Recombinant Prochlorococcus marinus subsp. pastoris UvrABC system protein B (uvrB), partial

What is the functional role of UvrB in the UvrABC repair system of Prochlorococcus marinus?

UvrB functions as a central component of the nucleotide excision repair (NER) pathway in Prochlorococcus marinus subsp. pastoris. Within the UvrABC system, UvrB works in concert with UvrA to recognize DNA lesions such as pyrimidine dimers or DNA intra-strand cross-links . Specifically, UvrA and UvrB form a two-protein complex that identifies damaged DNA sites, after which UvrC generates a double incision on both sides of the lesion, and UvrD helicase removes the damaged single-strand DNA segment . This excision repair mechanism is crucial for Prochlorococcus to maintain genomic integrity in response to UV radiation damage, which is particularly relevant given its habitat in the upper layers of oceans where UV exposure is significant .

Unlike in some other bacteria where uvrA and uvrB are organized in an operon, genomic analyses reveal that in cyanobacteria including Prochlorococcus, these genes are not arranged in an operon structure . This distinct genomic organization may reflect evolutionary adaptations specific to photosynthetic organisms that routinely encounter oxidative stress and UV damage.

What are the structural characteristics of the recombinant UvrB protein from Prochlorococcus marinus subsp. pastoris?

The recombinant Prochlorococcus marinus subsp. pastoris UvrB protein is characterized as a partial protein with specific structural features optimized for DNA repair functions. According to available product information, the protein:

• Is identified by UniProt accession Q7V1I9

• Contains conserved domains characteristic of the UvrB family, including nucleotide-binding motifs and DNA interaction regions

• Functions as the Excinuclease ABC subunit B

• Is typically produced recombinantly in expression systems such as yeast

• Demonstrates a purity of >85% when analyzed by SDS-PAGE

The "partial" designation indicates that the recombinant protein doesn't represent the complete native sequence, but rather contains the functional domains necessary for research applications. This partial structure may be deliberately designed to improve solubility or expression efficiency while maintaining essential functional regions.

How does Prochlorococcus marinus maintain genomic integrity in high-UV marine environments?

Prochlorococcus marinus maintains genomic integrity through multiple DNA repair pathways that work in concert to address different types of DNA damage in marine environments with high UV exposure.

The UvrABC nucleotide excision repair system represents one critical pathway among at least five different branches of post-replication repair mechanisms identified in prokaryotes . In addition to the UvrABC pathway, Prochlorococcus employs:

• RecA-mediated homologous recombination repair for double-strand breaks

• RecFOR/RecBCD pathways for processing single-strand nicks and double-strand breaks respectively

• RuvABC system for Holliday junction resolution

• Photoreactivation through photolyase (encoded by phr genes)

Evidence from comparative genomic studies indicates that while UvrABC genes are universally distributed across cyanobacterial genomes, other repair pathways show greater variability . This suggests that the UvrABC system represents a core DNA repair mechanism in Prochlorococcus that has been conserved throughout evolution due to its essential function in addressing UV-induced DNA damage.

The robust DNA repair capability is particularly important for Prochlorococcus, which is found abundantly in tropical and subtropical oceans between 40°S and 40°N latitudes, where solar radiation is most intense .

What are the optimal storage conditions for maintaining activity of recombinant UvrB protein?

Optimal storage conditions for recombinant Prochlorococcus marinus subsp. pastoris UvrB protein are critical for maintaining its structural integrity and enzymatic activity. Based on established protocols :

Storage Temperature and Stability:

• For liquid form preparations: Store at -20°C to -80°C with an expected shelf life of approximately 6 months

• For lyophilized form preparations: Store at -20°C to -80°C with an extended shelf life of approximately 12 months

• Avoid repeated freeze-thaw cycles as this significantly reduces activity

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to collect contents at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being optimal) for long-term storage

• Divide into small aliquots to minimize freeze-thaw cycles

Critical Factors Affecting Shelf Life:

• Storage temperature (with colder temperatures generally providing better stability)

• Buffer composition (pH, salt concentration)

• Presence of stabilizing agents (glycerol, reducing agents)

• Protein concentration

• Intrinsic stability of the protein itself

Researchers should validate protein activity after extended storage using appropriate functional assays depending on their specific experimental applications.

What experimental approaches can be used to assess the DNA repair activity of UvrB in vitro?

Multiple experimental approaches can be employed to assess the DNA repair activity of recombinant UvrB from Prochlorococcus marinus subsp. pastoris:

1. Reconstituted UvrABC Incision Assay:

• Prepare DNA substrates containing specific lesions (UV-induced pyrimidine dimers or chemical adducts)

• Incubate with purified UvrA, UvrB, and UvrC proteins under appropriate buffer conditions

• Analyze the incision products by gel electrophoresis

• Quantify the percentage of DNA substrates cleaved as a measure of repair activity

2. DNA Binding Assays:

• Electrophoretic mobility shift assays (EMSA) to assess UvrB binding to damaged DNA

• Fluorescence anisotropy measurements using fluorescently labeled DNA substrates

• Surface plasmon resonance (SPR) to determine binding kinetics and affinity

3. ATPase Activity Assays:

• Measure ATP hydrolysis rates in the presence of damaged DNA, as UvrB has DNA-dependent ATPase activity

• Use colorimetric assays (e.g., malachite green) or radiometric assays with [γ-32P]ATP

4. Helicase Unwinding Assays:

• Prepare DNA substrates with partial duplex regions

• Monitor unwinding activity through fluorescence-based or gel-based methods

5. Single-Molecule Approaches:

• Atomic force microscopy (AFM) to visualize UvrB-DNA complexes

• FRET-based assays to monitor conformational changes during repair

When conducting these assays, it's essential to include appropriate positive controls (e.g., well-characterized UvrB proteins from E. coli) and negative controls (e.g., catalytically inactive UvrB mutants).

How can researchers optimize the expression and purification of recombinant UvrB from Prochlorococcus marinus?

Optimizing expression and purification of recombinant UvrB from Prochlorococcus marinus requires careful consideration of expression systems, growth conditions, and purification strategies:

Expression System Selection:

• Yeast expression systems are commonly used for this protein

• Consider evaluating E. coli-based systems with codon optimization for marine cyanobacterial sequences

• For challenging expressions, insect cell or mammalian cell systems may be considered

Expression Optimization:

• Modify induction parameters (temperature, inducer concentration, duration)

• Test different promoter strengths and vector systems

• Co-express with molecular chaperones if folding problems occur

• Optimize media composition and growth conditions

Purification Strategy:

• Implement affinity chromatography using appropriate tags (His-tag, GST, etc.)

• Include a tag removal step if the tag interferes with activity

• Further purify using ion-exchange and/or size-exclusion chromatography

• Verify purity using SDS-PAGE (aim for >85% purity)

Solubility Enhancement:

• Express as fusion proteins with solubility-enhancing partners

• Optimize buffer conditions during lysis and purification

• Consider adding stabilizing agents (glycerol, reducing agents)

• Test truncated constructs if full-length protein proves problematic

Quality Control Measurements:

• Assess protein homogeneity through dynamic light scattering

• Verify proper folding using circular dichroism spectroscopy

• Perform functional assays to confirm activity

• Conduct mass spectrometry analysis to confirm protein identity

These optimization strategies should be tailored to the specific requirements of the downstream applications for which the recombinant UvrB will be used.

How does UvrB from Prochlorococcus marinus compare structurally and functionally to UvrB homologs in other organisms?

Comparative analysis of UvrB from Prochlorococcus marinus with homologs from other organisms reveals both conserved features and unique adaptations:

Structural Comparisons:

• Core structural domains responsible for ATP binding and DNA interaction are conserved across prokaryotic UvrB proteins

• Prochlorococcus UvrB likely contains the characteristic β-hairpin structure responsible for DNA damage verification as seen in other UvrB proteins

• May exhibit specific adaptations in surface-exposed regions that facilitate interactions with other Prochlorococcus-specific repair proteins

Functional Comparisons:

• The fundamental mechanism of damage recognition and incision is conserved across prokaryotes

• Studies in E. coli have shown that UvrB functions in multiple repair pathways , and similar pathway integration likely exists in Prochlorococcus

• UV dose-response experiments have demonstrated that Prochlorococcus exhibits differential sensitivity to UV radiation compared to other cyanobacteria like Synechococcus , which may reflect differences in UvrABC system efficiency

Comparative Genomic Analysis:

• Unlike in E. coli where uvrA and uvrB form an operon, in cyanobacteria including Prochlorococcus, these genes are not organized in an operon structure

• In some cyanobacterial genomes, uvrA, uvrB, uvrC, and/or uvrD are clustered with other DNA repair genes such as phr or recN

• The UvrABC system genes are universally present across cyanobacterial genomes, suggesting their fundamental importance in DNA repair

These comparisons highlight that while the core functions of UvrB are conserved across prokaryotes, Prochlorococcus may have evolved specific adaptations in its UvrB protein to optimize DNA repair efficiency in its unique ecological niche.

What evolutionary patterns are observed in UvrB across Prochlorococcus ecotypes from different ocean depths?

The evolutionary patterns of UvrB across Prochlorococcus ecotypes reveal adaptations to distinct light regimes and UV exposure at different ocean depths:

Ecotype Differentiation and UvrB Evolution:

Prochlorococcus has evolved into distinct ecotypes adapted to different light conditions:

• High-light (HL) adapted ecotypes (e.g., MED4) dominate surface waters

• Low-light (LL) adapted ecotypes (e.g., SS120, MIT9313) thrive at deeper ocean levels

Analysis of DNA repair genes across these ecotypes shows:

Genomic Context Variations:

• Comparative genomic analyses reveal different genomic organizations of repair genes across ecotypes

• High-light adapted strains show evidence of genome streamlining with the UvrABC genes maintained as essential components

• Low-light adapted strains maintain more comprehensive DNA repair systems

Selection Pressures:

• Genes involved in DNA repair, including UvrB, show evidence of being under different selection pressures in different ecotypes

• In high-light ecotypes, UvrB may have evolved for rapid repair efficiency due to higher UV damage rates

• In low-light ecotypes, UvrB may have co-evolved with other repair systems for complementary functions

These evolutionary patterns suggest that UvrB has been differentially selected across Prochlorococcus ecotypes as part of their adaptation to specific light niches in the ocean water column.

What role does horizontal gene transfer play in the evolution of DNA repair systems in Prochlorococcus?

Horizontal gene transfer (HGT) has significantly influenced the evolution of DNA repair systems in Prochlorococcus, contributing to its remarkable ecological success and adaptation to diverse marine environments:

Evidence for HGT in Prochlorococcus DNA Repair:

• Comparative genomic analyses have revealed that Prochlorococcus strains contain unique arsenals of genes that have been horizontally acquired, contributing to their physiological diversity

• Specifically for DNA repair genes, phylogenetic incongruence analyses indicate that some repair genes show evolutionary histories distinct from core genome phylogeny

• The distribution pattern of certain DNA repair genes across Prochlorococcus strains suggests acquisition and loss events through horizontal transfer

Mechanisms and Sources of HGT:

• Phage-mediated transduction appears to be a significant mechanism for HGT in Prochlorococcus

• Gene transfer from heterotrophic bacteria sharing the same marine environment

• Recent studies have identified intercellular membrane nanotubes between marine cyanobacteria that may facilitate direct genetic exchange

Functional Consequences of HGT for DNA Repair:

• Acquisition of novel repair genes has likely expanded the repair capacity in some lineages

• Genes involved in pigment biosynthesis and binding have been horizontally acquired, contributing to light adaptation which indirectly affects UV exposure and DNA damage patterns

• HGT events may have contributed to niche-specific adaptations in DNA repair efficiency

A particularly notable example is found in the LLVII and LLVIII strains of Prochlorococcus, where horizontal acquisition of photosynthetic antenna genes coincides with adaptations to deep, low-light environments . The concomitant adjustments in DNA repair systems suggest co-evolution of light harvesting and DNA repair mechanisms to balance energy acquisition with genome protection.

The detection of such HGT events has been facilitated by advanced bioinformatic approaches specifically designed to identify genomic regions associated with horizontal gene transfer in prokaryotes .

How can UvrB be used as a model to study adaptive evolution in marine microbial populations?

UvrB provides an excellent model for studying adaptive evolution in marine microbial populations, offering insights into how environmental pressures shape genomic maintenance strategies:

Experimental Approaches for Studying Adaptive Evolution:

• Comparative Genomic Analysis:

  • Analyze UvrB sequence variation across Prochlorococcus strains from different oceanographic regions

  • Identify positively selected amino acid residues that may confer adaptive advantages

  • Compare UvrB evolution rates with other genes to detect selective pressures

• Experimental Evolution Studies:

  • Subject Prochlorococcus cultures to varying UV regimes to observe real-time evolution of UvrB

  • Sequence evolved populations to identify emergent mutations in UvrB and related repair genes

  • Measure repair efficiency changes in evolved populations

• Field-Based Approaches:

  • Sample natural Prochlorococcus populations along depth gradients to correlate UvrB variants with UV exposure

  • Analyze seasonal variations in UvrB allele frequencies in relation to changing light conditions

  • Compare UvrB diversity in different oceanic regions with varying UV penetration depths

Research Applications for Understanding Adaptive Evolution:

UvrB serves as a valuable marker for testing evolutionary theories because:

• It exhibits differential selective pressures at various ocean depths due to UV gradient

• Its function is directly linked to survival under environmental stress

• It shows evidence of both vertical inheritance and horizontal gene transfer

Recent studies have employed artificial neural network (ANN) analyses to identify genetic differences between high-light and low-light adapted Prochlorococcus strains . Similar approaches could be applied specifically to UvrB to develop predictive models for how DNA repair mechanisms evolve in response to changing environmental conditions.

This research direction not only illuminates evolutionary mechanisms but also provides insights into how marine microbes might respond to increased UV radiation resulting from climate change-induced stratification changes in ocean systems.

What is the relationship between UvrB function and the ecological distribution of Prochlorococcus in global oceans?

The function of UvrB in DNA repair has significant implications for the ecological distribution of Prochlorococcus across global oceans:

Geographic and Vertical Distribution Patterns:

Prochlorococcus is the most abundant photosynthetic organism on Earth, with a distribution primarily within the 40°S to 40°N latitudinal band and from the surface down to depths of 200m . This distribution pattern correlates with variations in UV exposure and consequent DNA damage:

• Surface waters (0-50m): Dominated by high-light adapted ecotypes with potentially enhanced UvrB-mediated repair efficiency

• Mid-depths (50-100m): Mixed ecotypes with balanced repair systems

• Deeper waters (>100m): Low-light adapted ecotypes with specialized repair strategies

Experimental Evidence of UV Sensitivity Differences:

Studies comparing Prochlorococcus and Synechococcus have demonstrated differential sensitivity to UV radiation:

• Prochlorococcus strains (PCC 9511 and SS120) showed greater sensitivity to high irradiance than Synechococcus strains

• Field measurements confirmed that natural Prochlorococcus populations exhibited lower resistance to UVR than Synechococcus in surface waters of both the Atlantic Ocean and Mediterranean Sea

Ecological Modeling Based on UvrB Function:

The efficiency of DNA repair mechanisms, including UvrB-mediated repair, influences:

• Vertical distribution limits in the water column

• Daily vertical migration patterns in response to changing UV exposure

• Seasonal abundance patterns in relation to solar intensity variations

This relationship can be visualized in a conceptual model:

Recent modeling approaches have successfully predicted the vertical distribution of Prochlorococcus using remote sensing data that accounts for light penetration in the water column . These models could be enhanced by incorporating knowledge of DNA repair efficiency across ecotypes to better predict Prochlorococcus distribution under changing climate scenarios.

How does the UvrABC system in Prochlorococcus interact with other DNA repair pathways under different environmental stressors?

The UvrABC system in Prochlorococcus interacts with multiple DNA repair pathways in a complex network that responds dynamically to different environmental stressors:

Integration with Other Repair Pathways:

UvrABC-mediated nucleotide excision repair (NER) functions within a network of repair systems including:

• Photoreactivation via photolyase (light-dependent direct reversal)

• Recombinational repair (RecA, RecFOR, RecBCD)

• Base excision repair

• SOS response system

• Translesion synthesis (TLS) using specialized polymerases

Pathway Selection Under Different Stressors:

Molecular Mechanisms of Pathway Coordination:

Research evidence suggests several coordination mechanisms:

• Transcriptional regulation through stress-specific response regulators

• Post-translational modifications affecting protein activity

• Substrate competition between pathways

• Physical interactions between components of different repair systems

In Prochlorococcus, these interactions have unique characteristics:

• Diel cycling of DNA repair gene expression synchronized with the cell cycle

• Differential sensitivity to hydrogen peroxide and oxidative stress compared to other cyanobacteria like Synechococcus

• Genome streamlining has potentially eliminated redundant repair pathways, making remaining systems like UvrABC more critical

Advanced research studying these interactions employs:

• RNA-seq to detect co-expression patterns under different stressors

• ChIP-seq to identify regulatory network connections

• Protein-protein interaction studies to map physical repair complexes

• Genetically modified strains with specific pathway components disabled to assess compensatory mechanisms

Understanding these pathway interactions is essential for predicting how Prochlorococcus populations will respond to multiple simultaneous stressors in changing ocean environments.

What emerging technologies could advance our understanding of UvrB function in Prochlorococcus?

Several cutting-edge technologies show promise for advancing our understanding of UvrB function in Prochlorococcus:

CRISPR-Based Approaches:

• Development of targeted genome editing for Prochlorococcus to create UvrB variants

• CRISPRi for conditional knockdown of UvrB to study essentiality under different conditions

• Base editing to introduce specific mutations in the uvrB gene to test structure-function relationships

Advanced Imaging Techniques:

• Super-resolution microscopy to visualize UvrB localization during DNA repair in single cells

• Live-cell imaging using fluorescently tagged UvrB to track repair dynamics in real-time

• Correlative light and electron microscopy to link UvrB activity with cellular ultrastructure

Single-Cell Technologies:

• Single-cell RNA-seq to measure uvrB expression heterogeneity within Prochlorococcus populations

• Single-cell proteomics to quantify UvrB protein levels and modifications

• Microfluidic approaches to isolate and study individual cells under controlled UV exposure

Computational and Systems Biology Approaches:

• Deep learning neural networks to predict UvrB function from sequence variations

• Molecular dynamics simulations to understand UvrB-DNA interactions at atomic resolution

• Systems biology modeling to integrate UvrB function within the complete DNA repair network

Synthetic Biology Applications:

• Designer UvrB variants with enhanced repair capabilities for studying evolutionary constraints

• Biosensor development using UvrB to detect environmental DNA damaging agents

• Synthetic circuitry to control UvrB expression for studying repair capacity limits

Field-Based Technologies:

• In situ gene expression assays for monitoring uvrB activation in natural populations

• Environmental DNA (eDNA) analysis to track UvrB variants across oceanic regions

• Integration with autonomous underwater vehicles for sampling across oceanographic features

These emerging technologies will help bridge laboratory findings with ecological understanding, providing new insights into how UvrB function contributes to Prochlorococcus success as the most abundant photosynthetic organism on Earth.

How might climate change affect UvrB-mediated DNA repair in Prochlorococcus populations?

Climate change is expected to significantly impact UvrB-mediated DNA repair in Prochlorococcus populations through multiple interacting factors:

Predicted Climate Change Impacts:

• Increased UV Radiation Exposure:

  • Stratification strengthening will trap Prochlorococcus cells closer to the surface

  • Ozone layer changes may increase harmful UV-B radiation reaching oceans

  • Clearer waters due to reduced particulate matter in some regions will increase UV penetration depth

• Temperature Effects:

  • Rising ocean temperatures will influence enzyme kinetics of UvrB

  • Thermal stress may compete for cellular resources needed for DNA repair

  • Range shifts of Prochlorococcus populations into new temperature regimes

• Ocean Acidification:

  • pH changes may alter protein stability and activity of UvrB

  • Potential synergistic effects with other stressors on DNA damage rates

• Nutrient Availability Changes:

  • Altered nutrient regimes may limit resources for DNA repair protein synthesis

  • Iron limitation could affect electron transport chains, increasing oxidative stress

Predicted Evolutionary Responses:

Research Approaches to Study These Effects:

• Experimental evolution under simulated future ocean conditions

• Mesocosm experiments with manipulated climate variables

• Comparative genomics of UvrB across existing climate gradients as proxies for future conditions

• Mathematical modeling of repair capacity versus damage rate under different scenarios

Preliminary studies comparing UV sensitivity between Prochlorococcus and Synechococcus suggest that Prochlorococcus may be more vulnerable to increased radiation , potentially making UvrB function a critical determinant of future ecological success under climate change scenarios.

What potential applications exist for genetically engineered UvrB variants in biotechnology and environmental monitoring?

Genetically engineered UvrB variants derived from Prochlorococcus research offer promising applications in both biotechnology and environmental monitoring:

Biotechnological Applications:

• Enhanced DNA Repair for Synthetic Biology:

  • Engineered UvrB variants could improve the genomic stability of synthetic organisms

  • Custom repair systems optimized for specific types of DNA damage in industrial microbes

  • UvrB components could be incorporated into minimal synthetic genomes

• Protein Engineering Platforms:

  • UvrB's DNA binding and verification mechanisms could be repurposed for targeted DNA modifications

  • Engineered variants could serve as scaffolds for novel DNA processing enzymes

  • Structure-guided design could create specialized DNA damage sensors

• Therapeutic Potential:

  • UvrB-inspired biomolecules for targeting and repairing damaged DNA in human cells

  • Potential applications in UV-induced skin damage protection

  • Disease models for understanding nucleotide excision repair deficiencies

Environmental Monitoring Applications:

• Biosensor Development:

  • Engineered UvrB-based biosensors for detecting genotoxic compounds in marine environments

  • Real-time monitoring systems for UV radiation impacts on aquatic ecosystems

  • Integration with autonomous underwater vehicles for broad spatial coverage

• Environmental DNA Damage Assessment:

  • UvrB-based assays to quantify DNA damage in natural microbial communities

  • Tools for assessing ecosystem health through DNA damage metrics

  • Standardized tests for environmental impact assessments

• Climate Change Research:

  • Reporter systems using UvrB to track changing UV stress in marine environments

  • Tools for monitoring evolutionary responses in repair systems

  • Integration with ocean observing systems for long-term monitoring

Technical Requirements for Development:

Progress in these applications requires:

• Detailed structure-function understanding of UvrB domains

• Optimization of expression and stability in diverse systems

• Development of high-throughput screening methods for engineered variants

• Integration with existing biosensor platforms

• Field-testing under relevant environmental conditions

These applications represent translational opportunities arising from fundamental research on UvrB in Prochlorococcus, highlighting how studies of environmentally adapted DNA repair systems can contribute to both fundamental science and applied technologies.