression: The phrB gene can be cloned and expressed in E. coli expression systems, as demonstrated in the purification of the recombinant protein with >90% purity .

• Affinity chromatography: His-tagged phrB protein is commonly purified using proprietary chromatography techniques, resulting in sterile filtered clear solutions with high purity (>90% as determined by SDS-PAGE) .

• Spectroscopic analysis: Techniques such as Fourier Transform Infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (¹H-NMR) can be used to characterize the protein, similar to methodologies used for other recombinant E. coli proteins .

• Stability assessment: Thermogravimetric analysis (TGA) can be employed to study thermal properties, providing insights into the protein's stability under various conditions .

How should recombinant phrB protein be stored to maintain activity?

For optimal storage of phrB recombinant protein, the following conditions are recommended:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this timeframe .

• Long-term storage: Store frozen at -20°C for extended periods .

• Stability enhancement: For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) to maintain stability and activity .

• Avoid freeze-thaw cycles: Repeated freezing and thawing should be avoided as this can compromise protein integrity and activity .

The commercial preparation is typically formulated in a buffer containing 20mM Tris-HCl (pH 8.0), 0.2M NaCl, 50% glycerol, and 1mM DTT at a concentration of 0.25mg/ml, which helps maintain stability during storage .

How does the phrB photolyase mechanism repair UV-induced DNA damage at the molecular level?

The phrB photolyase repairs UV-induced DNA damage through a light-dependent mechanism involving several distinct steps:

• Substrate recognition: The enzyme specifically targets cyclobutyl pyrimidine dimers (CPDs) that form between adjacent pyrimidine bases on the same DNA strand after UV exposure .

• Light activation: The photolyase absorbs light in the 300-600 nm range through its chromophore cofactors .

• Catalytic action: Upon activation, the enzyme catalyzes the monomerization of cyclobutyl pyrimidine dimers specifically in the cis-syn configuration .

• Direct reversal: Unlike many other DNA repair mechanisms that remove and replace damaged bases, phrB directly reverses the damage, restoring the original DNA sequence without introducing potential mutations .

This mechanism represents a highly specific and energy-efficient repair system that helps maintain genomic integrity following UV exposure, contributing to E. coli's ability to survive in environments with UV radiation.

What are the implications of E. coli's strictly asexual reproduction for studying phrB evolution?

Studying phrB evolution in E. coli laboratory strains presents unique advantages due to their strictly asexual reproduction patterns:

• Mutation tracking: In asexual organisms like the E. coli strains used in long-term evolution experiments, new mutations provide the sole source of genetic variation available to selection . This allows precise tracking of mutations in the phrB gene and their phenotypic effects.

• No standing variation: Since experimental populations are often founded from a single clone, there is no standing genetic variation at the start . This means any parallelism observed in phrB evolution necessarily involves both independent origin and fate of variants.

• Natural selection dynamics: In these experimental systems, selection is natural rather than artificial - the environment selects rather than researchers choosing particular individuals to reproduce based on traits . This allows observation of how natural selection acts on phrB variants under controlled conditions.

• Clonal interference: In strictly clonal E. coli populations, beneficial mutations in phrB would compete with other beneficial mutations elsewhere in the genome, affecting fixation probabilities and evolutionary trajectories .

These characteristics make E. coli an excellent model for studying the fundamental evolutionary processes acting on DNA repair genes like phrB without the confounding effects of recombination.

How do mutations in the phrB gene interact with other DNA repair pathways in E. coli?

The interaction between phrB mutations and other DNA repair pathways demonstrates the complex redundancy in bacterial DNA damage response:

• Synergistic effects: Mutations in phrB often have more pronounced effects when combined with mutations in other DNA repair pathways, particularly the nucleotide excision repair (NER) pathway .

• Compensatory mechanisms: In long-term experimental evolution, bacteria can develop compensatory adaptations when one repair pathway is compromised, potentially through upregulation of alternative pathways .

• Differential response to damage types: While phrB specifically addresses pyrimidine dimers through photoreactivation, other pathways like base excision repair handle different types of DNA damage. Mutations affecting multiple pathways can result in sensitization to a broader spectrum of DNA-damaging agents .

• Genomic stability implications: Long-term experimental evolution studies with E. coli demonstrate that DNA repair capabilities significantly influence the rate and spectrum of mutations that accumulate over time, with important consequences for genomic stability and adaptation .

Research methodologies to study these interactions include construction of strains with multiple mutations across repair pathways, competitive fitness assays, and genomic analysis of mutation patterns in repair-deficient strains.

What is the relationship between phrB activity and antibiotic resistance in E. coli?

The relationship between DNA repair mechanisms like phrB and antibiotic resistance presents a complex research area:

• Stress response connection: Both UV damage repair and antibiotic stress responses involve overlapping cellular pathways. Research on drug-resistant E. coli isolates shows they often harbor multiple resistance genes alongside various DNA repair capabilities .

• Mutation rate effects: In strains with compromised DNA repair (including potential phrB mutations), increased mutation rates could accelerate the evolution of antibiotic resistance. This relationship is particularly relevant in environments with both UV exposure and antibiotic pressure .

• Resistance gene associations: Studies of E. coli isolates from environmental sources (such as duck farms) have identified correlations between virulence genes and resistance profiles, suggesting complex interactions between pathogenicity and stress response systems .

• Environmental adaptation: The extensive drug resistance spectrum observed in environmental E. coli isolates (with resistance rates exceeding 70% for multiple antibiotics including Penicillin, Amoxicillin, Ampicillin, and others) demonstrates how bacteria employ multiple molecular mechanisms to survive in challenging environments .

Research approaches to investigate this relationship include analyzing correlations between drug resistance phenotypes and DNA repair capabilities across clinical and environmental isolates, and experimental evolution studies under combined UV and antibiotic selection pressures.

How can phrB be utilized in recombinant DNA technology applications?

The phrB photolyase has several potential applications in recombinant DNA technology:

• DNA damage repair tool: Purified phrB can be used to repair UV-damaged DNA in vitro, potentially improving the quality of DNA templates for sensitive applications like sequencing or cloning .

• Expression system component: As demonstrated by work with other recombinant proteins, phrB expression systems can serve as models for optimizing protein production in E. coli .

• Stress resistance engineering: Similar to how the vgb gene (encoding Vitreoscilla hemoglobin) improved production in recombinant E. coli systems, phrB could potentially be utilized to enhance the UV resistance of production strains in industrial biotechnology .

• Protein engineering platform: The well-characterized structure and function of phrB makes it a suitable candidate for protein engineering efforts aimed at creating modified photolyases with enhanced properties for biotechnological applications .

Several methodological approaches support these applications, including:

• Recombinant protein expression optimization using vectors like pETDuet1

• Purification strategies achieving >90% purity through affinity chromatography

• Stability formulations containing appropriate buffers and cryoprotectants

• Functional validation through spectroscopic and enzymatic assays

What factors influence phrB expression and activity in different environmental conditions?

The expression and activity of phrB in E. coli is influenced by multiple environmental factors:

• Light conditions: As a photolyase, phrB activity is fundamentally dependent on light in the 300-600 nm range, with its effectiveness varying based on light intensity and wavelength .

• Growth phase effects: Long-term evolution experiments with E. coli suggest that growth phase transitions impact the expression of various stress-response genes, potentially including DNA repair mechanisms like phrB .

• Environmental stressors: Exposure to DNA-damaging agents likely alters phrB expression as part of the cellular stress response, similar to how environmental strains develop complex resistance profiles in response to antimicrobial pressure .

• Nutrient availability: Research on recombinant E. coli strains demonstrates that media composition and nutritional status significantly affect protein production and activity, which would apply to native phrB expression as well .

• Temperature effects: Thermal stability studies of recombinant proteins from E. coli indicate that temperature is a critical factor affecting enzyme activity and stability, with implications for phrB function across different environmental conditions .

Research methodologies to investigate these factors include quantitative PCR for expression analysis, reporter gene constructs to monitor promoter activity, and enzymatic assays under varied environmental conditions.

What are the most effective experimental designs for studying phrB function in vivo?

Effective experimental designs for studying phrB function in vivo include:

These experimental designs benefit from E. coli's rapid generation time, the ability to store and revive organisms, and the capacity to place genetic markers in specific backgrounds due to its clonal nature .

How do the kinetics of phrB-mediated DNA repair compare with other repair mechanisms?

The kinetics of phrB-mediated photorepair compared to other DNA repair mechanisms reveal important differences in efficiency and energy requirements:

• Direct reversal advantage: The phrB photolyase performs direct reversal of pyrimidine dimers, which is kinetically faster than excision repair pathways that require multiple enzymatic steps to remove and replace damaged DNA .

• Light dependency: Unlike nucleotide excision repair (NER) and base excision repair (BER) that function in both light and dark conditions, phrB photorepair has the kinetic limitation of requiring light activation (300-600 nm) .

• Energy efficiency: Photorepair is energetically more efficient than excision repair mechanisms, requiring only light energy rather than ATP consumption for multiple enzymatic steps .

• Damage specificity: phrB specifically targets cyclobutyl pyrimidine dimers in the cis-syn configuration, giving it high specificity but limiting its repair spectrum compared to more general mechanisms like NER .

• Experimental approaches: Kinetic studies typically employ techniques such as:

  • UV survival assays comparing wild-type and phrB mutant strains

  • Time-course experiments measuring damage removal under various light conditions

  • In vitro enzyme kinetics using purified phrB and synthetic substrates

  • Comparative genomics examining the prevalence of different repair mechanisms across bacterial species

These kinetic differences explain why bacteria maintain multiple DNA repair pathways, with the rapid and efficient phrB system complementing the more versatile but slower excision repair mechanisms.

What novel recombinant systems could be developed using phrB for biotechnological applications?

Several innovative recombinant systems could be developed using phrB for biotechnological applications:

• Enhanced bioproduction systems: Similar to how the vgb gene improved polyhydroxybutyrate (PHB) production in recombinant E. coli (achieving up to 213.30% more production) , phrB could be utilized to create UV-resistant production strains for outdoor bioreactors or environmental applications.

• DNA repair enhancement modules: Recombinant systems expressing optimized phrB variants could be developed to protect industrial microorganisms from UV damage, potentially increasing process stability and yield in bioproduction settings.

• Multi-gene expression systems: The strategies used to construct pETDuet1-based expression vectors combining multiple genes (as shown with phaA2, phaB1, phaC1, and vgb) could be applied to create systems where phrB works synergistically with other stress-response proteins.

• Biosensor development: phrB could be incorporated into biosensor systems for UV exposure monitoring, with the repair activity linked to reporter gene expression.

• Environmental bioremediation tools: Recombinant microorganisms with enhanced phrB expression could be engineered for improved survival in environments with high UV exposure, such as surface water bioremediation applications.

These applications would benefit from the established protocols for recombinant protein expression in E. coli, with purity levels exceeding 90% and well-defined storage conditions to maintain stability .

How can phrB research contribute to understanding bacterial adaptation to environmental stressors?

Research on phrB provides valuable insights into bacterial adaptation to environmental stressors:

• Long-term evolutionary dynamics: Studies of E. coli evolution over 20,000+ generations demonstrate how DNA repair mechanisms contribute to genomic stability and adaptive capacity under various stressors .

• Antimicrobial resistance connections: The complex relationship between DNA repair capabilities and antibiotic resistance profiles, as seen in environmental E. coli isolates with varied resistance genes (like oqxA with 93.3% prevalence) and virulence factors (like fimC with 93.3% prevalence) , illustrates how bacteria integrate multiple survival strategies.

• UV adaptation mechanisms: By studying phrB variants across E. coli strains from different environments, researchers can understand how bacteria adapt to varied UV exposure levels in different ecological niches .

• Methodological frameworks: The combination of experimental evolution approaches , molecular typing techniques , and recombinant expression systems provides powerful tools for investigating bacterial adaptation to multiple stressors simultaneously.

• Practical applications: Insights from phrB research can inform strategies for controlling bacterial populations in environments where UV exposure and antimicrobials are used as control measures, with potential applications in water treatment, food safety, and clinical settings .

This research area exemplifies how detailed molecular understanding of specific bacterial systems like phrB contributes to broader ecological and evolutionary insights with practical implications for biotechnology and public health.